Research ArticleAntiproliferative Effects of Methanolic Extracts ofCryptocarya concinna Hance Roots on Oral Cancer Ca9-22 andCAL 27 Cell Lines Involving Apoptosis ROS Induction andMitochondrial Depolarization
Hurng-Wern Huang1 Yi-An Chung1 Hsun-Shuo Chang23 Jen-Yang Tang456
Ih-Sheng Chen23 and Hsueh-Wei Chang678
1 Institute of Biomedical Science National Sun Yat-Sen University Kaohsiung 80424 Taiwan2Graduate Institute of Natural Products College of Pharmacy Kaohsiung Medical University Kaohsiung 80708 Taiwan3 School of Pharmacy College of Pharmacy Kaohsiung Medical University Kaohsiung 80708 Taiwan4Department of Radiation Oncology Faculty of Medicine College of Medicine Kaohsiung Medical UniversityKaohsiung 80708 Taiwan
5Department of Radiation Oncology Kaohsiung Medical University Hospital Kaohsiung 80708 Taiwan6Cancer Center Translational Research Center Kaohsiung Medical University Hospital Kaohsiung Medical UniversityKaohsiung 80708 Taiwan
7 Institute of Medical Science and Technology National Sun Yat-sen University Kaohsiung 80424 Taiwan8Department of Biomedical Science and Environmental Biology Kaohsiung Medical University Kaohsiung 80708 Taiwan
Correspondence should be addressed to Ih-Sheng Chen m635013kmuedutw and Hsueh-Wei Chang changhwkmuedutw
Received 16 July 2014 Accepted 25 August 2014 Published 15 October 2014
Academic Editor Shao-Yu Chen
Copyright copy 2014 Hurng-Wern Huang et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited
Cryptocarya-derived natural products were reported to have several biological effects such as the antiproliferation of some cancersThe possible antioral cancer effect of Cryptocarya-derived substances was little addressed as yet In this study we firstly used themethanolic extracts of C concinna Hance roots (MECCrt) to evaluate its potential function in antioral cancer bioactivity Wefound that MECCrt significantly reduced cell viability of two oral cancer Ca9-22 and CAL 27 cell lines in dose-responsive manners(119875 lt 001) The percentages of sub-G1 phase and annexin V-positive of MECCrt-treated Ca9-22 and CAL 27 cell lines significantlyaccumulated (119875 lt 001) in a dose-responsive manner as evidenced by flow cytometry These apoptotic effects were associated withthe findings that intracellular ROS generation was induced in MECCrt-treated Ca9-22 and CAL 27 cell lines in dose-responsiveand time-dependent manners (119875 lt 001) In a dose-responsive manner MECCrt also significantly reduced the mitochondrialmembrane potential in these two cell lines (119875 lt 001ndash005) In conclusion we demonstrated thatMECCrtmay have antiproliferativepotential against oral cancer cells involving apoptosis ROS generation and mitochondria membrane depolarization
1 Introduction
Oral squamous cell carcinoma (OSCC) is a type of cancer thatfrequently occurs in oral cavity Although it is comparativelyeasy to clinically inspect by a dentist or to detect by someOSCC tumormarkers [1 2] this carcinoma is usually ignored
by patients especially for the early stage Subsequently OSCCis frequently diagnosed at advanced stages which then leadto high mortality [3] Therefore the drug development ofantioral cancer is still necessary and remains to be a challenge
Natural products have improved the drug discoveryfor anticancer therapy [4] For example some anticancer
Hindawi Publishing Corporatione Scientific World JournalVolume 2014 Article ID 180462 10 pageshttpdxdoiorg1011552014180462
2 The Scientific World Journal
drugs derived from natural products were approved by theUnited States Food and Drug Administration [5] In basicresearches natural products with antioral cancer effects haveincreasingly being reported This holds for the ethanolic andmethanolic extracts of red algaGracilaria tenuistipitata [6 7]crude extracts of Selaginella tamariscina (oriental medicinalherb) [8] green tea [9] goniothalamin from Goniothalamusspecies [10] and 4120573-hydroxywithanolide E fromgolden berry[11]
Cryptocarya plants (family Lauraceae) comprising about350 species worldwide are widely distributed in the tropicsand subtropics [12] This plant group is well known forits common secondary metabolites containing alkaloidsflavonoids and 120572-pyrones [12ndash15] Several biological effectsof Cryptocarya-derived natural products have been reportedthat include anti-dengue virus [16] anti-HIV [17] anti-tuberculosis [18] antiplasmodial [19] antitrypanosomal [20]and anti-inflammatory [21] function
Anticancer effects of crude extracts of Cryptocarya plantare known as well For example the ethanolic extracts of fruitand trunk bark of C obovata showed 56 and 23 growthinhibition of human KB cells at 10 120583gmL respectively [22]Methanolic extracts of the leaves of C griffithiana providecytotoxicity forhuman HL60 promyelocytic leukemia cells[23]
Recently accumulating findings for anticancer effectsof pure compounds isolated from Cryptocarya plants werereported especially from methanolic extracts For examplecompounds isolated frommethanol extracts of the trunk barkof C infectoria [24] the trunk bark of C costata [25] andthe wood of C konishii [26] were reported to be cytotoxicto leukemia cells Compounds from methanolic extracts ofleaves of C chinensis were shown to be cytotoxic to humanlung cancer and glioblastoma cells [27] These drugs wereisolated from the trunk bark wood and leaves ofCryptocaryasp However the bioactivity of the roots of Cryptocaryaplants remained little investigated particularly with respectto antioral cancer
Because C concinna Hance is an evergreen plant com-monly distributed in low-altitude forests in Taiwan [28] it iseasy to preparemethanolic extracts of the roots ofC concinnaHance (namely forMECCrt)We therefore chose twoOSCCcell lines that is Ca9-22 and CAL 27 to evaluate the possibleanticancer function of MECCrt and investigate their drugmechanisms in terms of cell viability cell cycle distributionapoptosis reactive oxygen species (ROS) generation andmitochondrial depolarization
2 Materials and Methods
21 Cell Cultures and Methanolic Extracts of C concinnaTwo human OSCC cell lines Ca9-22 and CAL 27 purchasedfrom the Cell Bank RIKEN BioResource Center (TsukubaJapan) and the American Type Culture Collection (ATCCVirginia USA) respectively were incubated in DMEMF12(3 2) medium (Gibco Grand Island NY USA) supple-mented with 10 fetal bovine serum (Gibco) 100UmLpenicillin 100 120583gmL streptomycin and 003 glutamine
These two cell lines were humidly incubated at 37∘C with 5CO2in the humid atmosphere
C concinna was identified by one of the authors (Ih-Sheng Chen) and its roots were collected at Mudan PingtungCounty Taiwan in May 2004 A voucher specimen (Chen6153) has been deposited in the Herbarium of the Schoolof Pharmacy College of Pharmacy Kaohsiung MedicalUniversity The dried roots of C concinna were processedby slicing and cold methanol-extraction for three times atroom temperature Finally the solutionwas evaporated underreduced pressure to yield the methanolic extract (MECCrt)MECCrt was stored at minus20∘C and dissolved in dimethylsulfoxide (DMSO) before treatment
22 Cell Viability Cell viability was measured by theCellTiter 96 AQueous one solution cell proliferation assay(MTS) (Promega Corporation Madison WI USA) as pre-viously described [11] Ca9-22 and CAL 27 cell lines wereseeded at a density of 1 times 105 and 2 times 105 cells per well ina 6-well plate respectively After plating for 24 h these cellswere incubated with different concentrations of MECCrt for24 h and finally subjected to a MTS assay applying an ELISAreader at 490 nm
23 Cell Cycle Progression and Sub-G1 Population Propidiumiodide (PI Sigma St Louis MO USA) was added to stainthe cellular DNA content [29] In brief 3 times 105 cells perwell in 6 well plates were plated for 24 h and then treatedwith vehicle (DMSO 1120583L2mL culture medium) as a controlor 5 10 15 20 and 25 120583gmL of MECCrt for 24 h Afterexposure termination cells were centrifuged washed twicewith PBS fixed overnight with 70 ethanol and centrifugedSubsequently the cell pellets were resuspended in 50120583gmLPI reagent and stand for 30min at 37∘C in darkness Cellcycle distribution was evaluated by a flow cytometer (BDAccuri C6 Becton-Dickinson Mansfield MA USA) and aBD Accuri C6 Software (version 10264)
24 Apoptosis To validate apoptosis in MECCrt-treated oralcancer cells annexin V (Strong Biotect Corporation TaipeiTaiwan) [30]PI (Sigma St Louis MO USA) method wasused [31] Briefly 3 times 105 cells per well in 6 well plates wereplated for 24 h and then treated with vehicle or indicated con-centrations of MECCrt for 24 h Subsequently apoptotic cellswere stained for 30minwith 100120583L binding buffer containing2 120583L of annexin-V-fluorescein isothiocyanate (FITC) stock(025 120583g120583L) and 2120583L of PI stock (1mgmL) Finally it wassuspended with 400 120583L PBS for analysis of a flow cytometer(BD Accuri C6 Becton-Dickinson) and its software
25 Intracellular ROS The dye 2101584071015840-dichlorodihydroflu-orescein diacetate (DCFH-DA) was used to detect ROS byits fluorescence change [7] Cells at the density of 3 times 105 in2mL medium per well in 6 well plates were plated for 24 hDifferent concentrations of MECCrt were added to Ca9-22cells for 6 h and 12 h After washing with PBS 100 nMDCFH-DA in PBS was added to cells in 6 well plates at cell cultureincubator for 30min After trypsinization PBS washing and
The Scientific World Journal 3
120
100
80
60
40
20
0
0 5 10 2015
Cel
l via
bilit
y (
)Ca9-22
MECCrt (120583gmL)
lowastlowast
lowastlowast
lowastlowast
lowastlowast
(a)
0 5 10 2015
Cel
l via
bilit
y (
)
MECCrt (120583gmL)
CAL 27140
120
100
80
60
40
20
0
lowastlowast
lowastlowast
lowastlowastlowastlowast
(b)
Figure 1 Cell viability of two oral cancer cells was inhibited by MECCrt Oral cancer Ca9-22 and CAL 27 cell lines were treated with variousconcentrations ofMECCrt (0 5 10 15 and 20 120583gmL) for 24 hThe cell viability was measured by theMTS assay Data means plusmn SDs (119899 = 18)lowastlowast
119875 lt 001 against vehicle
centrifugation cell pellets were resuspended in 1mL PBSbefore analyzing by a flow cytometer (BDAccuri C6 Becton-Dickinson) and its software
26 Mitochondrial Membrane Potential MitoProbeDiOC
2(3) assay kit (Invitrogen Eugene OR USA) was
applied to analyze mitochondrial membrane potential(MMP) as described previously [10] Briefly 3 times 105 cellsin 2mL medium per well in 6 well plates were plated for24 h After MECCrt treatment 10 120583L of 10 120583M DiOC
2(3)
was added per well and incubated in a cell culture incubatorfor 20min After being harvested cells were washedand resuspended in 1mL PBS for analysis using a flowcytometer (BD Accuri C6 Becton-Dickinson) and itssoftware
27 Statistical Analysis The significance of differences wasdetermined by Studentrsquos 119905-test compared with the test datawith the vehicle controls Data are expressed as means plusmn SDs
3 Results
31 Antiproliferation in MECCrt-Treated Two Oral CancerCell Lines Based on MTS assay (Figure 1) the relative cellviability () of oral cancer Ca9-22 cells at indicated concen-trations of MECCrt (0 5 10 15 and 20120583gmL) was 1000 plusmn07 933 plusmn 23 714 plusmn 30 576 plusmn 16 and 484 plusmn 12 after24 h respectively The relative cell viability () of CAL 27cells at indicated concentrations of MECCrt (0 5 10 15 and20120583gmL) was 1000 plusmn 081193 plusmn 49 869 plusmn 100 298 plusmn 62and 284 plusmn 55 respectively The MTS-based cell viabilities ofMECCrt-treated twooral cancerCa9-22 andCAL27 cell linessignificantly reduced in a dose-responsive manner (119875 lt 001compared to the vehicle)
32 Sub-G1 Population in MECCrt-Treated Two Oral Can-cer Cell Lines The MECCrt-treated effects of cell cycledistribution profiles are demonstrated in Figure 2(a) AfterMECCrt treatment (Figure 2(b)) the sub-G1 populations ()ofMECCrt- (0 5 10 15 20 and 25 120583gmL) treated oral cancerCa9-22 cells were 50 plusmn 03 67 plusmn 01 181 plusmn 06 179 plusmn 07165plusmn03 and 224plusmn13 and those ofMECCrt-treatedCAL 27cells were 67plusmn17 51plusmn11 79plusmn01 283plusmn10 526plusmn02 and691 plusmn 01 respectively These sub-G1 changes significantlyaccumulated in a dose-responsive manner (119875 lt 001)
33 Apoptosis of MECCrt-Treated Two Oral Cancer Cell LinesTo validate the possible outcome of apoptosis in MECCrt-induced sub-G1 accumulation of these two oral cancer cellsannexin VPI profiles of flow cytometry were generated(Figure 3(a)) In Figure 3(b) the percentages of annexin V-positive intensities forMECCrt (0 5 10 15 20 and 25120583gmL)treatment of Ca9-22 cells were 93 plusmn 02 86 plusmn 03 117 plusmn 09223 plusmn 08 400 plusmn 02 and 544 plusmn 17 and those of MECCrt-treated CAL 27 cells were 248 plusmn 01 175 plusmn 03 207 plusmn 04598plusmn17 794plusmn02 and 853plusmn05 respectively AccordinglyMECCrt treatments significantly increased in annexin V-positive intensities of two oral cancer Ca9-22 and CAL 27 celllines in a dose-responsive manner (119875 lt 001)
34 ROSGeneration inMECCrt-Treated TwoOral Cancer CellLines To validate the role of ROS in the MECCrt-inducedapoptosis of two oral cancer cell lines a DCFH-DA assay offlow cytometry was chosen Figures 4(a) and 4(b) show therelative ROS-positive staining () of two oral cancer Ca9-22 and CAL 27 cell lines for the different concentrations ofMECCrt treatment for 6 and 12 h incubation After MECCrttreatment for 6 h the relative ROS-positive staining () of 05 10 15 20 and 25 120583gmL MECCrt-treated Ca9-22 cells was
4 The Scientific World Journal
Cou
ntCa
9-22
Cou
ntCa
9-22
0 120583gmL 5120583gmL 10120583gmL
15120583gmL 20120583gmL 25120583gmL
400
300
200
100
0
DNA content DNA content DNA content
400
300
200
100
0
Sub-G147G1
492S
177G2M288
Sub-G168G1
529S
182G2M232
Sub-G1171
G1370
S127G2M285
Sub-G1158
G1307
S179G2M265
Sub-G1141
G1266
S212G2M235
Sub-G1200
G1293
S201G2M208
(a)
Cou
ntC
ount
0120583gmL 5120583gmL 10120583gmL
15120583gmL 20120583gmL 25120583gmL
400
300
200
100
0
DNA content DNA content DNA content
400
300
200
100
0
Sub-G148G1
539S
201G2M216
Sub-G151G1
548S
214G2M192
Sub-G1G1
539S
201G2M216
Sub-G1279
G1322
S230G2M184
Sub-G1535
G1248
S122G2M109
Sub-G1699
G1181
SG2M
48
78
54
CAL 27
CAL 27
(b)
Sub-
G1
()
MECCrt (120583gmL)
lowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowast20
20
10
10
5
5
0
0
25
25
15
15
(c)
Sub-
G1
()
lowastlowast
lowastlowast
lowastlowast
0
10
20
30
40
50
60
70
80
MECCrt (120583gmL)201050 2515
(d)
Figure 2 The sub-G1 accumulation of two oral cancer cells was induced by MECCrt Oral cancer Ca9-22 and CAL 27 cell lines were treatedwith 0 5 10 15 20 and 25 120583gmL of MECCrt for 24 h ((a) and (b)) Representative cell cycle distribution profiles of flow cytometry forMECCrt-treated oral cancer Ca9-22 and CAL 27 cells and vehicles at 24 h respectively ((c) and (d)) Statistics analyses for the percentages ofsub-G1 population in (a) and (b) respectively Data means plusmn SDs (119899 = 3) lowastlowast119875 lt 001 against vehicle
1000 plusmn 18 1067 plusmn 04 1312 plusmn 08 1406 plusmn 16 1506 plusmn 03and 1831 plusmn 78 and that of MECCrt-treated CAL 27 cells was1000 plusmn 08 1141 plusmn 14 1423 plusmn 15 1611 plusmn 07 1798 plusmn 11and 1851 plusmn 14 respectively After MECCrt treatment for12 h the relative ROS-positive staining () of 0 5 10 1520 and 25120583gmL MECCrt-treated Ca9-22 cells was 1000 plusmn23 1241 plusmn 04 1664 plusmn 12 1848 plusmn 06 1933 plusmn 03 and2005 plusmn 00 and that of MECCrt-treated CAL 27 cells was1000plusmn09 1407plusmn14 1771plusmn03 1938plusmn01 1986plusmn01 and1994 plusmn 01 respectively Accordingly MECCrt treatmentssignificantly increased in both dose-responsive and time-dependent manners in these two oral cancer cell lines (119875 lt005) (Figures 4(c) and 4(d))
35 MMP Depolarization in MECCrt-Treated Two Oral Can-cer Cell Lines Figures 5(a) and 5(b) show the MMP profilesof DiOC
2(3)-positive intensities for the vehicle andMECCrt-
treated oral cancer cell lines in 24-hour treatments Treated
with MECCrt (0 5 10 15 20 and 25120583gmL) for 24 hthe DiOC
2(3)-positive () intensities of Ca9-22 cells were
1000 plusmn 29 961 plusmn 24 940 plusmn 16 768 plusmn 14 456 plusmn 14and 250 plusmn 11 respectively Similarly the percentages ofDiOC
2(3)-positive () intensities ofMECCrt-treatedCAL27
cells were 1000 plusmn 15 1142 plusmn 07 1089 plusmn 13 513 plusmn 05273 plusmn 05 and 75 plusmn 04 respectively Accordingly MECCrtsignificantly reduced DiOC
2(3)-positive intensities of two
oral cancer Ca9-22 and CAL 27 cell lines in a dose-responsivemanner (119875 lt 001ndash005)
4 Discussion
We discovered for the first time that methanolic extracts ofthe roots ofC concinnaHance have an antiproliferative effecton two oral cancer cell lines The proliferation inhibitingfunction of MECCrt against oral cancer Ca9-22 and CAL 27cell lines was dose-responsive (Figure 1)
The Scientific World Journal 5
104
105
106
1027
104
105
106
1027
1031
104
105
10610
6410
3110
410
510
610
6410
3110
410
510
610
64
Ca9
-22
0 120583gmL 5120583gmL 10120583gmL
15120583gmL 20120583gmL 25120583gmLAnnexin V intensity Annexin V intensity Annexin V intensity
PI in
tens
ityCa
9-22
PI in
tens
ity
(a)
15120583gmL 20120583gmL 25120583gmLAnnexin V intensity Annexin V intensity Annexin V intensity
104
105
106
1027
104
105
106
1027
1031
104
105
10610
6410
3110
410
510
610
6410
3110
410
510
610
64
0 120583gmL 5120583gmL 10120583gmL
PI in
tens
ityPI
inte
nsity
CAL 27
CAL 27
(b)
Apop
totic
cells
()
lowastlowast
lowastlowast
lowastlowast
lowastlowast
60
50
40
30
20
10
0
0 5 10 15 20 25
MECCrt (120583gmL)
(c)
Apop
totic
cells
()
lowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowast
10
20
30
40
50
60
70
80
90
0 5 10 15 20 25
MECCrt (120583gmL)
(d)
Figure 3 Apoptosis of two oral cancer cells was induced byMECCrt Oral cancer Ca9-22 and CAL 27 cell lines were treated with 0ndash25120583gmLof MECCrt for 24 h ((a) and (b)) Representative results of annexin VPI double staining of flow cytometry for MECCrt-treated oral cancerCa9-22 and CAL 27 cell lines and vehicle controls at 24 h respectively ((c) and (d)) Quantification analysis of apoptosis for MECCrt-treatedoral cancer Ca9-22 and CAL 27 cell lines in (a) and (b) respectively Data means plusmn SDs (119899 = 3) lowastlowast119875 lt 001 against vehicle
6 The Scientific World Journal
Cou
ntCa
9-22
0 120583gmL 5120583gmL 10120583gmL
15120583gmL
400
300
200
100
0
Cou
ntCa
9-22
400
300
200
100
0
400
300
200
100
0
400
300
200
100
0
10 5000 10000 10 5000 10000 10 5000 10000 10 5000 10000 10 5000 10000 10 5000 10000
ROS intensity20120583gmL
ROS intensity ROS intensity25120583gmL 15120583gmL
ROS intensity20120583gmL
ROS intensity ROS intensity25120583gmL
6h 6h 6h0120583gmL 5120583gmL 10120583gmL12h 12h 12h
V2-L500
V2-R V2-L V2-R V2-L V2-R
V2-L V2-R V2-L V2-R V2-L V2-R
V3-L V3-R V3-L V3-R
V3-L V3-R
V3-L V3-R
V3-L V3-R V3-L V3-R
500 500 500
916 962 996
172 828618382471 529 347 653
298 702 255 745 119 881 84 38 04
(a)
Cou
nt
0120583gmL 5120583gmL 10120583gmL
15120583gmL
400
300
200
100
0
Cou
nt
400
300
200
100
0
400
300
200
100
0
400
300
200
100
0
10 5000 10000 10 5000 10000 10 5000 10000 10 5000 10000 10 5000 10000 10 5000 10000
ROS intensity20120583gmL
ROS intensity ROS intensity25120583gmL 15120583gmL
ROS intensity20120583gmL
ROS intensity ROS intensity25120583gmL
6h 6h 6h0120583gmL 5120583gmL 10120583gmL12h 12h 12h
V2-L500
V2-R V2-L V2-R V2-L V2-R
V2-L V2-R V2-L V2-R V2-L V2-R
V2-L V2-R V2-L V2-R
V2-L V2-R
V2-L V2-R
V2-L V2-R V2-L V2-R
500 500 500
970 993 998
112 888700300497 503 386 614
298 702 173 827 921 3079 07 02
CAL 27
CAL 27
(b)
lowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowast
Relat
ive R
OS-
posit
ive (
)
220
200
180
160
140
120
100
80
0 5 10 15 20 25
6h12h
MECCrt (120583gmL)
(c)
lowastlowastlowastlowastlowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowastlowastlowast
lowastlowastlowastlowast
Relat
ive R
OS-
posit
ive (
)
220
200
180
160
140
120
100
80
0 5 10 15 20 25 25
6h12h
MECCrt (120583gmL)
(d)
Figure 4 Reactive oxygen species (ROS) generation of two oral cancer cell lines was induced by MECCrt Oral cancer Ca9-22 and CAL 27cell lines were treated with different concentrations (0ndash25120583gmL) of MECCrt for 6 and 12 h ((a) (b)) Representative ROS profiles of flowcytometry for MECCrt-treated oral cancer Ca9-22 and CAL 27 cell lines ((c) and (d)) Statistics analysis of relative ROS intensity in (a) and(b) respectively Data means plusmn SDs (119899 = 3) lowastlowast119875 lt 001 against vehicle
The Scientific World Journal 7
500V1-L V1-R
500 517V1-L V1-R
483 539V1-L V1-R
461
620V1-L V1-R
380 770V1-L V1-R
230 872V1-L V1-R
128
Cou
ntCa
9-22
Cou
ntCa
9-22
0 120583gmL 5120583gmL 10120583gmL
15120583gmL 20120583gmL 25120583gmLMMP intensity MMP intensity MMP intensity
1200
1000
500
0
1200
1000
500
0
10110
210
310
410
510
610
7210
110
210
310
410
510
610
7210
110
210
310
410
510
610
72
(a)
500V2-L V2-R
500 440V2-L V2-R
560 455V2-L V2-R
545
744V2-L V2-R
256 868V2-L V2-R
132 964V2-L V2-R
Cou
ntC
ount
0120583gmL 5120583gmL 10120583gmL
15120583gmL 20120583gmL 25120583gmLMMP intensity MMP intensity MMP intensity
1200
1000
500
0
1200
1000
500
0
10110
210
310
410
510
610
7210
110
210
310
410
510
610
7210
110
210
310
410
510
610
72
36
CAL 27
CAL 27
(b)
lowast
lowastlowast
lowastlowast
lowastlowast
120
100
80
60
40
20
0
Relat
ive M
MP-
posit
ive (
)
0 5 10 15 20 25
MECCrt (120583gmL)
(c)
lowastlowastlowastlowast
lowastlowast
lowastlowast
lowastlowast
120
140
100
80
60
40
20
0
Relat
ive M
MP-
posit
ive (
)
0 5 10 15 20 25
MECCrt (120583gmL)
(d)
Figure 5 Depolarization of mitochondrial membrane potential (MMP) of Ca9-22 and CAL 27 oral cancer cell lines was induced byMECCrt Oral cancer Ca9-22 and CAL 27 cell lines were treated with different concentrations (0ndash25120583gmL) of MECCrt for 24 h ((a) (b))RepresentativeMMPprofiles of flow cytometry forMECCrt-treated oral cancer Ca9-22 andCAL 27 cells ((c) and (d))Quantification analysisof relative MMP intensity in (a) and (b) respectively Data means plusmn SDs (119899 = 3) lowast119875 lt 005 and lowastlowast119875 lt 001 against vehicle
8 The Scientific World Journal
The anticancer effects for other Cryptocarya-derivedcompounds from methanolic extracts of nonroot parts havebeen reported earlier For example for murine leukemiaP-388 cells the IC
50values of 2101584041015840-dihydroxy-5101584061015840-
dimethoxychalcone and isodidymocarpin isolated fromtree bark of C costata were 57 and 111 120583M [25] and IC
50
values of the chalcone derivative (desmethylinfectocaryone)and phenolic compound (infectocaryone) isolated fromwood of C konishii were 217 and 08 120583M [26] at 48 hrespectively For compounds from leaves of C chinensis theIC50
values of infectocaryone and cryptocaryanone A wereat the 120583M level for human lung cancer NCI-H460 cells andglioblastoma SF-268 cells [27] These Cryptocarya-derivedcompounds from methanolic extracts of nonroot partsshowed the IC
50values ranging from 08 to 11 120583M This is
close to our preliminary result that the IC50
of the clinicalanticancer drug cisplatin at 24 h treatment in oral cancerCa9-22 cells is 306 120583gmL (102 120583M) (data not shown) Inthe present study the IC
50values of the MECCrt in oral
cancer Ca9-22 and CAL 27 cell lines at 24 h were 1867 and1322 120583gmL respectively Although the IC
50values of the
MECCrt were about 3-4 folds of cisplatin for oral cancercells its crude extract nature has to be concerned Thereforeit is warranted to further investigate the particular bioactivecomponents that are included in the methanolic extracts ofCryptocarya concinnaHance roots
Moreover the anticancer effect for trunk bark ofC infectoria-derived methanol extracts was reported tobe cytotoxic to KB cells [24] KB cells were regardedas oral epidermal carcinoma however it was recentlyvalidated to have marker chromosomes and DNAfinger printings of human cervical cancer HeLacells (httpwwwncbinlmnihgovmeshDb=meshampterm=KB+Cells) [32] Accordingly the anticancer effect of oralcancer by the bioactive compounds from Cryptocarya plantremains unclear Conversely we here demonstrate theantioral cancer effect of methanolic extracts of a Cryptocaryaspecies for the first time using two OSCC cell lines Ca9-22and CAL 27
In several anticancer drugs [6 7 10 11 33ndash36] ROSgeneration is one of the common strategies to inhibit cancercell proliferation ROS plays a vital role in early stagesof apoptosis [37] and leads to MMP depolarization [3839] Escaping apoptosis is demonstrated to be involved inthe drug resistance of cancer cells [40 41] To enhanceapoptotic induction of anticancer drugs may interfere thedrug resistance if there In the present study we observed thatapoptosis was inducible by MECCrt in two OSCC cell linesas it was demonstrated by sub-G1 monitoring and annexinVPI assayWe also found that MECCrt significantly inducedthe ROS level and reduced the MMP level in two oral cancercell lines in dose-responsive ways These findings suggestthat oxidative stress may be involved in theMECCrt-inducedantiproliferative effect in two oral cancer Ca9-22 and CAL 27cell lines However the role of oxidative stress in MECCrtneed to be further examined by the ROS scavenger such asN-acetylcysteine [42] to confirm if raised ROS has played acritical role in the process of apoptosis Furthermore the ROSmay generate nonapoptotic effect like autophagy described
in literature [43 44] Therefore it was warranted to furtherinvestigate the role of autophagy in MECCrt-treated oralcancer cell lines in future
5 Conclusions
We demonstrated the antiproliferative and apoptotic effectsof MECCrt through ROS generation and mitochondrialdepolarization in twoOSCC cell linesTherefore these resultssuggest that MECCrt has anticancer potential for oral cancertherapy
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was supported by funds of the NSC102-2314-B-384-002 MOST103-2320-B-037-008 the 103CM-KMU-09the National Sun Yat-sen University-KMU Joint ResearchProject (NSYSU-KMU 103-p014) the kmtth-102-011 andthe Health and welfare surcharge of tobacco products theMinistry of Health and Welfare Taiwan Republic of China(MOHW103-TD-B-111-05)The authors also acknowledgeDrHans-Uwe Dahms for the English editing
References
[1] C-Y Yen C-H Chen C-H Chang et al ldquoMatrix metallopro-teinases (MMP) 1 andMMP10 but notMMP12 are potential oralcancer markersrdquo Biomarkers vol 14 no 4 pp 244ndash249 2009
[2] C-Y Yen C-Y HuangM-F Hou et al ldquoEvaluating the perfor-mance of fibronectin 1 (FN1) integrin12057241205731 (ITGA4) syndecan-2 (SDC2) and glycoprotein CD44 as the potential biomarkersof oral squamous cell carcinoma (OSCC)rdquo Biomarkers vol 18no 1 pp 63ndash72 2013
[3] H Myoung S-P Hong P-Y Yun J-H Lee and M-J KimldquoAnti-cancer effect of genistein in oral squamous cell carcinomawith respect to angiogenesis and in vitro invasionrdquo CancerScience vol 94 no 2 pp 215ndash220 2003
[4] H K Kim E G Wilson Y H Choi and R VerpoorteldquoMetabolomics a tool for anticancer lead-finding from naturalproductsrdquo Planta Medica vol 76 no 11 pp 1094ndash1102 2010
[5] A D Kinghorn L Pan J N Fletcher and H Chai ldquoThe rele-vance of higher plants in lead compound discovery programsrdquoJournal of Natural Products vol 74 no 6 pp 1539ndash1555 2011
[6] C-C Yeh C-N Tseng J-I Yang et al ldquoAntiproliferation andinduction of apoptosis in Ca9-22 oral cancer cells by ethanolicextract of Gracilaria tenuistipitatardquoMolecules vol 17 no 9 pp10916ndash10927 2012
[7] C-C Yeh J-I Yang J-C Lee et al ldquoAnti-proliferative effect ofmethanolic extract of Gracilaria tenuistipitata on oral cancercells involves apoptosis DNA damage and oxidative stressrdquoBMC Complementary and Alternative Medicine vol 12 article142 2012
[8] J S Yang C W Lin C H Hsin M J Hsieh and Y C ChangldquoSelaginella tamariscina attenuates metastasis via Akt pathways
The Scientific World Journal 9
in oral cancer cellsrdquo PLoS ONE vol 8 no 6 Article ID e680352013
[9] B Narotzki A Z Reznick D Aizenbud and Y Levy ldquoGreentea a promising natural product in oral healthrdquoArchives of OralBiology vol 57 no 5 pp 429ndash435 2012
[10] C-Y Yen C-C Chiu R-W Haung et al ldquoAntiprolifera-tive effects of goniothalamin on Ca9-22 oral cancer cellsthrough apoptosis DNAdamage andROS inductionrdquoMutationResearch vol 747 no 2 pp 253ndash258 2012
[11] C-C Chiu J-W Haung F-R Chang et al ldquoGolden berry-derived 4120573-hydroxywithanolide E for selectively killing oralcancer cells by generating ROS DNA damage and apoptoticpathwaysrdquo PLoS ONE vol 8 no 5 Article ID e64739 2013
[12] M T Davies-Coleman andD E A Rivett ldquoNaturally occurring6-substituted 56-dihydro-120572-pyronesrdquo Fortschritte der ChemieOrganischer Naturstoffe vol 55 pp 1ndash35 1989
[13] A Toribio A Bonfils E Delannay et al ldquoNovel seco-dibenzopyrrocoline alkaloid from Cryptocarya oubatchensisrdquoOrganic Letters vol 8 no 17 pp 3825ndash3828 2006
[14] A J Cavalheiro and M Yoshida ldquo6-[120596-arylalkenyl]-56-dihydro-120572-pyrones from Cryptocarya moschata (Lauraceae)rdquoPhytochemistry vol 53 no 7 pp 811ndash819 2000
[15] L D Juliawaty M Kitajima H Takayama S A Achmad andN Aimi ldquoA new type of stilbene-related secondary metaboliteidenburgene from Cryptocarya idenburgensisrdquo Chemical andPharmaceutical Bulletin vol 48 no 11 pp 1726ndash1728 2000
[16] P-M Allard E T H Dau C Eydoux et al ldquoAlkylatedflavanones from the bark of Cryptocarya chartacea as denguevirus NS5 polymerase inhibitorsrdquo Journal of Natural Productsvol 74 no 11 pp 2446ndash2453 2011
[17] T-S Wu C-R Su and K-H Lee ldquoCytotoxic and anti-HIVphenanthroindolizidine alkaloids from Cryptocarya chinensisrdquoNatural Product Communications vol 7 no 6 pp 725ndash727 2012
[18] T-H Chou J-J Chen C-F Peng M-J Cheng and I-S ChenldquoNew flavanones from the leaves of Cryptocarya chinensis andtheir antituberculosis activityrdquo Chemistry and Biodiversity vol8 no 11 pp 2015ndash2024 2011
[19] A A Nasrullah A Zahari J Mohamad and K AwangldquoAntiplasmodial alkaloids from the bark of Cryptocarya nigra(Lauraceae)rdquoMolecules vol 18 no 7 pp 8009ndash8017 2013
[20] R A Davis O Demirkiran M L Sykes et al ldquo7101584081015840-Dihydroobolactone a typanocidal 120572-pyrone from the rainforesttree Cryptocarya obovatardquo Bioorganic andMedicinal ChemistryLetters vol 20 no 14 pp 4057ndash4059 2010
[21] R Feng Z K Guo C M Yan E G Li R X Tan and H MGe ldquoAnti-inflammatory flavonoids from Cryptocarya chingiirdquoPhytochemistry vol 76 pp 98ndash105 2012
[22] V Dumontet N Van Hung M-T Adeline et al ldquoCytotoxicflavonoids and 120572-pyrones from Cryptocarya obovatardquo Journalof Natural Products vol 67 no 5 pp 858ndash862 2004
[23] C Y Ong S K Ling R M Ali et al ldquoSystematic analysis of invitro photo-cytotoxic activity in extracts from terrestrial plantsin Peninsula Malaysia for photodynamic therapyrdquo Journal ofPhotochemistry and Photobiology B vol 96 no 3 pp 216ndash2222009
[24] V Dumontet C Gaspard N van Hung et al ldquoNew cytotoxicflavonoids fromCryptocarya infectoriardquoTetrahedron vol 57 no29 pp 6189ndash6196 2001
[25] H Usman E H Hakim T Harlim et al ldquoCytotoxic chalconesand flavanones from the tree bark of Cryptocarya costatardquoZeitschrift fur Naturforschung C vol 61 no 3-4 pp 184ndash1882006
[26] F Kurniadewi L D Juliawaty Y M Syah et al ldquoPhenolic com-pounds from Cryptocarya konishii their cytotoxic and tyrosinekinase inhibitory propertiesrdquo Journal of Natural Medicines vol64 no 2 pp 121ndash125 2010
[27] T-H Chou J-J Chen S-J Lee M Y Chiang C-W Yang andI-S Chen ldquoCytotoxic flavonoids from the leaves ofCryptocaryachinensisrdquo Journal of Natural Products vol 73 no 9 pp 1470ndash1475 2010
[28] J C Liao ldquoLauraceae in flora of Taiwanrdquo in Editorial Committeeof the Flora of Taiwan vol 2 pp 448ndash451 Taipei Taiwan 1996
[29] B H Chen H W Chang H M Huang et al ldquo(-)-anonaineinduces DNA damage and inhibits growth and migration ofhuman lung carcinomaH1299 cellsrdquo Journal of Agricultural andFood Chemistry vol 59 no 6 pp 2284ndash2290 2011
[30] M-Y Lin Y-R Lee S-Y Chiang et al ldquoCortexMoutan inducesbladder cancer cell death via apoptosis and retards tumorgrowth inmouse bladdersrdquoEvidence-based Complementary andAlternative Medicine vol 2013 Article ID 207279 8 pages 2013
[31] C-C Chiu P-L Liu K-J Huang et al ldquoGoniothalamininhibits growth of human lung cancer cells through DNAdamage apoptosis and reduced migration abilityrdquo Journal ofAgricultural and Food Chemistry vol 59 no 8 pp 4288ndash42932011
[32] J Masters ldquoFalse cell linesrdquoCarcinogenesis vol 23 no 2 p 3712002
[33] H Ding C Han D Guo et al ldquoSelective induction of apoptosisof human oral cancer cell lines by avocado extracts via a ROS-mediated mechanismrdquo Nutrition and Cancer vol 61 no 3 pp348ndash356 2009
[34] J-C Lee M-F Hou H-W Huang et al ldquoMarine algal natu-ral products with anti-oxidative anti-inflammatory and anti-cancer propertiesrdquoCancer Cell International vol 13 no 1 article55 2013
[35] C Gorrini I S Harris and TWMak ldquoModulation of oxidativestress as an anticancer strategyrdquoNature Reviews DrugDiscoveryvol 12 no 12 pp 931ndash947 2013
[36] D Trachootham J Alexandre and P Huang ldquoTargeting can-cer cells by ROS-mediated mechanisms a radical therapeuticapproachrdquo Nature Reviews Drug Discovery vol 8 no 7 pp579ndash591 2009
[37] A K Samhan-Arias F J Martın-Romero and C Gutierrez-Merino ldquoKaempferol blocks oxidative stress in cerebellar gran-ule cells and reveals a key role for reactive oxygen speciesproduction at the plasma membrane in the commitment toapoptosisrdquo Free Radical Biology and Medicine vol 37 no 1 pp48ndash61 2004
[38] S-H Oh and S-C Lim ldquoA rapid and transient ROS generationby cadmium triggers apoptosis via caspase-dependent pathwayin HepG2 cells and this is inhibited through N-acetylcysteine-mediated catalase upregulationrdquo Toxicology and Applied Phar-macology vol 212 no 3 pp 212ndash223 2006
[39] R A Ehlers A Hernandez L S Bloemendal R T EthridgeB Farrow and B M Evers ldquoMitochondrial DNA damage andaltered membrane potential (Δ120595) in pancreatic acinar cellsinduced by reactive oxygen speciesrdquo Surgery vol 126 no 2 pp148ndash155 1999
[40] S McKenzie and N Kyprianou ldquoApoptosis evasion the role ofsurvival pathways in prostate cancer progression and therapeu-tic resistancerdquo Journal of Cellular Biochemistry vol 97 no 1 pp18ndash32 2006
10 The Scientific World Journal
[41] S Fulda ldquoEvasion of apoptosis as a cellular stress response incancerrdquo International Journal of Cell Biology vol 2010 ArticleID 370835 6 pages 2010
[42] H Lin X-B Liu J-J Yu F Hua and Z-W Hu ldquoAntioxidantN-acetylcysteine attenuates hepatocarcinogenesis by inhibitingROSER stress in TLR2 deficient mouserdquo PLoS ONE vol 8 no10 Article ID e74130 2013
[43] R Scherz-Shouval and Z Elazar ldquoROS mitochondria and theregulation of autophagyrdquoTrends in Cell Biology vol 17 no 9 pp422ndash427 2007
[44] R Scherz-Shouval and Z Elazar ldquoRegulation of autophagy byROS physiology and pathologyrdquoTrends in Biochemical Sciencesvol 36 no 1 pp 30ndash38 2011
Submit your manuscripts athttpwwwhindawicom
PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom
Volume 2014
ToxinsJournal of
VaccinesJournal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
AntibioticsInternational Journal of
ToxicologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Drug DeliveryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in Pharmacological Sciences
Tropical MedicineJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
AddictionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Autoimmune Diseases
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anesthesiology Research and Practice
ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Pharmaceutics
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
2 The Scientific World Journal
drugs derived from natural products were approved by theUnited States Food and Drug Administration [5] In basicresearches natural products with antioral cancer effects haveincreasingly being reported This holds for the ethanolic andmethanolic extracts of red algaGracilaria tenuistipitata [6 7]crude extracts of Selaginella tamariscina (oriental medicinalherb) [8] green tea [9] goniothalamin from Goniothalamusspecies [10] and 4120573-hydroxywithanolide E fromgolden berry[11]
Cryptocarya plants (family Lauraceae) comprising about350 species worldwide are widely distributed in the tropicsand subtropics [12] This plant group is well known forits common secondary metabolites containing alkaloidsflavonoids and 120572-pyrones [12ndash15] Several biological effectsof Cryptocarya-derived natural products have been reportedthat include anti-dengue virus [16] anti-HIV [17] anti-tuberculosis [18] antiplasmodial [19] antitrypanosomal [20]and anti-inflammatory [21] function
Anticancer effects of crude extracts of Cryptocarya plantare known as well For example the ethanolic extracts of fruitand trunk bark of C obovata showed 56 and 23 growthinhibition of human KB cells at 10 120583gmL respectively [22]Methanolic extracts of the leaves of C griffithiana providecytotoxicity forhuman HL60 promyelocytic leukemia cells[23]
Recently accumulating findings for anticancer effectsof pure compounds isolated from Cryptocarya plants werereported especially from methanolic extracts For examplecompounds isolated frommethanol extracts of the trunk barkof C infectoria [24] the trunk bark of C costata [25] andthe wood of C konishii [26] were reported to be cytotoxicto leukemia cells Compounds from methanolic extracts ofleaves of C chinensis were shown to be cytotoxic to humanlung cancer and glioblastoma cells [27] These drugs wereisolated from the trunk bark wood and leaves ofCryptocaryasp However the bioactivity of the roots of Cryptocaryaplants remained little investigated particularly with respectto antioral cancer
Because C concinna Hance is an evergreen plant com-monly distributed in low-altitude forests in Taiwan [28] it iseasy to preparemethanolic extracts of the roots ofC concinnaHance (namely forMECCrt)We therefore chose twoOSCCcell lines that is Ca9-22 and CAL 27 to evaluate the possibleanticancer function of MECCrt and investigate their drugmechanisms in terms of cell viability cell cycle distributionapoptosis reactive oxygen species (ROS) generation andmitochondrial depolarization
2 Materials and Methods
21 Cell Cultures and Methanolic Extracts of C concinnaTwo human OSCC cell lines Ca9-22 and CAL 27 purchasedfrom the Cell Bank RIKEN BioResource Center (TsukubaJapan) and the American Type Culture Collection (ATCCVirginia USA) respectively were incubated in DMEMF12(3 2) medium (Gibco Grand Island NY USA) supple-mented with 10 fetal bovine serum (Gibco) 100UmLpenicillin 100 120583gmL streptomycin and 003 glutamine
These two cell lines were humidly incubated at 37∘C with 5CO2in the humid atmosphere
C concinna was identified by one of the authors (Ih-Sheng Chen) and its roots were collected at Mudan PingtungCounty Taiwan in May 2004 A voucher specimen (Chen6153) has been deposited in the Herbarium of the Schoolof Pharmacy College of Pharmacy Kaohsiung MedicalUniversity The dried roots of C concinna were processedby slicing and cold methanol-extraction for three times atroom temperature Finally the solutionwas evaporated underreduced pressure to yield the methanolic extract (MECCrt)MECCrt was stored at minus20∘C and dissolved in dimethylsulfoxide (DMSO) before treatment
22 Cell Viability Cell viability was measured by theCellTiter 96 AQueous one solution cell proliferation assay(MTS) (Promega Corporation Madison WI USA) as pre-viously described [11] Ca9-22 and CAL 27 cell lines wereseeded at a density of 1 times 105 and 2 times 105 cells per well ina 6-well plate respectively After plating for 24 h these cellswere incubated with different concentrations of MECCrt for24 h and finally subjected to a MTS assay applying an ELISAreader at 490 nm
23 Cell Cycle Progression and Sub-G1 Population Propidiumiodide (PI Sigma St Louis MO USA) was added to stainthe cellular DNA content [29] In brief 3 times 105 cells perwell in 6 well plates were plated for 24 h and then treatedwith vehicle (DMSO 1120583L2mL culture medium) as a controlor 5 10 15 20 and 25 120583gmL of MECCrt for 24 h Afterexposure termination cells were centrifuged washed twicewith PBS fixed overnight with 70 ethanol and centrifugedSubsequently the cell pellets were resuspended in 50120583gmLPI reagent and stand for 30min at 37∘C in darkness Cellcycle distribution was evaluated by a flow cytometer (BDAccuri C6 Becton-Dickinson Mansfield MA USA) and aBD Accuri C6 Software (version 10264)
24 Apoptosis To validate apoptosis in MECCrt-treated oralcancer cells annexin V (Strong Biotect Corporation TaipeiTaiwan) [30]PI (Sigma St Louis MO USA) method wasused [31] Briefly 3 times 105 cells per well in 6 well plates wereplated for 24 h and then treated with vehicle or indicated con-centrations of MECCrt for 24 h Subsequently apoptotic cellswere stained for 30minwith 100120583L binding buffer containing2 120583L of annexin-V-fluorescein isothiocyanate (FITC) stock(025 120583g120583L) and 2120583L of PI stock (1mgmL) Finally it wassuspended with 400 120583L PBS for analysis of a flow cytometer(BD Accuri C6 Becton-Dickinson) and its software
25 Intracellular ROS The dye 2101584071015840-dichlorodihydroflu-orescein diacetate (DCFH-DA) was used to detect ROS byits fluorescence change [7] Cells at the density of 3 times 105 in2mL medium per well in 6 well plates were plated for 24 hDifferent concentrations of MECCrt were added to Ca9-22cells for 6 h and 12 h After washing with PBS 100 nMDCFH-DA in PBS was added to cells in 6 well plates at cell cultureincubator for 30min After trypsinization PBS washing and
The Scientific World Journal 3
120
100
80
60
40
20
0
0 5 10 2015
Cel
l via
bilit
y (
)Ca9-22
MECCrt (120583gmL)
lowastlowast
lowastlowast
lowastlowast
lowastlowast
(a)
0 5 10 2015
Cel
l via
bilit
y (
)
MECCrt (120583gmL)
CAL 27140
120
100
80
60
40
20
0
lowastlowast
lowastlowast
lowastlowastlowastlowast
(b)
Figure 1 Cell viability of two oral cancer cells was inhibited by MECCrt Oral cancer Ca9-22 and CAL 27 cell lines were treated with variousconcentrations ofMECCrt (0 5 10 15 and 20 120583gmL) for 24 hThe cell viability was measured by theMTS assay Data means plusmn SDs (119899 = 18)lowastlowast
119875 lt 001 against vehicle
centrifugation cell pellets were resuspended in 1mL PBSbefore analyzing by a flow cytometer (BDAccuri C6 Becton-Dickinson) and its software
26 Mitochondrial Membrane Potential MitoProbeDiOC
2(3) assay kit (Invitrogen Eugene OR USA) was
applied to analyze mitochondrial membrane potential(MMP) as described previously [10] Briefly 3 times 105 cellsin 2mL medium per well in 6 well plates were plated for24 h After MECCrt treatment 10 120583L of 10 120583M DiOC
2(3)
was added per well and incubated in a cell culture incubatorfor 20min After being harvested cells were washedand resuspended in 1mL PBS for analysis using a flowcytometer (BD Accuri C6 Becton-Dickinson) and itssoftware
27 Statistical Analysis The significance of differences wasdetermined by Studentrsquos 119905-test compared with the test datawith the vehicle controls Data are expressed as means plusmn SDs
3 Results
31 Antiproliferation in MECCrt-Treated Two Oral CancerCell Lines Based on MTS assay (Figure 1) the relative cellviability () of oral cancer Ca9-22 cells at indicated concen-trations of MECCrt (0 5 10 15 and 20120583gmL) was 1000 plusmn07 933 plusmn 23 714 plusmn 30 576 plusmn 16 and 484 plusmn 12 after24 h respectively The relative cell viability () of CAL 27cells at indicated concentrations of MECCrt (0 5 10 15 and20120583gmL) was 1000 plusmn 081193 plusmn 49 869 plusmn 100 298 plusmn 62and 284 plusmn 55 respectively The MTS-based cell viabilities ofMECCrt-treated twooral cancerCa9-22 andCAL27 cell linessignificantly reduced in a dose-responsive manner (119875 lt 001compared to the vehicle)
32 Sub-G1 Population in MECCrt-Treated Two Oral Can-cer Cell Lines The MECCrt-treated effects of cell cycledistribution profiles are demonstrated in Figure 2(a) AfterMECCrt treatment (Figure 2(b)) the sub-G1 populations ()ofMECCrt- (0 5 10 15 20 and 25 120583gmL) treated oral cancerCa9-22 cells were 50 plusmn 03 67 plusmn 01 181 plusmn 06 179 plusmn 07165plusmn03 and 224plusmn13 and those ofMECCrt-treatedCAL 27cells were 67plusmn17 51plusmn11 79plusmn01 283plusmn10 526plusmn02 and691 plusmn 01 respectively These sub-G1 changes significantlyaccumulated in a dose-responsive manner (119875 lt 001)
33 Apoptosis of MECCrt-Treated Two Oral Cancer Cell LinesTo validate the possible outcome of apoptosis in MECCrt-induced sub-G1 accumulation of these two oral cancer cellsannexin VPI profiles of flow cytometry were generated(Figure 3(a)) In Figure 3(b) the percentages of annexin V-positive intensities forMECCrt (0 5 10 15 20 and 25120583gmL)treatment of Ca9-22 cells were 93 plusmn 02 86 plusmn 03 117 plusmn 09223 plusmn 08 400 plusmn 02 and 544 plusmn 17 and those of MECCrt-treated CAL 27 cells were 248 plusmn 01 175 plusmn 03 207 plusmn 04598plusmn17 794plusmn02 and 853plusmn05 respectively AccordinglyMECCrt treatments significantly increased in annexin V-positive intensities of two oral cancer Ca9-22 and CAL 27 celllines in a dose-responsive manner (119875 lt 001)
34 ROSGeneration inMECCrt-Treated TwoOral Cancer CellLines To validate the role of ROS in the MECCrt-inducedapoptosis of two oral cancer cell lines a DCFH-DA assay offlow cytometry was chosen Figures 4(a) and 4(b) show therelative ROS-positive staining () of two oral cancer Ca9-22 and CAL 27 cell lines for the different concentrations ofMECCrt treatment for 6 and 12 h incubation After MECCrttreatment for 6 h the relative ROS-positive staining () of 05 10 15 20 and 25 120583gmL MECCrt-treated Ca9-22 cells was
4 The Scientific World Journal
Cou
ntCa
9-22
Cou
ntCa
9-22
0 120583gmL 5120583gmL 10120583gmL
15120583gmL 20120583gmL 25120583gmL
400
300
200
100
0
DNA content DNA content DNA content
400
300
200
100
0
Sub-G147G1
492S
177G2M288
Sub-G168G1
529S
182G2M232
Sub-G1171
G1370
S127G2M285
Sub-G1158
G1307
S179G2M265
Sub-G1141
G1266
S212G2M235
Sub-G1200
G1293
S201G2M208
(a)
Cou
ntC
ount
0120583gmL 5120583gmL 10120583gmL
15120583gmL 20120583gmL 25120583gmL
400
300
200
100
0
DNA content DNA content DNA content
400
300
200
100
0
Sub-G148G1
539S
201G2M216
Sub-G151G1
548S
214G2M192
Sub-G1G1
539S
201G2M216
Sub-G1279
G1322
S230G2M184
Sub-G1535
G1248
S122G2M109
Sub-G1699
G1181
SG2M
48
78
54
CAL 27
CAL 27
(b)
Sub-
G1
()
MECCrt (120583gmL)
lowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowast20
20
10
10
5
5
0
0
25
25
15
15
(c)
Sub-
G1
()
lowastlowast
lowastlowast
lowastlowast
0
10
20
30
40
50
60
70
80
MECCrt (120583gmL)201050 2515
(d)
Figure 2 The sub-G1 accumulation of two oral cancer cells was induced by MECCrt Oral cancer Ca9-22 and CAL 27 cell lines were treatedwith 0 5 10 15 20 and 25 120583gmL of MECCrt for 24 h ((a) and (b)) Representative cell cycle distribution profiles of flow cytometry forMECCrt-treated oral cancer Ca9-22 and CAL 27 cells and vehicles at 24 h respectively ((c) and (d)) Statistics analyses for the percentages ofsub-G1 population in (a) and (b) respectively Data means plusmn SDs (119899 = 3) lowastlowast119875 lt 001 against vehicle
1000 plusmn 18 1067 plusmn 04 1312 plusmn 08 1406 plusmn 16 1506 plusmn 03and 1831 plusmn 78 and that of MECCrt-treated CAL 27 cells was1000 plusmn 08 1141 plusmn 14 1423 plusmn 15 1611 plusmn 07 1798 plusmn 11and 1851 plusmn 14 respectively After MECCrt treatment for12 h the relative ROS-positive staining () of 0 5 10 1520 and 25120583gmL MECCrt-treated Ca9-22 cells was 1000 plusmn23 1241 plusmn 04 1664 plusmn 12 1848 plusmn 06 1933 plusmn 03 and2005 plusmn 00 and that of MECCrt-treated CAL 27 cells was1000plusmn09 1407plusmn14 1771plusmn03 1938plusmn01 1986plusmn01 and1994 plusmn 01 respectively Accordingly MECCrt treatmentssignificantly increased in both dose-responsive and time-dependent manners in these two oral cancer cell lines (119875 lt005) (Figures 4(c) and 4(d))
35 MMP Depolarization in MECCrt-Treated Two Oral Can-cer Cell Lines Figures 5(a) and 5(b) show the MMP profilesof DiOC
2(3)-positive intensities for the vehicle andMECCrt-
treated oral cancer cell lines in 24-hour treatments Treated
with MECCrt (0 5 10 15 20 and 25120583gmL) for 24 hthe DiOC
2(3)-positive () intensities of Ca9-22 cells were
1000 plusmn 29 961 plusmn 24 940 plusmn 16 768 plusmn 14 456 plusmn 14and 250 plusmn 11 respectively Similarly the percentages ofDiOC
2(3)-positive () intensities ofMECCrt-treatedCAL27
cells were 1000 plusmn 15 1142 plusmn 07 1089 plusmn 13 513 plusmn 05273 plusmn 05 and 75 plusmn 04 respectively Accordingly MECCrtsignificantly reduced DiOC
2(3)-positive intensities of two
oral cancer Ca9-22 and CAL 27 cell lines in a dose-responsivemanner (119875 lt 001ndash005)
4 Discussion
We discovered for the first time that methanolic extracts ofthe roots ofC concinnaHance have an antiproliferative effecton two oral cancer cell lines The proliferation inhibitingfunction of MECCrt against oral cancer Ca9-22 and CAL 27cell lines was dose-responsive (Figure 1)
The Scientific World Journal 5
104
105
106
1027
104
105
106
1027
1031
104
105
10610
6410
3110
410
510
610
6410
3110
410
510
610
64
Ca9
-22
0 120583gmL 5120583gmL 10120583gmL
15120583gmL 20120583gmL 25120583gmLAnnexin V intensity Annexin V intensity Annexin V intensity
PI in
tens
ityCa
9-22
PI in
tens
ity
(a)
15120583gmL 20120583gmL 25120583gmLAnnexin V intensity Annexin V intensity Annexin V intensity
104
105
106
1027
104
105
106
1027
1031
104
105
10610
6410
3110
410
510
610
6410
3110
410
510
610
64
0 120583gmL 5120583gmL 10120583gmL
PI in
tens
ityPI
inte
nsity
CAL 27
CAL 27
(b)
Apop
totic
cells
()
lowastlowast
lowastlowast
lowastlowast
lowastlowast
60
50
40
30
20
10
0
0 5 10 15 20 25
MECCrt (120583gmL)
(c)
Apop
totic
cells
()
lowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowast
10
20
30
40
50
60
70
80
90
0 5 10 15 20 25
MECCrt (120583gmL)
(d)
Figure 3 Apoptosis of two oral cancer cells was induced byMECCrt Oral cancer Ca9-22 and CAL 27 cell lines were treated with 0ndash25120583gmLof MECCrt for 24 h ((a) and (b)) Representative results of annexin VPI double staining of flow cytometry for MECCrt-treated oral cancerCa9-22 and CAL 27 cell lines and vehicle controls at 24 h respectively ((c) and (d)) Quantification analysis of apoptosis for MECCrt-treatedoral cancer Ca9-22 and CAL 27 cell lines in (a) and (b) respectively Data means plusmn SDs (119899 = 3) lowastlowast119875 lt 001 against vehicle
6 The Scientific World Journal
Cou
ntCa
9-22
0 120583gmL 5120583gmL 10120583gmL
15120583gmL
400
300
200
100
0
Cou
ntCa
9-22
400
300
200
100
0
400
300
200
100
0
400
300
200
100
0
10 5000 10000 10 5000 10000 10 5000 10000 10 5000 10000 10 5000 10000 10 5000 10000
ROS intensity20120583gmL
ROS intensity ROS intensity25120583gmL 15120583gmL
ROS intensity20120583gmL
ROS intensity ROS intensity25120583gmL
6h 6h 6h0120583gmL 5120583gmL 10120583gmL12h 12h 12h
V2-L500
V2-R V2-L V2-R V2-L V2-R
V2-L V2-R V2-L V2-R V2-L V2-R
V3-L V3-R V3-L V3-R
V3-L V3-R
V3-L V3-R
V3-L V3-R V3-L V3-R
500 500 500
916 962 996
172 828618382471 529 347 653
298 702 255 745 119 881 84 38 04
(a)
Cou
nt
0120583gmL 5120583gmL 10120583gmL
15120583gmL
400
300
200
100
0
Cou
nt
400
300
200
100
0
400
300
200
100
0
400
300
200
100
0
10 5000 10000 10 5000 10000 10 5000 10000 10 5000 10000 10 5000 10000 10 5000 10000
ROS intensity20120583gmL
ROS intensity ROS intensity25120583gmL 15120583gmL
ROS intensity20120583gmL
ROS intensity ROS intensity25120583gmL
6h 6h 6h0120583gmL 5120583gmL 10120583gmL12h 12h 12h
V2-L500
V2-R V2-L V2-R V2-L V2-R
V2-L V2-R V2-L V2-R V2-L V2-R
V2-L V2-R V2-L V2-R
V2-L V2-R
V2-L V2-R
V2-L V2-R V2-L V2-R
500 500 500
970 993 998
112 888700300497 503 386 614
298 702 173 827 921 3079 07 02
CAL 27
CAL 27
(b)
lowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowast
Relat
ive R
OS-
posit
ive (
)
220
200
180
160
140
120
100
80
0 5 10 15 20 25
6h12h
MECCrt (120583gmL)
(c)
lowastlowastlowastlowastlowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowastlowastlowast
lowastlowastlowastlowast
Relat
ive R
OS-
posit
ive (
)
220
200
180
160
140
120
100
80
0 5 10 15 20 25 25
6h12h
MECCrt (120583gmL)
(d)
Figure 4 Reactive oxygen species (ROS) generation of two oral cancer cell lines was induced by MECCrt Oral cancer Ca9-22 and CAL 27cell lines were treated with different concentrations (0ndash25120583gmL) of MECCrt for 6 and 12 h ((a) (b)) Representative ROS profiles of flowcytometry for MECCrt-treated oral cancer Ca9-22 and CAL 27 cell lines ((c) and (d)) Statistics analysis of relative ROS intensity in (a) and(b) respectively Data means plusmn SDs (119899 = 3) lowastlowast119875 lt 001 against vehicle
The Scientific World Journal 7
500V1-L V1-R
500 517V1-L V1-R
483 539V1-L V1-R
461
620V1-L V1-R
380 770V1-L V1-R
230 872V1-L V1-R
128
Cou
ntCa
9-22
Cou
ntCa
9-22
0 120583gmL 5120583gmL 10120583gmL
15120583gmL 20120583gmL 25120583gmLMMP intensity MMP intensity MMP intensity
1200
1000
500
0
1200
1000
500
0
10110
210
310
410
510
610
7210
110
210
310
410
510
610
7210
110
210
310
410
510
610
72
(a)
500V2-L V2-R
500 440V2-L V2-R
560 455V2-L V2-R
545
744V2-L V2-R
256 868V2-L V2-R
132 964V2-L V2-R
Cou
ntC
ount
0120583gmL 5120583gmL 10120583gmL
15120583gmL 20120583gmL 25120583gmLMMP intensity MMP intensity MMP intensity
1200
1000
500
0
1200
1000
500
0
10110
210
310
410
510
610
7210
110
210
310
410
510
610
7210
110
210
310
410
510
610
72
36
CAL 27
CAL 27
(b)
lowast
lowastlowast
lowastlowast
lowastlowast
120
100
80
60
40
20
0
Relat
ive M
MP-
posit
ive (
)
0 5 10 15 20 25
MECCrt (120583gmL)
(c)
lowastlowastlowastlowast
lowastlowast
lowastlowast
lowastlowast
120
140
100
80
60
40
20
0
Relat
ive M
MP-
posit
ive (
)
0 5 10 15 20 25
MECCrt (120583gmL)
(d)
Figure 5 Depolarization of mitochondrial membrane potential (MMP) of Ca9-22 and CAL 27 oral cancer cell lines was induced byMECCrt Oral cancer Ca9-22 and CAL 27 cell lines were treated with different concentrations (0ndash25120583gmL) of MECCrt for 24 h ((a) (b))RepresentativeMMPprofiles of flow cytometry forMECCrt-treated oral cancer Ca9-22 andCAL 27 cells ((c) and (d))Quantification analysisof relative MMP intensity in (a) and (b) respectively Data means plusmn SDs (119899 = 3) lowast119875 lt 005 and lowastlowast119875 lt 001 against vehicle
8 The Scientific World Journal
The anticancer effects for other Cryptocarya-derivedcompounds from methanolic extracts of nonroot parts havebeen reported earlier For example for murine leukemiaP-388 cells the IC
50values of 2101584041015840-dihydroxy-5101584061015840-
dimethoxychalcone and isodidymocarpin isolated fromtree bark of C costata were 57 and 111 120583M [25] and IC
50
values of the chalcone derivative (desmethylinfectocaryone)and phenolic compound (infectocaryone) isolated fromwood of C konishii were 217 and 08 120583M [26] at 48 hrespectively For compounds from leaves of C chinensis theIC50
values of infectocaryone and cryptocaryanone A wereat the 120583M level for human lung cancer NCI-H460 cells andglioblastoma SF-268 cells [27] These Cryptocarya-derivedcompounds from methanolic extracts of nonroot partsshowed the IC
50values ranging from 08 to 11 120583M This is
close to our preliminary result that the IC50
of the clinicalanticancer drug cisplatin at 24 h treatment in oral cancerCa9-22 cells is 306 120583gmL (102 120583M) (data not shown) Inthe present study the IC
50values of the MECCrt in oral
cancer Ca9-22 and CAL 27 cell lines at 24 h were 1867 and1322 120583gmL respectively Although the IC
50values of the
MECCrt were about 3-4 folds of cisplatin for oral cancercells its crude extract nature has to be concerned Thereforeit is warranted to further investigate the particular bioactivecomponents that are included in the methanolic extracts ofCryptocarya concinnaHance roots
Moreover the anticancer effect for trunk bark ofC infectoria-derived methanol extracts was reported tobe cytotoxic to KB cells [24] KB cells were regardedas oral epidermal carcinoma however it was recentlyvalidated to have marker chromosomes and DNAfinger printings of human cervical cancer HeLacells (httpwwwncbinlmnihgovmeshDb=meshampterm=KB+Cells) [32] Accordingly the anticancer effect of oralcancer by the bioactive compounds from Cryptocarya plantremains unclear Conversely we here demonstrate theantioral cancer effect of methanolic extracts of a Cryptocaryaspecies for the first time using two OSCC cell lines Ca9-22and CAL 27
In several anticancer drugs [6 7 10 11 33ndash36] ROSgeneration is one of the common strategies to inhibit cancercell proliferation ROS plays a vital role in early stagesof apoptosis [37] and leads to MMP depolarization [3839] Escaping apoptosis is demonstrated to be involved inthe drug resistance of cancer cells [40 41] To enhanceapoptotic induction of anticancer drugs may interfere thedrug resistance if there In the present study we observed thatapoptosis was inducible by MECCrt in two OSCC cell linesas it was demonstrated by sub-G1 monitoring and annexinVPI assayWe also found that MECCrt significantly inducedthe ROS level and reduced the MMP level in two oral cancercell lines in dose-responsive ways These findings suggestthat oxidative stress may be involved in theMECCrt-inducedantiproliferative effect in two oral cancer Ca9-22 and CAL 27cell lines However the role of oxidative stress in MECCrtneed to be further examined by the ROS scavenger such asN-acetylcysteine [42] to confirm if raised ROS has played acritical role in the process of apoptosis Furthermore the ROSmay generate nonapoptotic effect like autophagy described
in literature [43 44] Therefore it was warranted to furtherinvestigate the role of autophagy in MECCrt-treated oralcancer cell lines in future
5 Conclusions
We demonstrated the antiproliferative and apoptotic effectsof MECCrt through ROS generation and mitochondrialdepolarization in twoOSCC cell linesTherefore these resultssuggest that MECCrt has anticancer potential for oral cancertherapy
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was supported by funds of the NSC102-2314-B-384-002 MOST103-2320-B-037-008 the 103CM-KMU-09the National Sun Yat-sen University-KMU Joint ResearchProject (NSYSU-KMU 103-p014) the kmtth-102-011 andthe Health and welfare surcharge of tobacco products theMinistry of Health and Welfare Taiwan Republic of China(MOHW103-TD-B-111-05)The authors also acknowledgeDrHans-Uwe Dahms for the English editing
References
[1] C-Y Yen C-H Chen C-H Chang et al ldquoMatrix metallopro-teinases (MMP) 1 andMMP10 but notMMP12 are potential oralcancer markersrdquo Biomarkers vol 14 no 4 pp 244ndash249 2009
[2] C-Y Yen C-Y HuangM-F Hou et al ldquoEvaluating the perfor-mance of fibronectin 1 (FN1) integrin12057241205731 (ITGA4) syndecan-2 (SDC2) and glycoprotein CD44 as the potential biomarkersof oral squamous cell carcinoma (OSCC)rdquo Biomarkers vol 18no 1 pp 63ndash72 2013
[3] H Myoung S-P Hong P-Y Yun J-H Lee and M-J KimldquoAnti-cancer effect of genistein in oral squamous cell carcinomawith respect to angiogenesis and in vitro invasionrdquo CancerScience vol 94 no 2 pp 215ndash220 2003
[4] H K Kim E G Wilson Y H Choi and R VerpoorteldquoMetabolomics a tool for anticancer lead-finding from naturalproductsrdquo Planta Medica vol 76 no 11 pp 1094ndash1102 2010
[5] A D Kinghorn L Pan J N Fletcher and H Chai ldquoThe rele-vance of higher plants in lead compound discovery programsrdquoJournal of Natural Products vol 74 no 6 pp 1539ndash1555 2011
[6] C-C Yeh C-N Tseng J-I Yang et al ldquoAntiproliferation andinduction of apoptosis in Ca9-22 oral cancer cells by ethanolicextract of Gracilaria tenuistipitatardquoMolecules vol 17 no 9 pp10916ndash10927 2012
[7] C-C Yeh J-I Yang J-C Lee et al ldquoAnti-proliferative effect ofmethanolic extract of Gracilaria tenuistipitata on oral cancercells involves apoptosis DNA damage and oxidative stressrdquoBMC Complementary and Alternative Medicine vol 12 article142 2012
[8] J S Yang C W Lin C H Hsin M J Hsieh and Y C ChangldquoSelaginella tamariscina attenuates metastasis via Akt pathways
The Scientific World Journal 9
in oral cancer cellsrdquo PLoS ONE vol 8 no 6 Article ID e680352013
[9] B Narotzki A Z Reznick D Aizenbud and Y Levy ldquoGreentea a promising natural product in oral healthrdquoArchives of OralBiology vol 57 no 5 pp 429ndash435 2012
[10] C-Y Yen C-C Chiu R-W Haung et al ldquoAntiprolifera-tive effects of goniothalamin on Ca9-22 oral cancer cellsthrough apoptosis DNAdamage andROS inductionrdquoMutationResearch vol 747 no 2 pp 253ndash258 2012
[11] C-C Chiu J-W Haung F-R Chang et al ldquoGolden berry-derived 4120573-hydroxywithanolide E for selectively killing oralcancer cells by generating ROS DNA damage and apoptoticpathwaysrdquo PLoS ONE vol 8 no 5 Article ID e64739 2013
[12] M T Davies-Coleman andD E A Rivett ldquoNaturally occurring6-substituted 56-dihydro-120572-pyronesrdquo Fortschritte der ChemieOrganischer Naturstoffe vol 55 pp 1ndash35 1989
[13] A Toribio A Bonfils E Delannay et al ldquoNovel seco-dibenzopyrrocoline alkaloid from Cryptocarya oubatchensisrdquoOrganic Letters vol 8 no 17 pp 3825ndash3828 2006
[14] A J Cavalheiro and M Yoshida ldquo6-[120596-arylalkenyl]-56-dihydro-120572-pyrones from Cryptocarya moschata (Lauraceae)rdquoPhytochemistry vol 53 no 7 pp 811ndash819 2000
[15] L D Juliawaty M Kitajima H Takayama S A Achmad andN Aimi ldquoA new type of stilbene-related secondary metaboliteidenburgene from Cryptocarya idenburgensisrdquo Chemical andPharmaceutical Bulletin vol 48 no 11 pp 1726ndash1728 2000
[16] P-M Allard E T H Dau C Eydoux et al ldquoAlkylatedflavanones from the bark of Cryptocarya chartacea as denguevirus NS5 polymerase inhibitorsrdquo Journal of Natural Productsvol 74 no 11 pp 2446ndash2453 2011
[17] T-S Wu C-R Su and K-H Lee ldquoCytotoxic and anti-HIVphenanthroindolizidine alkaloids from Cryptocarya chinensisrdquoNatural Product Communications vol 7 no 6 pp 725ndash727 2012
[18] T-H Chou J-J Chen C-F Peng M-J Cheng and I-S ChenldquoNew flavanones from the leaves of Cryptocarya chinensis andtheir antituberculosis activityrdquo Chemistry and Biodiversity vol8 no 11 pp 2015ndash2024 2011
[19] A A Nasrullah A Zahari J Mohamad and K AwangldquoAntiplasmodial alkaloids from the bark of Cryptocarya nigra(Lauraceae)rdquoMolecules vol 18 no 7 pp 8009ndash8017 2013
[20] R A Davis O Demirkiran M L Sykes et al ldquo7101584081015840-Dihydroobolactone a typanocidal 120572-pyrone from the rainforesttree Cryptocarya obovatardquo Bioorganic andMedicinal ChemistryLetters vol 20 no 14 pp 4057ndash4059 2010
[21] R Feng Z K Guo C M Yan E G Li R X Tan and H MGe ldquoAnti-inflammatory flavonoids from Cryptocarya chingiirdquoPhytochemistry vol 76 pp 98ndash105 2012
[22] V Dumontet N Van Hung M-T Adeline et al ldquoCytotoxicflavonoids and 120572-pyrones from Cryptocarya obovatardquo Journalof Natural Products vol 67 no 5 pp 858ndash862 2004
[23] C Y Ong S K Ling R M Ali et al ldquoSystematic analysis of invitro photo-cytotoxic activity in extracts from terrestrial plantsin Peninsula Malaysia for photodynamic therapyrdquo Journal ofPhotochemistry and Photobiology B vol 96 no 3 pp 216ndash2222009
[24] V Dumontet C Gaspard N van Hung et al ldquoNew cytotoxicflavonoids fromCryptocarya infectoriardquoTetrahedron vol 57 no29 pp 6189ndash6196 2001
[25] H Usman E H Hakim T Harlim et al ldquoCytotoxic chalconesand flavanones from the tree bark of Cryptocarya costatardquoZeitschrift fur Naturforschung C vol 61 no 3-4 pp 184ndash1882006
[26] F Kurniadewi L D Juliawaty Y M Syah et al ldquoPhenolic com-pounds from Cryptocarya konishii their cytotoxic and tyrosinekinase inhibitory propertiesrdquo Journal of Natural Medicines vol64 no 2 pp 121ndash125 2010
[27] T-H Chou J-J Chen S-J Lee M Y Chiang C-W Yang andI-S Chen ldquoCytotoxic flavonoids from the leaves ofCryptocaryachinensisrdquo Journal of Natural Products vol 73 no 9 pp 1470ndash1475 2010
[28] J C Liao ldquoLauraceae in flora of Taiwanrdquo in Editorial Committeeof the Flora of Taiwan vol 2 pp 448ndash451 Taipei Taiwan 1996
[29] B H Chen H W Chang H M Huang et al ldquo(-)-anonaineinduces DNA damage and inhibits growth and migration ofhuman lung carcinomaH1299 cellsrdquo Journal of Agricultural andFood Chemistry vol 59 no 6 pp 2284ndash2290 2011
[30] M-Y Lin Y-R Lee S-Y Chiang et al ldquoCortexMoutan inducesbladder cancer cell death via apoptosis and retards tumorgrowth inmouse bladdersrdquoEvidence-based Complementary andAlternative Medicine vol 2013 Article ID 207279 8 pages 2013
[31] C-C Chiu P-L Liu K-J Huang et al ldquoGoniothalamininhibits growth of human lung cancer cells through DNAdamage apoptosis and reduced migration abilityrdquo Journal ofAgricultural and Food Chemistry vol 59 no 8 pp 4288ndash42932011
[32] J Masters ldquoFalse cell linesrdquoCarcinogenesis vol 23 no 2 p 3712002
[33] H Ding C Han D Guo et al ldquoSelective induction of apoptosisof human oral cancer cell lines by avocado extracts via a ROS-mediated mechanismrdquo Nutrition and Cancer vol 61 no 3 pp348ndash356 2009
[34] J-C Lee M-F Hou H-W Huang et al ldquoMarine algal natu-ral products with anti-oxidative anti-inflammatory and anti-cancer propertiesrdquoCancer Cell International vol 13 no 1 article55 2013
[35] C Gorrini I S Harris and TWMak ldquoModulation of oxidativestress as an anticancer strategyrdquoNature Reviews DrugDiscoveryvol 12 no 12 pp 931ndash947 2013
[36] D Trachootham J Alexandre and P Huang ldquoTargeting can-cer cells by ROS-mediated mechanisms a radical therapeuticapproachrdquo Nature Reviews Drug Discovery vol 8 no 7 pp579ndash591 2009
[37] A K Samhan-Arias F J Martın-Romero and C Gutierrez-Merino ldquoKaempferol blocks oxidative stress in cerebellar gran-ule cells and reveals a key role for reactive oxygen speciesproduction at the plasma membrane in the commitment toapoptosisrdquo Free Radical Biology and Medicine vol 37 no 1 pp48ndash61 2004
[38] S-H Oh and S-C Lim ldquoA rapid and transient ROS generationby cadmium triggers apoptosis via caspase-dependent pathwayin HepG2 cells and this is inhibited through N-acetylcysteine-mediated catalase upregulationrdquo Toxicology and Applied Phar-macology vol 212 no 3 pp 212ndash223 2006
[39] R A Ehlers A Hernandez L S Bloemendal R T EthridgeB Farrow and B M Evers ldquoMitochondrial DNA damage andaltered membrane potential (Δ120595) in pancreatic acinar cellsinduced by reactive oxygen speciesrdquo Surgery vol 126 no 2 pp148ndash155 1999
[40] S McKenzie and N Kyprianou ldquoApoptosis evasion the role ofsurvival pathways in prostate cancer progression and therapeu-tic resistancerdquo Journal of Cellular Biochemistry vol 97 no 1 pp18ndash32 2006
10 The Scientific World Journal
[41] S Fulda ldquoEvasion of apoptosis as a cellular stress response incancerrdquo International Journal of Cell Biology vol 2010 ArticleID 370835 6 pages 2010
[42] H Lin X-B Liu J-J Yu F Hua and Z-W Hu ldquoAntioxidantN-acetylcysteine attenuates hepatocarcinogenesis by inhibitingROSER stress in TLR2 deficient mouserdquo PLoS ONE vol 8 no10 Article ID e74130 2013
[43] R Scherz-Shouval and Z Elazar ldquoROS mitochondria and theregulation of autophagyrdquoTrends in Cell Biology vol 17 no 9 pp422ndash427 2007
[44] R Scherz-Shouval and Z Elazar ldquoRegulation of autophagy byROS physiology and pathologyrdquoTrends in Biochemical Sciencesvol 36 no 1 pp 30ndash38 2011
Submit your manuscripts athttpwwwhindawicom
PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom
Volume 2014
ToxinsJournal of
VaccinesJournal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
AntibioticsInternational Journal of
ToxicologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Drug DeliveryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in Pharmacological Sciences
Tropical MedicineJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
AddictionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Autoimmune Diseases
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anesthesiology Research and Practice
ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Pharmaceutics
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
The Scientific World Journal 3
120
100
80
60
40
20
0
0 5 10 2015
Cel
l via
bilit
y (
)Ca9-22
MECCrt (120583gmL)
lowastlowast
lowastlowast
lowastlowast
lowastlowast
(a)
0 5 10 2015
Cel
l via
bilit
y (
)
MECCrt (120583gmL)
CAL 27140
120
100
80
60
40
20
0
lowastlowast
lowastlowast
lowastlowastlowastlowast
(b)
Figure 1 Cell viability of two oral cancer cells was inhibited by MECCrt Oral cancer Ca9-22 and CAL 27 cell lines were treated with variousconcentrations ofMECCrt (0 5 10 15 and 20 120583gmL) for 24 hThe cell viability was measured by theMTS assay Data means plusmn SDs (119899 = 18)lowastlowast
119875 lt 001 against vehicle
centrifugation cell pellets were resuspended in 1mL PBSbefore analyzing by a flow cytometer (BDAccuri C6 Becton-Dickinson) and its software
26 Mitochondrial Membrane Potential MitoProbeDiOC
2(3) assay kit (Invitrogen Eugene OR USA) was
applied to analyze mitochondrial membrane potential(MMP) as described previously [10] Briefly 3 times 105 cellsin 2mL medium per well in 6 well plates were plated for24 h After MECCrt treatment 10 120583L of 10 120583M DiOC
2(3)
was added per well and incubated in a cell culture incubatorfor 20min After being harvested cells were washedand resuspended in 1mL PBS for analysis using a flowcytometer (BD Accuri C6 Becton-Dickinson) and itssoftware
27 Statistical Analysis The significance of differences wasdetermined by Studentrsquos 119905-test compared with the test datawith the vehicle controls Data are expressed as means plusmn SDs
3 Results
31 Antiproliferation in MECCrt-Treated Two Oral CancerCell Lines Based on MTS assay (Figure 1) the relative cellviability () of oral cancer Ca9-22 cells at indicated concen-trations of MECCrt (0 5 10 15 and 20120583gmL) was 1000 plusmn07 933 plusmn 23 714 plusmn 30 576 plusmn 16 and 484 plusmn 12 after24 h respectively The relative cell viability () of CAL 27cells at indicated concentrations of MECCrt (0 5 10 15 and20120583gmL) was 1000 plusmn 081193 plusmn 49 869 plusmn 100 298 plusmn 62and 284 plusmn 55 respectively The MTS-based cell viabilities ofMECCrt-treated twooral cancerCa9-22 andCAL27 cell linessignificantly reduced in a dose-responsive manner (119875 lt 001compared to the vehicle)
32 Sub-G1 Population in MECCrt-Treated Two Oral Can-cer Cell Lines The MECCrt-treated effects of cell cycledistribution profiles are demonstrated in Figure 2(a) AfterMECCrt treatment (Figure 2(b)) the sub-G1 populations ()ofMECCrt- (0 5 10 15 20 and 25 120583gmL) treated oral cancerCa9-22 cells were 50 plusmn 03 67 plusmn 01 181 plusmn 06 179 plusmn 07165plusmn03 and 224plusmn13 and those ofMECCrt-treatedCAL 27cells were 67plusmn17 51plusmn11 79plusmn01 283plusmn10 526plusmn02 and691 plusmn 01 respectively These sub-G1 changes significantlyaccumulated in a dose-responsive manner (119875 lt 001)
33 Apoptosis of MECCrt-Treated Two Oral Cancer Cell LinesTo validate the possible outcome of apoptosis in MECCrt-induced sub-G1 accumulation of these two oral cancer cellsannexin VPI profiles of flow cytometry were generated(Figure 3(a)) In Figure 3(b) the percentages of annexin V-positive intensities forMECCrt (0 5 10 15 20 and 25120583gmL)treatment of Ca9-22 cells were 93 plusmn 02 86 plusmn 03 117 plusmn 09223 plusmn 08 400 plusmn 02 and 544 plusmn 17 and those of MECCrt-treated CAL 27 cells were 248 plusmn 01 175 plusmn 03 207 plusmn 04598plusmn17 794plusmn02 and 853plusmn05 respectively AccordinglyMECCrt treatments significantly increased in annexin V-positive intensities of two oral cancer Ca9-22 and CAL 27 celllines in a dose-responsive manner (119875 lt 001)
34 ROSGeneration inMECCrt-Treated TwoOral Cancer CellLines To validate the role of ROS in the MECCrt-inducedapoptosis of two oral cancer cell lines a DCFH-DA assay offlow cytometry was chosen Figures 4(a) and 4(b) show therelative ROS-positive staining () of two oral cancer Ca9-22 and CAL 27 cell lines for the different concentrations ofMECCrt treatment for 6 and 12 h incubation After MECCrttreatment for 6 h the relative ROS-positive staining () of 05 10 15 20 and 25 120583gmL MECCrt-treated Ca9-22 cells was
4 The Scientific World Journal
Cou
ntCa
9-22
Cou
ntCa
9-22
0 120583gmL 5120583gmL 10120583gmL
15120583gmL 20120583gmL 25120583gmL
400
300
200
100
0
DNA content DNA content DNA content
400
300
200
100
0
Sub-G147G1
492S
177G2M288
Sub-G168G1
529S
182G2M232
Sub-G1171
G1370
S127G2M285
Sub-G1158
G1307
S179G2M265
Sub-G1141
G1266
S212G2M235
Sub-G1200
G1293
S201G2M208
(a)
Cou
ntC
ount
0120583gmL 5120583gmL 10120583gmL
15120583gmL 20120583gmL 25120583gmL
400
300
200
100
0
DNA content DNA content DNA content
400
300
200
100
0
Sub-G148G1
539S
201G2M216
Sub-G151G1
548S
214G2M192
Sub-G1G1
539S
201G2M216
Sub-G1279
G1322
S230G2M184
Sub-G1535
G1248
S122G2M109
Sub-G1699
G1181
SG2M
48
78
54
CAL 27
CAL 27
(b)
Sub-
G1
()
MECCrt (120583gmL)
lowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowast20
20
10
10
5
5
0
0
25
25
15
15
(c)
Sub-
G1
()
lowastlowast
lowastlowast
lowastlowast
0
10
20
30
40
50
60
70
80
MECCrt (120583gmL)201050 2515
(d)
Figure 2 The sub-G1 accumulation of two oral cancer cells was induced by MECCrt Oral cancer Ca9-22 and CAL 27 cell lines were treatedwith 0 5 10 15 20 and 25 120583gmL of MECCrt for 24 h ((a) and (b)) Representative cell cycle distribution profiles of flow cytometry forMECCrt-treated oral cancer Ca9-22 and CAL 27 cells and vehicles at 24 h respectively ((c) and (d)) Statistics analyses for the percentages ofsub-G1 population in (a) and (b) respectively Data means plusmn SDs (119899 = 3) lowastlowast119875 lt 001 against vehicle
1000 plusmn 18 1067 plusmn 04 1312 plusmn 08 1406 plusmn 16 1506 plusmn 03and 1831 plusmn 78 and that of MECCrt-treated CAL 27 cells was1000 plusmn 08 1141 plusmn 14 1423 plusmn 15 1611 plusmn 07 1798 plusmn 11and 1851 plusmn 14 respectively After MECCrt treatment for12 h the relative ROS-positive staining () of 0 5 10 1520 and 25120583gmL MECCrt-treated Ca9-22 cells was 1000 plusmn23 1241 plusmn 04 1664 plusmn 12 1848 plusmn 06 1933 plusmn 03 and2005 plusmn 00 and that of MECCrt-treated CAL 27 cells was1000plusmn09 1407plusmn14 1771plusmn03 1938plusmn01 1986plusmn01 and1994 plusmn 01 respectively Accordingly MECCrt treatmentssignificantly increased in both dose-responsive and time-dependent manners in these two oral cancer cell lines (119875 lt005) (Figures 4(c) and 4(d))
35 MMP Depolarization in MECCrt-Treated Two Oral Can-cer Cell Lines Figures 5(a) and 5(b) show the MMP profilesof DiOC
2(3)-positive intensities for the vehicle andMECCrt-
treated oral cancer cell lines in 24-hour treatments Treated
with MECCrt (0 5 10 15 20 and 25120583gmL) for 24 hthe DiOC
2(3)-positive () intensities of Ca9-22 cells were
1000 plusmn 29 961 plusmn 24 940 plusmn 16 768 plusmn 14 456 plusmn 14and 250 plusmn 11 respectively Similarly the percentages ofDiOC
2(3)-positive () intensities ofMECCrt-treatedCAL27
cells were 1000 plusmn 15 1142 plusmn 07 1089 plusmn 13 513 plusmn 05273 plusmn 05 and 75 plusmn 04 respectively Accordingly MECCrtsignificantly reduced DiOC
2(3)-positive intensities of two
oral cancer Ca9-22 and CAL 27 cell lines in a dose-responsivemanner (119875 lt 001ndash005)
4 Discussion
We discovered for the first time that methanolic extracts ofthe roots ofC concinnaHance have an antiproliferative effecton two oral cancer cell lines The proliferation inhibitingfunction of MECCrt against oral cancer Ca9-22 and CAL 27cell lines was dose-responsive (Figure 1)
The Scientific World Journal 5
104
105
106
1027
104
105
106
1027
1031
104
105
10610
6410
3110
410
510
610
6410
3110
410
510
610
64
Ca9
-22
0 120583gmL 5120583gmL 10120583gmL
15120583gmL 20120583gmL 25120583gmLAnnexin V intensity Annexin V intensity Annexin V intensity
PI in
tens
ityCa
9-22
PI in
tens
ity
(a)
15120583gmL 20120583gmL 25120583gmLAnnexin V intensity Annexin V intensity Annexin V intensity
104
105
106
1027
104
105
106
1027
1031
104
105
10610
6410
3110
410
510
610
6410
3110
410
510
610
64
0 120583gmL 5120583gmL 10120583gmL
PI in
tens
ityPI
inte
nsity
CAL 27
CAL 27
(b)
Apop
totic
cells
()
lowastlowast
lowastlowast
lowastlowast
lowastlowast
60
50
40
30
20
10
0
0 5 10 15 20 25
MECCrt (120583gmL)
(c)
Apop
totic
cells
()
lowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowast
10
20
30
40
50
60
70
80
90
0 5 10 15 20 25
MECCrt (120583gmL)
(d)
Figure 3 Apoptosis of two oral cancer cells was induced byMECCrt Oral cancer Ca9-22 and CAL 27 cell lines were treated with 0ndash25120583gmLof MECCrt for 24 h ((a) and (b)) Representative results of annexin VPI double staining of flow cytometry for MECCrt-treated oral cancerCa9-22 and CAL 27 cell lines and vehicle controls at 24 h respectively ((c) and (d)) Quantification analysis of apoptosis for MECCrt-treatedoral cancer Ca9-22 and CAL 27 cell lines in (a) and (b) respectively Data means plusmn SDs (119899 = 3) lowastlowast119875 lt 001 against vehicle
6 The Scientific World Journal
Cou
ntCa
9-22
0 120583gmL 5120583gmL 10120583gmL
15120583gmL
400
300
200
100
0
Cou
ntCa
9-22
400
300
200
100
0
400
300
200
100
0
400
300
200
100
0
10 5000 10000 10 5000 10000 10 5000 10000 10 5000 10000 10 5000 10000 10 5000 10000
ROS intensity20120583gmL
ROS intensity ROS intensity25120583gmL 15120583gmL
ROS intensity20120583gmL
ROS intensity ROS intensity25120583gmL
6h 6h 6h0120583gmL 5120583gmL 10120583gmL12h 12h 12h
V2-L500
V2-R V2-L V2-R V2-L V2-R
V2-L V2-R V2-L V2-R V2-L V2-R
V3-L V3-R V3-L V3-R
V3-L V3-R
V3-L V3-R
V3-L V3-R V3-L V3-R
500 500 500
916 962 996
172 828618382471 529 347 653
298 702 255 745 119 881 84 38 04
(a)
Cou
nt
0120583gmL 5120583gmL 10120583gmL
15120583gmL
400
300
200
100
0
Cou
nt
400
300
200
100
0
400
300
200
100
0
400
300
200
100
0
10 5000 10000 10 5000 10000 10 5000 10000 10 5000 10000 10 5000 10000 10 5000 10000
ROS intensity20120583gmL
ROS intensity ROS intensity25120583gmL 15120583gmL
ROS intensity20120583gmL
ROS intensity ROS intensity25120583gmL
6h 6h 6h0120583gmL 5120583gmL 10120583gmL12h 12h 12h
V2-L500
V2-R V2-L V2-R V2-L V2-R
V2-L V2-R V2-L V2-R V2-L V2-R
V2-L V2-R V2-L V2-R
V2-L V2-R
V2-L V2-R
V2-L V2-R V2-L V2-R
500 500 500
970 993 998
112 888700300497 503 386 614
298 702 173 827 921 3079 07 02
CAL 27
CAL 27
(b)
lowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowast
Relat
ive R
OS-
posit
ive (
)
220
200
180
160
140
120
100
80
0 5 10 15 20 25
6h12h
MECCrt (120583gmL)
(c)
lowastlowastlowastlowastlowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowastlowastlowast
lowastlowastlowastlowast
Relat
ive R
OS-
posit
ive (
)
220
200
180
160
140
120
100
80
0 5 10 15 20 25 25
6h12h
MECCrt (120583gmL)
(d)
Figure 4 Reactive oxygen species (ROS) generation of two oral cancer cell lines was induced by MECCrt Oral cancer Ca9-22 and CAL 27cell lines were treated with different concentrations (0ndash25120583gmL) of MECCrt for 6 and 12 h ((a) (b)) Representative ROS profiles of flowcytometry for MECCrt-treated oral cancer Ca9-22 and CAL 27 cell lines ((c) and (d)) Statistics analysis of relative ROS intensity in (a) and(b) respectively Data means plusmn SDs (119899 = 3) lowastlowast119875 lt 001 against vehicle
The Scientific World Journal 7
500V1-L V1-R
500 517V1-L V1-R
483 539V1-L V1-R
461
620V1-L V1-R
380 770V1-L V1-R
230 872V1-L V1-R
128
Cou
ntCa
9-22
Cou
ntCa
9-22
0 120583gmL 5120583gmL 10120583gmL
15120583gmL 20120583gmL 25120583gmLMMP intensity MMP intensity MMP intensity
1200
1000
500
0
1200
1000
500
0
10110
210
310
410
510
610
7210
110
210
310
410
510
610
7210
110
210
310
410
510
610
72
(a)
500V2-L V2-R
500 440V2-L V2-R
560 455V2-L V2-R
545
744V2-L V2-R
256 868V2-L V2-R
132 964V2-L V2-R
Cou
ntC
ount
0120583gmL 5120583gmL 10120583gmL
15120583gmL 20120583gmL 25120583gmLMMP intensity MMP intensity MMP intensity
1200
1000
500
0
1200
1000
500
0
10110
210
310
410
510
610
7210
110
210
310
410
510
610
7210
110
210
310
410
510
610
72
36
CAL 27
CAL 27
(b)
lowast
lowastlowast
lowastlowast
lowastlowast
120
100
80
60
40
20
0
Relat
ive M
MP-
posit
ive (
)
0 5 10 15 20 25
MECCrt (120583gmL)
(c)
lowastlowastlowastlowast
lowastlowast
lowastlowast
lowastlowast
120
140
100
80
60
40
20
0
Relat
ive M
MP-
posit
ive (
)
0 5 10 15 20 25
MECCrt (120583gmL)
(d)
Figure 5 Depolarization of mitochondrial membrane potential (MMP) of Ca9-22 and CAL 27 oral cancer cell lines was induced byMECCrt Oral cancer Ca9-22 and CAL 27 cell lines were treated with different concentrations (0ndash25120583gmL) of MECCrt for 24 h ((a) (b))RepresentativeMMPprofiles of flow cytometry forMECCrt-treated oral cancer Ca9-22 andCAL 27 cells ((c) and (d))Quantification analysisof relative MMP intensity in (a) and (b) respectively Data means plusmn SDs (119899 = 3) lowast119875 lt 005 and lowastlowast119875 lt 001 against vehicle
8 The Scientific World Journal
The anticancer effects for other Cryptocarya-derivedcompounds from methanolic extracts of nonroot parts havebeen reported earlier For example for murine leukemiaP-388 cells the IC
50values of 2101584041015840-dihydroxy-5101584061015840-
dimethoxychalcone and isodidymocarpin isolated fromtree bark of C costata were 57 and 111 120583M [25] and IC
50
values of the chalcone derivative (desmethylinfectocaryone)and phenolic compound (infectocaryone) isolated fromwood of C konishii were 217 and 08 120583M [26] at 48 hrespectively For compounds from leaves of C chinensis theIC50
values of infectocaryone and cryptocaryanone A wereat the 120583M level for human lung cancer NCI-H460 cells andglioblastoma SF-268 cells [27] These Cryptocarya-derivedcompounds from methanolic extracts of nonroot partsshowed the IC
50values ranging from 08 to 11 120583M This is
close to our preliminary result that the IC50
of the clinicalanticancer drug cisplatin at 24 h treatment in oral cancerCa9-22 cells is 306 120583gmL (102 120583M) (data not shown) Inthe present study the IC
50values of the MECCrt in oral
cancer Ca9-22 and CAL 27 cell lines at 24 h were 1867 and1322 120583gmL respectively Although the IC
50values of the
MECCrt were about 3-4 folds of cisplatin for oral cancercells its crude extract nature has to be concerned Thereforeit is warranted to further investigate the particular bioactivecomponents that are included in the methanolic extracts ofCryptocarya concinnaHance roots
Moreover the anticancer effect for trunk bark ofC infectoria-derived methanol extracts was reported tobe cytotoxic to KB cells [24] KB cells were regardedas oral epidermal carcinoma however it was recentlyvalidated to have marker chromosomes and DNAfinger printings of human cervical cancer HeLacells (httpwwwncbinlmnihgovmeshDb=meshampterm=KB+Cells) [32] Accordingly the anticancer effect of oralcancer by the bioactive compounds from Cryptocarya plantremains unclear Conversely we here demonstrate theantioral cancer effect of methanolic extracts of a Cryptocaryaspecies for the first time using two OSCC cell lines Ca9-22and CAL 27
In several anticancer drugs [6 7 10 11 33ndash36] ROSgeneration is one of the common strategies to inhibit cancercell proliferation ROS plays a vital role in early stagesof apoptosis [37] and leads to MMP depolarization [3839] Escaping apoptosis is demonstrated to be involved inthe drug resistance of cancer cells [40 41] To enhanceapoptotic induction of anticancer drugs may interfere thedrug resistance if there In the present study we observed thatapoptosis was inducible by MECCrt in two OSCC cell linesas it was demonstrated by sub-G1 monitoring and annexinVPI assayWe also found that MECCrt significantly inducedthe ROS level and reduced the MMP level in two oral cancercell lines in dose-responsive ways These findings suggestthat oxidative stress may be involved in theMECCrt-inducedantiproliferative effect in two oral cancer Ca9-22 and CAL 27cell lines However the role of oxidative stress in MECCrtneed to be further examined by the ROS scavenger such asN-acetylcysteine [42] to confirm if raised ROS has played acritical role in the process of apoptosis Furthermore the ROSmay generate nonapoptotic effect like autophagy described
in literature [43 44] Therefore it was warranted to furtherinvestigate the role of autophagy in MECCrt-treated oralcancer cell lines in future
5 Conclusions
We demonstrated the antiproliferative and apoptotic effectsof MECCrt through ROS generation and mitochondrialdepolarization in twoOSCC cell linesTherefore these resultssuggest that MECCrt has anticancer potential for oral cancertherapy
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was supported by funds of the NSC102-2314-B-384-002 MOST103-2320-B-037-008 the 103CM-KMU-09the National Sun Yat-sen University-KMU Joint ResearchProject (NSYSU-KMU 103-p014) the kmtth-102-011 andthe Health and welfare surcharge of tobacco products theMinistry of Health and Welfare Taiwan Republic of China(MOHW103-TD-B-111-05)The authors also acknowledgeDrHans-Uwe Dahms for the English editing
References
[1] C-Y Yen C-H Chen C-H Chang et al ldquoMatrix metallopro-teinases (MMP) 1 andMMP10 but notMMP12 are potential oralcancer markersrdquo Biomarkers vol 14 no 4 pp 244ndash249 2009
[2] C-Y Yen C-Y HuangM-F Hou et al ldquoEvaluating the perfor-mance of fibronectin 1 (FN1) integrin12057241205731 (ITGA4) syndecan-2 (SDC2) and glycoprotein CD44 as the potential biomarkersof oral squamous cell carcinoma (OSCC)rdquo Biomarkers vol 18no 1 pp 63ndash72 2013
[3] H Myoung S-P Hong P-Y Yun J-H Lee and M-J KimldquoAnti-cancer effect of genistein in oral squamous cell carcinomawith respect to angiogenesis and in vitro invasionrdquo CancerScience vol 94 no 2 pp 215ndash220 2003
[4] H K Kim E G Wilson Y H Choi and R VerpoorteldquoMetabolomics a tool for anticancer lead-finding from naturalproductsrdquo Planta Medica vol 76 no 11 pp 1094ndash1102 2010
[5] A D Kinghorn L Pan J N Fletcher and H Chai ldquoThe rele-vance of higher plants in lead compound discovery programsrdquoJournal of Natural Products vol 74 no 6 pp 1539ndash1555 2011
[6] C-C Yeh C-N Tseng J-I Yang et al ldquoAntiproliferation andinduction of apoptosis in Ca9-22 oral cancer cells by ethanolicextract of Gracilaria tenuistipitatardquoMolecules vol 17 no 9 pp10916ndash10927 2012
[7] C-C Yeh J-I Yang J-C Lee et al ldquoAnti-proliferative effect ofmethanolic extract of Gracilaria tenuistipitata on oral cancercells involves apoptosis DNA damage and oxidative stressrdquoBMC Complementary and Alternative Medicine vol 12 article142 2012
[8] J S Yang C W Lin C H Hsin M J Hsieh and Y C ChangldquoSelaginella tamariscina attenuates metastasis via Akt pathways
The Scientific World Journal 9
in oral cancer cellsrdquo PLoS ONE vol 8 no 6 Article ID e680352013
[9] B Narotzki A Z Reznick D Aizenbud and Y Levy ldquoGreentea a promising natural product in oral healthrdquoArchives of OralBiology vol 57 no 5 pp 429ndash435 2012
[10] C-Y Yen C-C Chiu R-W Haung et al ldquoAntiprolifera-tive effects of goniothalamin on Ca9-22 oral cancer cellsthrough apoptosis DNAdamage andROS inductionrdquoMutationResearch vol 747 no 2 pp 253ndash258 2012
[11] C-C Chiu J-W Haung F-R Chang et al ldquoGolden berry-derived 4120573-hydroxywithanolide E for selectively killing oralcancer cells by generating ROS DNA damage and apoptoticpathwaysrdquo PLoS ONE vol 8 no 5 Article ID e64739 2013
[12] M T Davies-Coleman andD E A Rivett ldquoNaturally occurring6-substituted 56-dihydro-120572-pyronesrdquo Fortschritte der ChemieOrganischer Naturstoffe vol 55 pp 1ndash35 1989
[13] A Toribio A Bonfils E Delannay et al ldquoNovel seco-dibenzopyrrocoline alkaloid from Cryptocarya oubatchensisrdquoOrganic Letters vol 8 no 17 pp 3825ndash3828 2006
[14] A J Cavalheiro and M Yoshida ldquo6-[120596-arylalkenyl]-56-dihydro-120572-pyrones from Cryptocarya moschata (Lauraceae)rdquoPhytochemistry vol 53 no 7 pp 811ndash819 2000
[15] L D Juliawaty M Kitajima H Takayama S A Achmad andN Aimi ldquoA new type of stilbene-related secondary metaboliteidenburgene from Cryptocarya idenburgensisrdquo Chemical andPharmaceutical Bulletin vol 48 no 11 pp 1726ndash1728 2000
[16] P-M Allard E T H Dau C Eydoux et al ldquoAlkylatedflavanones from the bark of Cryptocarya chartacea as denguevirus NS5 polymerase inhibitorsrdquo Journal of Natural Productsvol 74 no 11 pp 2446ndash2453 2011
[17] T-S Wu C-R Su and K-H Lee ldquoCytotoxic and anti-HIVphenanthroindolizidine alkaloids from Cryptocarya chinensisrdquoNatural Product Communications vol 7 no 6 pp 725ndash727 2012
[18] T-H Chou J-J Chen C-F Peng M-J Cheng and I-S ChenldquoNew flavanones from the leaves of Cryptocarya chinensis andtheir antituberculosis activityrdquo Chemistry and Biodiversity vol8 no 11 pp 2015ndash2024 2011
[19] A A Nasrullah A Zahari J Mohamad and K AwangldquoAntiplasmodial alkaloids from the bark of Cryptocarya nigra(Lauraceae)rdquoMolecules vol 18 no 7 pp 8009ndash8017 2013
[20] R A Davis O Demirkiran M L Sykes et al ldquo7101584081015840-Dihydroobolactone a typanocidal 120572-pyrone from the rainforesttree Cryptocarya obovatardquo Bioorganic andMedicinal ChemistryLetters vol 20 no 14 pp 4057ndash4059 2010
[21] R Feng Z K Guo C M Yan E G Li R X Tan and H MGe ldquoAnti-inflammatory flavonoids from Cryptocarya chingiirdquoPhytochemistry vol 76 pp 98ndash105 2012
[22] V Dumontet N Van Hung M-T Adeline et al ldquoCytotoxicflavonoids and 120572-pyrones from Cryptocarya obovatardquo Journalof Natural Products vol 67 no 5 pp 858ndash862 2004
[23] C Y Ong S K Ling R M Ali et al ldquoSystematic analysis of invitro photo-cytotoxic activity in extracts from terrestrial plantsin Peninsula Malaysia for photodynamic therapyrdquo Journal ofPhotochemistry and Photobiology B vol 96 no 3 pp 216ndash2222009
[24] V Dumontet C Gaspard N van Hung et al ldquoNew cytotoxicflavonoids fromCryptocarya infectoriardquoTetrahedron vol 57 no29 pp 6189ndash6196 2001
[25] H Usman E H Hakim T Harlim et al ldquoCytotoxic chalconesand flavanones from the tree bark of Cryptocarya costatardquoZeitschrift fur Naturforschung C vol 61 no 3-4 pp 184ndash1882006
[26] F Kurniadewi L D Juliawaty Y M Syah et al ldquoPhenolic com-pounds from Cryptocarya konishii their cytotoxic and tyrosinekinase inhibitory propertiesrdquo Journal of Natural Medicines vol64 no 2 pp 121ndash125 2010
[27] T-H Chou J-J Chen S-J Lee M Y Chiang C-W Yang andI-S Chen ldquoCytotoxic flavonoids from the leaves ofCryptocaryachinensisrdquo Journal of Natural Products vol 73 no 9 pp 1470ndash1475 2010
[28] J C Liao ldquoLauraceae in flora of Taiwanrdquo in Editorial Committeeof the Flora of Taiwan vol 2 pp 448ndash451 Taipei Taiwan 1996
[29] B H Chen H W Chang H M Huang et al ldquo(-)-anonaineinduces DNA damage and inhibits growth and migration ofhuman lung carcinomaH1299 cellsrdquo Journal of Agricultural andFood Chemistry vol 59 no 6 pp 2284ndash2290 2011
[30] M-Y Lin Y-R Lee S-Y Chiang et al ldquoCortexMoutan inducesbladder cancer cell death via apoptosis and retards tumorgrowth inmouse bladdersrdquoEvidence-based Complementary andAlternative Medicine vol 2013 Article ID 207279 8 pages 2013
[31] C-C Chiu P-L Liu K-J Huang et al ldquoGoniothalamininhibits growth of human lung cancer cells through DNAdamage apoptosis and reduced migration abilityrdquo Journal ofAgricultural and Food Chemistry vol 59 no 8 pp 4288ndash42932011
[32] J Masters ldquoFalse cell linesrdquoCarcinogenesis vol 23 no 2 p 3712002
[33] H Ding C Han D Guo et al ldquoSelective induction of apoptosisof human oral cancer cell lines by avocado extracts via a ROS-mediated mechanismrdquo Nutrition and Cancer vol 61 no 3 pp348ndash356 2009
[34] J-C Lee M-F Hou H-W Huang et al ldquoMarine algal natu-ral products with anti-oxidative anti-inflammatory and anti-cancer propertiesrdquoCancer Cell International vol 13 no 1 article55 2013
[35] C Gorrini I S Harris and TWMak ldquoModulation of oxidativestress as an anticancer strategyrdquoNature Reviews DrugDiscoveryvol 12 no 12 pp 931ndash947 2013
[36] D Trachootham J Alexandre and P Huang ldquoTargeting can-cer cells by ROS-mediated mechanisms a radical therapeuticapproachrdquo Nature Reviews Drug Discovery vol 8 no 7 pp579ndash591 2009
[37] A K Samhan-Arias F J Martın-Romero and C Gutierrez-Merino ldquoKaempferol blocks oxidative stress in cerebellar gran-ule cells and reveals a key role for reactive oxygen speciesproduction at the plasma membrane in the commitment toapoptosisrdquo Free Radical Biology and Medicine vol 37 no 1 pp48ndash61 2004
[38] S-H Oh and S-C Lim ldquoA rapid and transient ROS generationby cadmium triggers apoptosis via caspase-dependent pathwayin HepG2 cells and this is inhibited through N-acetylcysteine-mediated catalase upregulationrdquo Toxicology and Applied Phar-macology vol 212 no 3 pp 212ndash223 2006
[39] R A Ehlers A Hernandez L S Bloemendal R T EthridgeB Farrow and B M Evers ldquoMitochondrial DNA damage andaltered membrane potential (Δ120595) in pancreatic acinar cellsinduced by reactive oxygen speciesrdquo Surgery vol 126 no 2 pp148ndash155 1999
[40] S McKenzie and N Kyprianou ldquoApoptosis evasion the role ofsurvival pathways in prostate cancer progression and therapeu-tic resistancerdquo Journal of Cellular Biochemistry vol 97 no 1 pp18ndash32 2006
10 The Scientific World Journal
[41] S Fulda ldquoEvasion of apoptosis as a cellular stress response incancerrdquo International Journal of Cell Biology vol 2010 ArticleID 370835 6 pages 2010
[42] H Lin X-B Liu J-J Yu F Hua and Z-W Hu ldquoAntioxidantN-acetylcysteine attenuates hepatocarcinogenesis by inhibitingROSER stress in TLR2 deficient mouserdquo PLoS ONE vol 8 no10 Article ID e74130 2013
[43] R Scherz-Shouval and Z Elazar ldquoROS mitochondria and theregulation of autophagyrdquoTrends in Cell Biology vol 17 no 9 pp422ndash427 2007
[44] R Scherz-Shouval and Z Elazar ldquoRegulation of autophagy byROS physiology and pathologyrdquoTrends in Biochemical Sciencesvol 36 no 1 pp 30ndash38 2011
Submit your manuscripts athttpwwwhindawicom
PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom
Volume 2014
ToxinsJournal of
VaccinesJournal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
AntibioticsInternational Journal of
ToxicologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Drug DeliveryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in Pharmacological Sciences
Tropical MedicineJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
AddictionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Autoimmune Diseases
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anesthesiology Research and Practice
ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Pharmaceutics
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
4 The Scientific World Journal
Cou
ntCa
9-22
Cou
ntCa
9-22
0 120583gmL 5120583gmL 10120583gmL
15120583gmL 20120583gmL 25120583gmL
400
300
200
100
0
DNA content DNA content DNA content
400
300
200
100
0
Sub-G147G1
492S
177G2M288
Sub-G168G1
529S
182G2M232
Sub-G1171
G1370
S127G2M285
Sub-G1158
G1307
S179G2M265
Sub-G1141
G1266
S212G2M235
Sub-G1200
G1293
S201G2M208
(a)
Cou
ntC
ount
0120583gmL 5120583gmL 10120583gmL
15120583gmL 20120583gmL 25120583gmL
400
300
200
100
0
DNA content DNA content DNA content
400
300
200
100
0
Sub-G148G1
539S
201G2M216
Sub-G151G1
548S
214G2M192
Sub-G1G1
539S
201G2M216
Sub-G1279
G1322
S230G2M184
Sub-G1535
G1248
S122G2M109
Sub-G1699
G1181
SG2M
48
78
54
CAL 27
CAL 27
(b)
Sub-
G1
()
MECCrt (120583gmL)
lowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowast20
20
10
10
5
5
0
0
25
25
15
15
(c)
Sub-
G1
()
lowastlowast
lowastlowast
lowastlowast
0
10
20
30
40
50
60
70
80
MECCrt (120583gmL)201050 2515
(d)
Figure 2 The sub-G1 accumulation of two oral cancer cells was induced by MECCrt Oral cancer Ca9-22 and CAL 27 cell lines were treatedwith 0 5 10 15 20 and 25 120583gmL of MECCrt for 24 h ((a) and (b)) Representative cell cycle distribution profiles of flow cytometry forMECCrt-treated oral cancer Ca9-22 and CAL 27 cells and vehicles at 24 h respectively ((c) and (d)) Statistics analyses for the percentages ofsub-G1 population in (a) and (b) respectively Data means plusmn SDs (119899 = 3) lowastlowast119875 lt 001 against vehicle
1000 plusmn 18 1067 plusmn 04 1312 plusmn 08 1406 plusmn 16 1506 plusmn 03and 1831 plusmn 78 and that of MECCrt-treated CAL 27 cells was1000 plusmn 08 1141 plusmn 14 1423 plusmn 15 1611 plusmn 07 1798 plusmn 11and 1851 plusmn 14 respectively After MECCrt treatment for12 h the relative ROS-positive staining () of 0 5 10 1520 and 25120583gmL MECCrt-treated Ca9-22 cells was 1000 plusmn23 1241 plusmn 04 1664 plusmn 12 1848 plusmn 06 1933 plusmn 03 and2005 plusmn 00 and that of MECCrt-treated CAL 27 cells was1000plusmn09 1407plusmn14 1771plusmn03 1938plusmn01 1986plusmn01 and1994 plusmn 01 respectively Accordingly MECCrt treatmentssignificantly increased in both dose-responsive and time-dependent manners in these two oral cancer cell lines (119875 lt005) (Figures 4(c) and 4(d))
35 MMP Depolarization in MECCrt-Treated Two Oral Can-cer Cell Lines Figures 5(a) and 5(b) show the MMP profilesof DiOC
2(3)-positive intensities for the vehicle andMECCrt-
treated oral cancer cell lines in 24-hour treatments Treated
with MECCrt (0 5 10 15 20 and 25120583gmL) for 24 hthe DiOC
2(3)-positive () intensities of Ca9-22 cells were
1000 plusmn 29 961 plusmn 24 940 plusmn 16 768 plusmn 14 456 plusmn 14and 250 plusmn 11 respectively Similarly the percentages ofDiOC
2(3)-positive () intensities ofMECCrt-treatedCAL27
cells were 1000 plusmn 15 1142 plusmn 07 1089 plusmn 13 513 plusmn 05273 plusmn 05 and 75 plusmn 04 respectively Accordingly MECCrtsignificantly reduced DiOC
2(3)-positive intensities of two
oral cancer Ca9-22 and CAL 27 cell lines in a dose-responsivemanner (119875 lt 001ndash005)
4 Discussion
We discovered for the first time that methanolic extracts ofthe roots ofC concinnaHance have an antiproliferative effecton two oral cancer cell lines The proliferation inhibitingfunction of MECCrt against oral cancer Ca9-22 and CAL 27cell lines was dose-responsive (Figure 1)
The Scientific World Journal 5
104
105
106
1027
104
105
106
1027
1031
104
105
10610
6410
3110
410
510
610
6410
3110
410
510
610
64
Ca9
-22
0 120583gmL 5120583gmL 10120583gmL
15120583gmL 20120583gmL 25120583gmLAnnexin V intensity Annexin V intensity Annexin V intensity
PI in
tens
ityCa
9-22
PI in
tens
ity
(a)
15120583gmL 20120583gmL 25120583gmLAnnexin V intensity Annexin V intensity Annexin V intensity
104
105
106
1027
104
105
106
1027
1031
104
105
10610
6410
3110
410
510
610
6410
3110
410
510
610
64
0 120583gmL 5120583gmL 10120583gmL
PI in
tens
ityPI
inte
nsity
CAL 27
CAL 27
(b)
Apop
totic
cells
()
lowastlowast
lowastlowast
lowastlowast
lowastlowast
60
50
40
30
20
10
0
0 5 10 15 20 25
MECCrt (120583gmL)
(c)
Apop
totic
cells
()
lowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowast
10
20
30
40
50
60
70
80
90
0 5 10 15 20 25
MECCrt (120583gmL)
(d)
Figure 3 Apoptosis of two oral cancer cells was induced byMECCrt Oral cancer Ca9-22 and CAL 27 cell lines were treated with 0ndash25120583gmLof MECCrt for 24 h ((a) and (b)) Representative results of annexin VPI double staining of flow cytometry for MECCrt-treated oral cancerCa9-22 and CAL 27 cell lines and vehicle controls at 24 h respectively ((c) and (d)) Quantification analysis of apoptosis for MECCrt-treatedoral cancer Ca9-22 and CAL 27 cell lines in (a) and (b) respectively Data means plusmn SDs (119899 = 3) lowastlowast119875 lt 001 against vehicle
6 The Scientific World Journal
Cou
ntCa
9-22
0 120583gmL 5120583gmL 10120583gmL
15120583gmL
400
300
200
100
0
Cou
ntCa
9-22
400
300
200
100
0
400
300
200
100
0
400
300
200
100
0
10 5000 10000 10 5000 10000 10 5000 10000 10 5000 10000 10 5000 10000 10 5000 10000
ROS intensity20120583gmL
ROS intensity ROS intensity25120583gmL 15120583gmL
ROS intensity20120583gmL
ROS intensity ROS intensity25120583gmL
6h 6h 6h0120583gmL 5120583gmL 10120583gmL12h 12h 12h
V2-L500
V2-R V2-L V2-R V2-L V2-R
V2-L V2-R V2-L V2-R V2-L V2-R
V3-L V3-R V3-L V3-R
V3-L V3-R
V3-L V3-R
V3-L V3-R V3-L V3-R
500 500 500
916 962 996
172 828618382471 529 347 653
298 702 255 745 119 881 84 38 04
(a)
Cou
nt
0120583gmL 5120583gmL 10120583gmL
15120583gmL
400
300
200
100
0
Cou
nt
400
300
200
100
0
400
300
200
100
0
400
300
200
100
0
10 5000 10000 10 5000 10000 10 5000 10000 10 5000 10000 10 5000 10000 10 5000 10000
ROS intensity20120583gmL
ROS intensity ROS intensity25120583gmL 15120583gmL
ROS intensity20120583gmL
ROS intensity ROS intensity25120583gmL
6h 6h 6h0120583gmL 5120583gmL 10120583gmL12h 12h 12h
V2-L500
V2-R V2-L V2-R V2-L V2-R
V2-L V2-R V2-L V2-R V2-L V2-R
V2-L V2-R V2-L V2-R
V2-L V2-R
V2-L V2-R
V2-L V2-R V2-L V2-R
500 500 500
970 993 998
112 888700300497 503 386 614
298 702 173 827 921 3079 07 02
CAL 27
CAL 27
(b)
lowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowast
Relat
ive R
OS-
posit
ive (
)
220
200
180
160
140
120
100
80
0 5 10 15 20 25
6h12h
MECCrt (120583gmL)
(c)
lowastlowastlowastlowastlowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowastlowastlowast
lowastlowastlowastlowast
Relat
ive R
OS-
posit
ive (
)
220
200
180
160
140
120
100
80
0 5 10 15 20 25 25
6h12h
MECCrt (120583gmL)
(d)
Figure 4 Reactive oxygen species (ROS) generation of two oral cancer cell lines was induced by MECCrt Oral cancer Ca9-22 and CAL 27cell lines were treated with different concentrations (0ndash25120583gmL) of MECCrt for 6 and 12 h ((a) (b)) Representative ROS profiles of flowcytometry for MECCrt-treated oral cancer Ca9-22 and CAL 27 cell lines ((c) and (d)) Statistics analysis of relative ROS intensity in (a) and(b) respectively Data means plusmn SDs (119899 = 3) lowastlowast119875 lt 001 against vehicle
The Scientific World Journal 7
500V1-L V1-R
500 517V1-L V1-R
483 539V1-L V1-R
461
620V1-L V1-R
380 770V1-L V1-R
230 872V1-L V1-R
128
Cou
ntCa
9-22
Cou
ntCa
9-22
0 120583gmL 5120583gmL 10120583gmL
15120583gmL 20120583gmL 25120583gmLMMP intensity MMP intensity MMP intensity
1200
1000
500
0
1200
1000
500
0
10110
210
310
410
510
610
7210
110
210
310
410
510
610
7210
110
210
310
410
510
610
72
(a)
500V2-L V2-R
500 440V2-L V2-R
560 455V2-L V2-R
545
744V2-L V2-R
256 868V2-L V2-R
132 964V2-L V2-R
Cou
ntC
ount
0120583gmL 5120583gmL 10120583gmL
15120583gmL 20120583gmL 25120583gmLMMP intensity MMP intensity MMP intensity
1200
1000
500
0
1200
1000
500
0
10110
210
310
410
510
610
7210
110
210
310
410
510
610
7210
110
210
310
410
510
610
72
36
CAL 27
CAL 27
(b)
lowast
lowastlowast
lowastlowast
lowastlowast
120
100
80
60
40
20
0
Relat
ive M
MP-
posit
ive (
)
0 5 10 15 20 25
MECCrt (120583gmL)
(c)
lowastlowastlowastlowast
lowastlowast
lowastlowast
lowastlowast
120
140
100
80
60
40
20
0
Relat
ive M
MP-
posit
ive (
)
0 5 10 15 20 25
MECCrt (120583gmL)
(d)
Figure 5 Depolarization of mitochondrial membrane potential (MMP) of Ca9-22 and CAL 27 oral cancer cell lines was induced byMECCrt Oral cancer Ca9-22 and CAL 27 cell lines were treated with different concentrations (0ndash25120583gmL) of MECCrt for 24 h ((a) (b))RepresentativeMMPprofiles of flow cytometry forMECCrt-treated oral cancer Ca9-22 andCAL 27 cells ((c) and (d))Quantification analysisof relative MMP intensity in (a) and (b) respectively Data means plusmn SDs (119899 = 3) lowast119875 lt 005 and lowastlowast119875 lt 001 against vehicle
8 The Scientific World Journal
The anticancer effects for other Cryptocarya-derivedcompounds from methanolic extracts of nonroot parts havebeen reported earlier For example for murine leukemiaP-388 cells the IC
50values of 2101584041015840-dihydroxy-5101584061015840-
dimethoxychalcone and isodidymocarpin isolated fromtree bark of C costata were 57 and 111 120583M [25] and IC
50
values of the chalcone derivative (desmethylinfectocaryone)and phenolic compound (infectocaryone) isolated fromwood of C konishii were 217 and 08 120583M [26] at 48 hrespectively For compounds from leaves of C chinensis theIC50
values of infectocaryone and cryptocaryanone A wereat the 120583M level for human lung cancer NCI-H460 cells andglioblastoma SF-268 cells [27] These Cryptocarya-derivedcompounds from methanolic extracts of nonroot partsshowed the IC
50values ranging from 08 to 11 120583M This is
close to our preliminary result that the IC50
of the clinicalanticancer drug cisplatin at 24 h treatment in oral cancerCa9-22 cells is 306 120583gmL (102 120583M) (data not shown) Inthe present study the IC
50values of the MECCrt in oral
cancer Ca9-22 and CAL 27 cell lines at 24 h were 1867 and1322 120583gmL respectively Although the IC
50values of the
MECCrt were about 3-4 folds of cisplatin for oral cancercells its crude extract nature has to be concerned Thereforeit is warranted to further investigate the particular bioactivecomponents that are included in the methanolic extracts ofCryptocarya concinnaHance roots
Moreover the anticancer effect for trunk bark ofC infectoria-derived methanol extracts was reported tobe cytotoxic to KB cells [24] KB cells were regardedas oral epidermal carcinoma however it was recentlyvalidated to have marker chromosomes and DNAfinger printings of human cervical cancer HeLacells (httpwwwncbinlmnihgovmeshDb=meshampterm=KB+Cells) [32] Accordingly the anticancer effect of oralcancer by the bioactive compounds from Cryptocarya plantremains unclear Conversely we here demonstrate theantioral cancer effect of methanolic extracts of a Cryptocaryaspecies for the first time using two OSCC cell lines Ca9-22and CAL 27
In several anticancer drugs [6 7 10 11 33ndash36] ROSgeneration is one of the common strategies to inhibit cancercell proliferation ROS plays a vital role in early stagesof apoptosis [37] and leads to MMP depolarization [3839] Escaping apoptosis is demonstrated to be involved inthe drug resistance of cancer cells [40 41] To enhanceapoptotic induction of anticancer drugs may interfere thedrug resistance if there In the present study we observed thatapoptosis was inducible by MECCrt in two OSCC cell linesas it was demonstrated by sub-G1 monitoring and annexinVPI assayWe also found that MECCrt significantly inducedthe ROS level and reduced the MMP level in two oral cancercell lines in dose-responsive ways These findings suggestthat oxidative stress may be involved in theMECCrt-inducedantiproliferative effect in two oral cancer Ca9-22 and CAL 27cell lines However the role of oxidative stress in MECCrtneed to be further examined by the ROS scavenger such asN-acetylcysteine [42] to confirm if raised ROS has played acritical role in the process of apoptosis Furthermore the ROSmay generate nonapoptotic effect like autophagy described
in literature [43 44] Therefore it was warranted to furtherinvestigate the role of autophagy in MECCrt-treated oralcancer cell lines in future
5 Conclusions
We demonstrated the antiproliferative and apoptotic effectsof MECCrt through ROS generation and mitochondrialdepolarization in twoOSCC cell linesTherefore these resultssuggest that MECCrt has anticancer potential for oral cancertherapy
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was supported by funds of the NSC102-2314-B-384-002 MOST103-2320-B-037-008 the 103CM-KMU-09the National Sun Yat-sen University-KMU Joint ResearchProject (NSYSU-KMU 103-p014) the kmtth-102-011 andthe Health and welfare surcharge of tobacco products theMinistry of Health and Welfare Taiwan Republic of China(MOHW103-TD-B-111-05)The authors also acknowledgeDrHans-Uwe Dahms for the English editing
References
[1] C-Y Yen C-H Chen C-H Chang et al ldquoMatrix metallopro-teinases (MMP) 1 andMMP10 but notMMP12 are potential oralcancer markersrdquo Biomarkers vol 14 no 4 pp 244ndash249 2009
[2] C-Y Yen C-Y HuangM-F Hou et al ldquoEvaluating the perfor-mance of fibronectin 1 (FN1) integrin12057241205731 (ITGA4) syndecan-2 (SDC2) and glycoprotein CD44 as the potential biomarkersof oral squamous cell carcinoma (OSCC)rdquo Biomarkers vol 18no 1 pp 63ndash72 2013
[3] H Myoung S-P Hong P-Y Yun J-H Lee and M-J KimldquoAnti-cancer effect of genistein in oral squamous cell carcinomawith respect to angiogenesis and in vitro invasionrdquo CancerScience vol 94 no 2 pp 215ndash220 2003
[4] H K Kim E G Wilson Y H Choi and R VerpoorteldquoMetabolomics a tool for anticancer lead-finding from naturalproductsrdquo Planta Medica vol 76 no 11 pp 1094ndash1102 2010
[5] A D Kinghorn L Pan J N Fletcher and H Chai ldquoThe rele-vance of higher plants in lead compound discovery programsrdquoJournal of Natural Products vol 74 no 6 pp 1539ndash1555 2011
[6] C-C Yeh C-N Tseng J-I Yang et al ldquoAntiproliferation andinduction of apoptosis in Ca9-22 oral cancer cells by ethanolicextract of Gracilaria tenuistipitatardquoMolecules vol 17 no 9 pp10916ndash10927 2012
[7] C-C Yeh J-I Yang J-C Lee et al ldquoAnti-proliferative effect ofmethanolic extract of Gracilaria tenuistipitata on oral cancercells involves apoptosis DNA damage and oxidative stressrdquoBMC Complementary and Alternative Medicine vol 12 article142 2012
[8] J S Yang C W Lin C H Hsin M J Hsieh and Y C ChangldquoSelaginella tamariscina attenuates metastasis via Akt pathways
The Scientific World Journal 9
in oral cancer cellsrdquo PLoS ONE vol 8 no 6 Article ID e680352013
[9] B Narotzki A Z Reznick D Aizenbud and Y Levy ldquoGreentea a promising natural product in oral healthrdquoArchives of OralBiology vol 57 no 5 pp 429ndash435 2012
[10] C-Y Yen C-C Chiu R-W Haung et al ldquoAntiprolifera-tive effects of goniothalamin on Ca9-22 oral cancer cellsthrough apoptosis DNAdamage andROS inductionrdquoMutationResearch vol 747 no 2 pp 253ndash258 2012
[11] C-C Chiu J-W Haung F-R Chang et al ldquoGolden berry-derived 4120573-hydroxywithanolide E for selectively killing oralcancer cells by generating ROS DNA damage and apoptoticpathwaysrdquo PLoS ONE vol 8 no 5 Article ID e64739 2013
[12] M T Davies-Coleman andD E A Rivett ldquoNaturally occurring6-substituted 56-dihydro-120572-pyronesrdquo Fortschritte der ChemieOrganischer Naturstoffe vol 55 pp 1ndash35 1989
[13] A Toribio A Bonfils E Delannay et al ldquoNovel seco-dibenzopyrrocoline alkaloid from Cryptocarya oubatchensisrdquoOrganic Letters vol 8 no 17 pp 3825ndash3828 2006
[14] A J Cavalheiro and M Yoshida ldquo6-[120596-arylalkenyl]-56-dihydro-120572-pyrones from Cryptocarya moschata (Lauraceae)rdquoPhytochemistry vol 53 no 7 pp 811ndash819 2000
[15] L D Juliawaty M Kitajima H Takayama S A Achmad andN Aimi ldquoA new type of stilbene-related secondary metaboliteidenburgene from Cryptocarya idenburgensisrdquo Chemical andPharmaceutical Bulletin vol 48 no 11 pp 1726ndash1728 2000
[16] P-M Allard E T H Dau C Eydoux et al ldquoAlkylatedflavanones from the bark of Cryptocarya chartacea as denguevirus NS5 polymerase inhibitorsrdquo Journal of Natural Productsvol 74 no 11 pp 2446ndash2453 2011
[17] T-S Wu C-R Su and K-H Lee ldquoCytotoxic and anti-HIVphenanthroindolizidine alkaloids from Cryptocarya chinensisrdquoNatural Product Communications vol 7 no 6 pp 725ndash727 2012
[18] T-H Chou J-J Chen C-F Peng M-J Cheng and I-S ChenldquoNew flavanones from the leaves of Cryptocarya chinensis andtheir antituberculosis activityrdquo Chemistry and Biodiversity vol8 no 11 pp 2015ndash2024 2011
[19] A A Nasrullah A Zahari J Mohamad and K AwangldquoAntiplasmodial alkaloids from the bark of Cryptocarya nigra(Lauraceae)rdquoMolecules vol 18 no 7 pp 8009ndash8017 2013
[20] R A Davis O Demirkiran M L Sykes et al ldquo7101584081015840-Dihydroobolactone a typanocidal 120572-pyrone from the rainforesttree Cryptocarya obovatardquo Bioorganic andMedicinal ChemistryLetters vol 20 no 14 pp 4057ndash4059 2010
[21] R Feng Z K Guo C M Yan E G Li R X Tan and H MGe ldquoAnti-inflammatory flavonoids from Cryptocarya chingiirdquoPhytochemistry vol 76 pp 98ndash105 2012
[22] V Dumontet N Van Hung M-T Adeline et al ldquoCytotoxicflavonoids and 120572-pyrones from Cryptocarya obovatardquo Journalof Natural Products vol 67 no 5 pp 858ndash862 2004
[23] C Y Ong S K Ling R M Ali et al ldquoSystematic analysis of invitro photo-cytotoxic activity in extracts from terrestrial plantsin Peninsula Malaysia for photodynamic therapyrdquo Journal ofPhotochemistry and Photobiology B vol 96 no 3 pp 216ndash2222009
[24] V Dumontet C Gaspard N van Hung et al ldquoNew cytotoxicflavonoids fromCryptocarya infectoriardquoTetrahedron vol 57 no29 pp 6189ndash6196 2001
[25] H Usman E H Hakim T Harlim et al ldquoCytotoxic chalconesand flavanones from the tree bark of Cryptocarya costatardquoZeitschrift fur Naturforschung C vol 61 no 3-4 pp 184ndash1882006
[26] F Kurniadewi L D Juliawaty Y M Syah et al ldquoPhenolic com-pounds from Cryptocarya konishii their cytotoxic and tyrosinekinase inhibitory propertiesrdquo Journal of Natural Medicines vol64 no 2 pp 121ndash125 2010
[27] T-H Chou J-J Chen S-J Lee M Y Chiang C-W Yang andI-S Chen ldquoCytotoxic flavonoids from the leaves ofCryptocaryachinensisrdquo Journal of Natural Products vol 73 no 9 pp 1470ndash1475 2010
[28] J C Liao ldquoLauraceae in flora of Taiwanrdquo in Editorial Committeeof the Flora of Taiwan vol 2 pp 448ndash451 Taipei Taiwan 1996
[29] B H Chen H W Chang H M Huang et al ldquo(-)-anonaineinduces DNA damage and inhibits growth and migration ofhuman lung carcinomaH1299 cellsrdquo Journal of Agricultural andFood Chemistry vol 59 no 6 pp 2284ndash2290 2011
[30] M-Y Lin Y-R Lee S-Y Chiang et al ldquoCortexMoutan inducesbladder cancer cell death via apoptosis and retards tumorgrowth inmouse bladdersrdquoEvidence-based Complementary andAlternative Medicine vol 2013 Article ID 207279 8 pages 2013
[31] C-C Chiu P-L Liu K-J Huang et al ldquoGoniothalamininhibits growth of human lung cancer cells through DNAdamage apoptosis and reduced migration abilityrdquo Journal ofAgricultural and Food Chemistry vol 59 no 8 pp 4288ndash42932011
[32] J Masters ldquoFalse cell linesrdquoCarcinogenesis vol 23 no 2 p 3712002
[33] H Ding C Han D Guo et al ldquoSelective induction of apoptosisof human oral cancer cell lines by avocado extracts via a ROS-mediated mechanismrdquo Nutrition and Cancer vol 61 no 3 pp348ndash356 2009
[34] J-C Lee M-F Hou H-W Huang et al ldquoMarine algal natu-ral products with anti-oxidative anti-inflammatory and anti-cancer propertiesrdquoCancer Cell International vol 13 no 1 article55 2013
[35] C Gorrini I S Harris and TWMak ldquoModulation of oxidativestress as an anticancer strategyrdquoNature Reviews DrugDiscoveryvol 12 no 12 pp 931ndash947 2013
[36] D Trachootham J Alexandre and P Huang ldquoTargeting can-cer cells by ROS-mediated mechanisms a radical therapeuticapproachrdquo Nature Reviews Drug Discovery vol 8 no 7 pp579ndash591 2009
[37] A K Samhan-Arias F J Martın-Romero and C Gutierrez-Merino ldquoKaempferol blocks oxidative stress in cerebellar gran-ule cells and reveals a key role for reactive oxygen speciesproduction at the plasma membrane in the commitment toapoptosisrdquo Free Radical Biology and Medicine vol 37 no 1 pp48ndash61 2004
[38] S-H Oh and S-C Lim ldquoA rapid and transient ROS generationby cadmium triggers apoptosis via caspase-dependent pathwayin HepG2 cells and this is inhibited through N-acetylcysteine-mediated catalase upregulationrdquo Toxicology and Applied Phar-macology vol 212 no 3 pp 212ndash223 2006
[39] R A Ehlers A Hernandez L S Bloemendal R T EthridgeB Farrow and B M Evers ldquoMitochondrial DNA damage andaltered membrane potential (Δ120595) in pancreatic acinar cellsinduced by reactive oxygen speciesrdquo Surgery vol 126 no 2 pp148ndash155 1999
[40] S McKenzie and N Kyprianou ldquoApoptosis evasion the role ofsurvival pathways in prostate cancer progression and therapeu-tic resistancerdquo Journal of Cellular Biochemistry vol 97 no 1 pp18ndash32 2006
10 The Scientific World Journal
[41] S Fulda ldquoEvasion of apoptosis as a cellular stress response incancerrdquo International Journal of Cell Biology vol 2010 ArticleID 370835 6 pages 2010
[42] H Lin X-B Liu J-J Yu F Hua and Z-W Hu ldquoAntioxidantN-acetylcysteine attenuates hepatocarcinogenesis by inhibitingROSER stress in TLR2 deficient mouserdquo PLoS ONE vol 8 no10 Article ID e74130 2013
[43] R Scherz-Shouval and Z Elazar ldquoROS mitochondria and theregulation of autophagyrdquoTrends in Cell Biology vol 17 no 9 pp422ndash427 2007
[44] R Scherz-Shouval and Z Elazar ldquoRegulation of autophagy byROS physiology and pathologyrdquoTrends in Biochemical Sciencesvol 36 no 1 pp 30ndash38 2011
Submit your manuscripts athttpwwwhindawicom
PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom
Volume 2014
ToxinsJournal of
VaccinesJournal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
AntibioticsInternational Journal of
ToxicologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Drug DeliveryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in Pharmacological Sciences
Tropical MedicineJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
AddictionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Autoimmune Diseases
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anesthesiology Research and Practice
ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Pharmaceutics
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
The Scientific World Journal 5
104
105
106
1027
104
105
106
1027
1031
104
105
10610
6410
3110
410
510
610
6410
3110
410
510
610
64
Ca9
-22
0 120583gmL 5120583gmL 10120583gmL
15120583gmL 20120583gmL 25120583gmLAnnexin V intensity Annexin V intensity Annexin V intensity
PI in
tens
ityCa
9-22
PI in
tens
ity
(a)
15120583gmL 20120583gmL 25120583gmLAnnexin V intensity Annexin V intensity Annexin V intensity
104
105
106
1027
104
105
106
1027
1031
104
105
10610
6410
3110
410
510
610
6410
3110
410
510
610
64
0 120583gmL 5120583gmL 10120583gmL
PI in
tens
ityPI
inte
nsity
CAL 27
CAL 27
(b)
Apop
totic
cells
()
lowastlowast
lowastlowast
lowastlowast
lowastlowast
60
50
40
30
20
10
0
0 5 10 15 20 25
MECCrt (120583gmL)
(c)
Apop
totic
cells
()
lowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowast
10
20
30
40
50
60
70
80
90
0 5 10 15 20 25
MECCrt (120583gmL)
(d)
Figure 3 Apoptosis of two oral cancer cells was induced byMECCrt Oral cancer Ca9-22 and CAL 27 cell lines were treated with 0ndash25120583gmLof MECCrt for 24 h ((a) and (b)) Representative results of annexin VPI double staining of flow cytometry for MECCrt-treated oral cancerCa9-22 and CAL 27 cell lines and vehicle controls at 24 h respectively ((c) and (d)) Quantification analysis of apoptosis for MECCrt-treatedoral cancer Ca9-22 and CAL 27 cell lines in (a) and (b) respectively Data means plusmn SDs (119899 = 3) lowastlowast119875 lt 001 against vehicle
6 The Scientific World Journal
Cou
ntCa
9-22
0 120583gmL 5120583gmL 10120583gmL
15120583gmL
400
300
200
100
0
Cou
ntCa
9-22
400
300
200
100
0
400
300
200
100
0
400
300
200
100
0
10 5000 10000 10 5000 10000 10 5000 10000 10 5000 10000 10 5000 10000 10 5000 10000
ROS intensity20120583gmL
ROS intensity ROS intensity25120583gmL 15120583gmL
ROS intensity20120583gmL
ROS intensity ROS intensity25120583gmL
6h 6h 6h0120583gmL 5120583gmL 10120583gmL12h 12h 12h
V2-L500
V2-R V2-L V2-R V2-L V2-R
V2-L V2-R V2-L V2-R V2-L V2-R
V3-L V3-R V3-L V3-R
V3-L V3-R
V3-L V3-R
V3-L V3-R V3-L V3-R
500 500 500
916 962 996
172 828618382471 529 347 653
298 702 255 745 119 881 84 38 04
(a)
Cou
nt
0120583gmL 5120583gmL 10120583gmL
15120583gmL
400
300
200
100
0
Cou
nt
400
300
200
100
0
400
300
200
100
0
400
300
200
100
0
10 5000 10000 10 5000 10000 10 5000 10000 10 5000 10000 10 5000 10000 10 5000 10000
ROS intensity20120583gmL
ROS intensity ROS intensity25120583gmL 15120583gmL
ROS intensity20120583gmL
ROS intensity ROS intensity25120583gmL
6h 6h 6h0120583gmL 5120583gmL 10120583gmL12h 12h 12h
V2-L500
V2-R V2-L V2-R V2-L V2-R
V2-L V2-R V2-L V2-R V2-L V2-R
V2-L V2-R V2-L V2-R
V2-L V2-R
V2-L V2-R
V2-L V2-R V2-L V2-R
500 500 500
970 993 998
112 888700300497 503 386 614
298 702 173 827 921 3079 07 02
CAL 27
CAL 27
(b)
lowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowast
Relat
ive R
OS-
posit
ive (
)
220
200
180
160
140
120
100
80
0 5 10 15 20 25
6h12h
MECCrt (120583gmL)
(c)
lowastlowastlowastlowastlowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowastlowastlowast
lowastlowastlowastlowast
Relat
ive R
OS-
posit
ive (
)
220
200
180
160
140
120
100
80
0 5 10 15 20 25 25
6h12h
MECCrt (120583gmL)
(d)
Figure 4 Reactive oxygen species (ROS) generation of two oral cancer cell lines was induced by MECCrt Oral cancer Ca9-22 and CAL 27cell lines were treated with different concentrations (0ndash25120583gmL) of MECCrt for 6 and 12 h ((a) (b)) Representative ROS profiles of flowcytometry for MECCrt-treated oral cancer Ca9-22 and CAL 27 cell lines ((c) and (d)) Statistics analysis of relative ROS intensity in (a) and(b) respectively Data means plusmn SDs (119899 = 3) lowastlowast119875 lt 001 against vehicle
The Scientific World Journal 7
500V1-L V1-R
500 517V1-L V1-R
483 539V1-L V1-R
461
620V1-L V1-R
380 770V1-L V1-R
230 872V1-L V1-R
128
Cou
ntCa
9-22
Cou
ntCa
9-22
0 120583gmL 5120583gmL 10120583gmL
15120583gmL 20120583gmL 25120583gmLMMP intensity MMP intensity MMP intensity
1200
1000
500
0
1200
1000
500
0
10110
210
310
410
510
610
7210
110
210
310
410
510
610
7210
110
210
310
410
510
610
72
(a)
500V2-L V2-R
500 440V2-L V2-R
560 455V2-L V2-R
545
744V2-L V2-R
256 868V2-L V2-R
132 964V2-L V2-R
Cou
ntC
ount
0120583gmL 5120583gmL 10120583gmL
15120583gmL 20120583gmL 25120583gmLMMP intensity MMP intensity MMP intensity
1200
1000
500
0
1200
1000
500
0
10110
210
310
410
510
610
7210
110
210
310
410
510
610
7210
110
210
310
410
510
610
72
36
CAL 27
CAL 27
(b)
lowast
lowastlowast
lowastlowast
lowastlowast
120
100
80
60
40
20
0
Relat
ive M
MP-
posit
ive (
)
0 5 10 15 20 25
MECCrt (120583gmL)
(c)
lowastlowastlowastlowast
lowastlowast
lowastlowast
lowastlowast
120
140
100
80
60
40
20
0
Relat
ive M
MP-
posit
ive (
)
0 5 10 15 20 25
MECCrt (120583gmL)
(d)
Figure 5 Depolarization of mitochondrial membrane potential (MMP) of Ca9-22 and CAL 27 oral cancer cell lines was induced byMECCrt Oral cancer Ca9-22 and CAL 27 cell lines were treated with different concentrations (0ndash25120583gmL) of MECCrt for 24 h ((a) (b))RepresentativeMMPprofiles of flow cytometry forMECCrt-treated oral cancer Ca9-22 andCAL 27 cells ((c) and (d))Quantification analysisof relative MMP intensity in (a) and (b) respectively Data means plusmn SDs (119899 = 3) lowast119875 lt 005 and lowastlowast119875 lt 001 against vehicle
8 The Scientific World Journal
The anticancer effects for other Cryptocarya-derivedcompounds from methanolic extracts of nonroot parts havebeen reported earlier For example for murine leukemiaP-388 cells the IC
50values of 2101584041015840-dihydroxy-5101584061015840-
dimethoxychalcone and isodidymocarpin isolated fromtree bark of C costata were 57 and 111 120583M [25] and IC
50
values of the chalcone derivative (desmethylinfectocaryone)and phenolic compound (infectocaryone) isolated fromwood of C konishii were 217 and 08 120583M [26] at 48 hrespectively For compounds from leaves of C chinensis theIC50
values of infectocaryone and cryptocaryanone A wereat the 120583M level for human lung cancer NCI-H460 cells andglioblastoma SF-268 cells [27] These Cryptocarya-derivedcompounds from methanolic extracts of nonroot partsshowed the IC
50values ranging from 08 to 11 120583M This is
close to our preliminary result that the IC50
of the clinicalanticancer drug cisplatin at 24 h treatment in oral cancerCa9-22 cells is 306 120583gmL (102 120583M) (data not shown) Inthe present study the IC
50values of the MECCrt in oral
cancer Ca9-22 and CAL 27 cell lines at 24 h were 1867 and1322 120583gmL respectively Although the IC
50values of the
MECCrt were about 3-4 folds of cisplatin for oral cancercells its crude extract nature has to be concerned Thereforeit is warranted to further investigate the particular bioactivecomponents that are included in the methanolic extracts ofCryptocarya concinnaHance roots
Moreover the anticancer effect for trunk bark ofC infectoria-derived methanol extracts was reported tobe cytotoxic to KB cells [24] KB cells were regardedas oral epidermal carcinoma however it was recentlyvalidated to have marker chromosomes and DNAfinger printings of human cervical cancer HeLacells (httpwwwncbinlmnihgovmeshDb=meshampterm=KB+Cells) [32] Accordingly the anticancer effect of oralcancer by the bioactive compounds from Cryptocarya plantremains unclear Conversely we here demonstrate theantioral cancer effect of methanolic extracts of a Cryptocaryaspecies for the first time using two OSCC cell lines Ca9-22and CAL 27
In several anticancer drugs [6 7 10 11 33ndash36] ROSgeneration is one of the common strategies to inhibit cancercell proliferation ROS plays a vital role in early stagesof apoptosis [37] and leads to MMP depolarization [3839] Escaping apoptosis is demonstrated to be involved inthe drug resistance of cancer cells [40 41] To enhanceapoptotic induction of anticancer drugs may interfere thedrug resistance if there In the present study we observed thatapoptosis was inducible by MECCrt in two OSCC cell linesas it was demonstrated by sub-G1 monitoring and annexinVPI assayWe also found that MECCrt significantly inducedthe ROS level and reduced the MMP level in two oral cancercell lines in dose-responsive ways These findings suggestthat oxidative stress may be involved in theMECCrt-inducedantiproliferative effect in two oral cancer Ca9-22 and CAL 27cell lines However the role of oxidative stress in MECCrtneed to be further examined by the ROS scavenger such asN-acetylcysteine [42] to confirm if raised ROS has played acritical role in the process of apoptosis Furthermore the ROSmay generate nonapoptotic effect like autophagy described
in literature [43 44] Therefore it was warranted to furtherinvestigate the role of autophagy in MECCrt-treated oralcancer cell lines in future
5 Conclusions
We demonstrated the antiproliferative and apoptotic effectsof MECCrt through ROS generation and mitochondrialdepolarization in twoOSCC cell linesTherefore these resultssuggest that MECCrt has anticancer potential for oral cancertherapy
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was supported by funds of the NSC102-2314-B-384-002 MOST103-2320-B-037-008 the 103CM-KMU-09the National Sun Yat-sen University-KMU Joint ResearchProject (NSYSU-KMU 103-p014) the kmtth-102-011 andthe Health and welfare surcharge of tobacco products theMinistry of Health and Welfare Taiwan Republic of China(MOHW103-TD-B-111-05)The authors also acknowledgeDrHans-Uwe Dahms for the English editing
References
[1] C-Y Yen C-H Chen C-H Chang et al ldquoMatrix metallopro-teinases (MMP) 1 andMMP10 but notMMP12 are potential oralcancer markersrdquo Biomarkers vol 14 no 4 pp 244ndash249 2009
[2] C-Y Yen C-Y HuangM-F Hou et al ldquoEvaluating the perfor-mance of fibronectin 1 (FN1) integrin12057241205731 (ITGA4) syndecan-2 (SDC2) and glycoprotein CD44 as the potential biomarkersof oral squamous cell carcinoma (OSCC)rdquo Biomarkers vol 18no 1 pp 63ndash72 2013
[3] H Myoung S-P Hong P-Y Yun J-H Lee and M-J KimldquoAnti-cancer effect of genistein in oral squamous cell carcinomawith respect to angiogenesis and in vitro invasionrdquo CancerScience vol 94 no 2 pp 215ndash220 2003
[4] H K Kim E G Wilson Y H Choi and R VerpoorteldquoMetabolomics a tool for anticancer lead-finding from naturalproductsrdquo Planta Medica vol 76 no 11 pp 1094ndash1102 2010
[5] A D Kinghorn L Pan J N Fletcher and H Chai ldquoThe rele-vance of higher plants in lead compound discovery programsrdquoJournal of Natural Products vol 74 no 6 pp 1539ndash1555 2011
[6] C-C Yeh C-N Tseng J-I Yang et al ldquoAntiproliferation andinduction of apoptosis in Ca9-22 oral cancer cells by ethanolicextract of Gracilaria tenuistipitatardquoMolecules vol 17 no 9 pp10916ndash10927 2012
[7] C-C Yeh J-I Yang J-C Lee et al ldquoAnti-proliferative effect ofmethanolic extract of Gracilaria tenuistipitata on oral cancercells involves apoptosis DNA damage and oxidative stressrdquoBMC Complementary and Alternative Medicine vol 12 article142 2012
[8] J S Yang C W Lin C H Hsin M J Hsieh and Y C ChangldquoSelaginella tamariscina attenuates metastasis via Akt pathways
The Scientific World Journal 9
in oral cancer cellsrdquo PLoS ONE vol 8 no 6 Article ID e680352013
[9] B Narotzki A Z Reznick D Aizenbud and Y Levy ldquoGreentea a promising natural product in oral healthrdquoArchives of OralBiology vol 57 no 5 pp 429ndash435 2012
[10] C-Y Yen C-C Chiu R-W Haung et al ldquoAntiprolifera-tive effects of goniothalamin on Ca9-22 oral cancer cellsthrough apoptosis DNAdamage andROS inductionrdquoMutationResearch vol 747 no 2 pp 253ndash258 2012
[11] C-C Chiu J-W Haung F-R Chang et al ldquoGolden berry-derived 4120573-hydroxywithanolide E for selectively killing oralcancer cells by generating ROS DNA damage and apoptoticpathwaysrdquo PLoS ONE vol 8 no 5 Article ID e64739 2013
[12] M T Davies-Coleman andD E A Rivett ldquoNaturally occurring6-substituted 56-dihydro-120572-pyronesrdquo Fortschritte der ChemieOrganischer Naturstoffe vol 55 pp 1ndash35 1989
[13] A Toribio A Bonfils E Delannay et al ldquoNovel seco-dibenzopyrrocoline alkaloid from Cryptocarya oubatchensisrdquoOrganic Letters vol 8 no 17 pp 3825ndash3828 2006
[14] A J Cavalheiro and M Yoshida ldquo6-[120596-arylalkenyl]-56-dihydro-120572-pyrones from Cryptocarya moschata (Lauraceae)rdquoPhytochemistry vol 53 no 7 pp 811ndash819 2000
[15] L D Juliawaty M Kitajima H Takayama S A Achmad andN Aimi ldquoA new type of stilbene-related secondary metaboliteidenburgene from Cryptocarya idenburgensisrdquo Chemical andPharmaceutical Bulletin vol 48 no 11 pp 1726ndash1728 2000
[16] P-M Allard E T H Dau C Eydoux et al ldquoAlkylatedflavanones from the bark of Cryptocarya chartacea as denguevirus NS5 polymerase inhibitorsrdquo Journal of Natural Productsvol 74 no 11 pp 2446ndash2453 2011
[17] T-S Wu C-R Su and K-H Lee ldquoCytotoxic and anti-HIVphenanthroindolizidine alkaloids from Cryptocarya chinensisrdquoNatural Product Communications vol 7 no 6 pp 725ndash727 2012
[18] T-H Chou J-J Chen C-F Peng M-J Cheng and I-S ChenldquoNew flavanones from the leaves of Cryptocarya chinensis andtheir antituberculosis activityrdquo Chemistry and Biodiversity vol8 no 11 pp 2015ndash2024 2011
[19] A A Nasrullah A Zahari J Mohamad and K AwangldquoAntiplasmodial alkaloids from the bark of Cryptocarya nigra(Lauraceae)rdquoMolecules vol 18 no 7 pp 8009ndash8017 2013
[20] R A Davis O Demirkiran M L Sykes et al ldquo7101584081015840-Dihydroobolactone a typanocidal 120572-pyrone from the rainforesttree Cryptocarya obovatardquo Bioorganic andMedicinal ChemistryLetters vol 20 no 14 pp 4057ndash4059 2010
[21] R Feng Z K Guo C M Yan E G Li R X Tan and H MGe ldquoAnti-inflammatory flavonoids from Cryptocarya chingiirdquoPhytochemistry vol 76 pp 98ndash105 2012
[22] V Dumontet N Van Hung M-T Adeline et al ldquoCytotoxicflavonoids and 120572-pyrones from Cryptocarya obovatardquo Journalof Natural Products vol 67 no 5 pp 858ndash862 2004
[23] C Y Ong S K Ling R M Ali et al ldquoSystematic analysis of invitro photo-cytotoxic activity in extracts from terrestrial plantsin Peninsula Malaysia for photodynamic therapyrdquo Journal ofPhotochemistry and Photobiology B vol 96 no 3 pp 216ndash2222009
[24] V Dumontet C Gaspard N van Hung et al ldquoNew cytotoxicflavonoids fromCryptocarya infectoriardquoTetrahedron vol 57 no29 pp 6189ndash6196 2001
[25] H Usman E H Hakim T Harlim et al ldquoCytotoxic chalconesand flavanones from the tree bark of Cryptocarya costatardquoZeitschrift fur Naturforschung C vol 61 no 3-4 pp 184ndash1882006
[26] F Kurniadewi L D Juliawaty Y M Syah et al ldquoPhenolic com-pounds from Cryptocarya konishii their cytotoxic and tyrosinekinase inhibitory propertiesrdquo Journal of Natural Medicines vol64 no 2 pp 121ndash125 2010
[27] T-H Chou J-J Chen S-J Lee M Y Chiang C-W Yang andI-S Chen ldquoCytotoxic flavonoids from the leaves ofCryptocaryachinensisrdquo Journal of Natural Products vol 73 no 9 pp 1470ndash1475 2010
[28] J C Liao ldquoLauraceae in flora of Taiwanrdquo in Editorial Committeeof the Flora of Taiwan vol 2 pp 448ndash451 Taipei Taiwan 1996
[29] B H Chen H W Chang H M Huang et al ldquo(-)-anonaineinduces DNA damage and inhibits growth and migration ofhuman lung carcinomaH1299 cellsrdquo Journal of Agricultural andFood Chemistry vol 59 no 6 pp 2284ndash2290 2011
[30] M-Y Lin Y-R Lee S-Y Chiang et al ldquoCortexMoutan inducesbladder cancer cell death via apoptosis and retards tumorgrowth inmouse bladdersrdquoEvidence-based Complementary andAlternative Medicine vol 2013 Article ID 207279 8 pages 2013
[31] C-C Chiu P-L Liu K-J Huang et al ldquoGoniothalamininhibits growth of human lung cancer cells through DNAdamage apoptosis and reduced migration abilityrdquo Journal ofAgricultural and Food Chemistry vol 59 no 8 pp 4288ndash42932011
[32] J Masters ldquoFalse cell linesrdquoCarcinogenesis vol 23 no 2 p 3712002
[33] H Ding C Han D Guo et al ldquoSelective induction of apoptosisof human oral cancer cell lines by avocado extracts via a ROS-mediated mechanismrdquo Nutrition and Cancer vol 61 no 3 pp348ndash356 2009
[34] J-C Lee M-F Hou H-W Huang et al ldquoMarine algal natu-ral products with anti-oxidative anti-inflammatory and anti-cancer propertiesrdquoCancer Cell International vol 13 no 1 article55 2013
[35] C Gorrini I S Harris and TWMak ldquoModulation of oxidativestress as an anticancer strategyrdquoNature Reviews DrugDiscoveryvol 12 no 12 pp 931ndash947 2013
[36] D Trachootham J Alexandre and P Huang ldquoTargeting can-cer cells by ROS-mediated mechanisms a radical therapeuticapproachrdquo Nature Reviews Drug Discovery vol 8 no 7 pp579ndash591 2009
[37] A K Samhan-Arias F J Martın-Romero and C Gutierrez-Merino ldquoKaempferol blocks oxidative stress in cerebellar gran-ule cells and reveals a key role for reactive oxygen speciesproduction at the plasma membrane in the commitment toapoptosisrdquo Free Radical Biology and Medicine vol 37 no 1 pp48ndash61 2004
[38] S-H Oh and S-C Lim ldquoA rapid and transient ROS generationby cadmium triggers apoptosis via caspase-dependent pathwayin HepG2 cells and this is inhibited through N-acetylcysteine-mediated catalase upregulationrdquo Toxicology and Applied Phar-macology vol 212 no 3 pp 212ndash223 2006
[39] R A Ehlers A Hernandez L S Bloemendal R T EthridgeB Farrow and B M Evers ldquoMitochondrial DNA damage andaltered membrane potential (Δ120595) in pancreatic acinar cellsinduced by reactive oxygen speciesrdquo Surgery vol 126 no 2 pp148ndash155 1999
[40] S McKenzie and N Kyprianou ldquoApoptosis evasion the role ofsurvival pathways in prostate cancer progression and therapeu-tic resistancerdquo Journal of Cellular Biochemistry vol 97 no 1 pp18ndash32 2006
10 The Scientific World Journal
[41] S Fulda ldquoEvasion of apoptosis as a cellular stress response incancerrdquo International Journal of Cell Biology vol 2010 ArticleID 370835 6 pages 2010
[42] H Lin X-B Liu J-J Yu F Hua and Z-W Hu ldquoAntioxidantN-acetylcysteine attenuates hepatocarcinogenesis by inhibitingROSER stress in TLR2 deficient mouserdquo PLoS ONE vol 8 no10 Article ID e74130 2013
[43] R Scherz-Shouval and Z Elazar ldquoROS mitochondria and theregulation of autophagyrdquoTrends in Cell Biology vol 17 no 9 pp422ndash427 2007
[44] R Scherz-Shouval and Z Elazar ldquoRegulation of autophagy byROS physiology and pathologyrdquoTrends in Biochemical Sciencesvol 36 no 1 pp 30ndash38 2011
Submit your manuscripts athttpwwwhindawicom
PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom
Volume 2014
ToxinsJournal of
VaccinesJournal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
AntibioticsInternational Journal of
ToxicologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Drug DeliveryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in Pharmacological Sciences
Tropical MedicineJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
AddictionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Autoimmune Diseases
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anesthesiology Research and Practice
ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Pharmaceutics
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
6 The Scientific World Journal
Cou
ntCa
9-22
0 120583gmL 5120583gmL 10120583gmL
15120583gmL
400
300
200
100
0
Cou
ntCa
9-22
400
300
200
100
0
400
300
200
100
0
400
300
200
100
0
10 5000 10000 10 5000 10000 10 5000 10000 10 5000 10000 10 5000 10000 10 5000 10000
ROS intensity20120583gmL
ROS intensity ROS intensity25120583gmL 15120583gmL
ROS intensity20120583gmL
ROS intensity ROS intensity25120583gmL
6h 6h 6h0120583gmL 5120583gmL 10120583gmL12h 12h 12h
V2-L500
V2-R V2-L V2-R V2-L V2-R
V2-L V2-R V2-L V2-R V2-L V2-R
V3-L V3-R V3-L V3-R
V3-L V3-R
V3-L V3-R
V3-L V3-R V3-L V3-R
500 500 500
916 962 996
172 828618382471 529 347 653
298 702 255 745 119 881 84 38 04
(a)
Cou
nt
0120583gmL 5120583gmL 10120583gmL
15120583gmL
400
300
200
100
0
Cou
nt
400
300
200
100
0
400
300
200
100
0
400
300
200
100
0
10 5000 10000 10 5000 10000 10 5000 10000 10 5000 10000 10 5000 10000 10 5000 10000
ROS intensity20120583gmL
ROS intensity ROS intensity25120583gmL 15120583gmL
ROS intensity20120583gmL
ROS intensity ROS intensity25120583gmL
6h 6h 6h0120583gmL 5120583gmL 10120583gmL12h 12h 12h
V2-L500
V2-R V2-L V2-R V2-L V2-R
V2-L V2-R V2-L V2-R V2-L V2-R
V2-L V2-R V2-L V2-R
V2-L V2-R
V2-L V2-R
V2-L V2-R V2-L V2-R
500 500 500
970 993 998
112 888700300497 503 386 614
298 702 173 827 921 3079 07 02
CAL 27
CAL 27
(b)
lowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowast
Relat
ive R
OS-
posit
ive (
)
220
200
180
160
140
120
100
80
0 5 10 15 20 25
6h12h
MECCrt (120583gmL)
(c)
lowastlowastlowastlowastlowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowastlowastlowast
lowastlowastlowastlowast
Relat
ive R
OS-
posit
ive (
)
220
200
180
160
140
120
100
80
0 5 10 15 20 25 25
6h12h
MECCrt (120583gmL)
(d)
Figure 4 Reactive oxygen species (ROS) generation of two oral cancer cell lines was induced by MECCrt Oral cancer Ca9-22 and CAL 27cell lines were treated with different concentrations (0ndash25120583gmL) of MECCrt for 6 and 12 h ((a) (b)) Representative ROS profiles of flowcytometry for MECCrt-treated oral cancer Ca9-22 and CAL 27 cell lines ((c) and (d)) Statistics analysis of relative ROS intensity in (a) and(b) respectively Data means plusmn SDs (119899 = 3) lowastlowast119875 lt 001 against vehicle
The Scientific World Journal 7
500V1-L V1-R
500 517V1-L V1-R
483 539V1-L V1-R
461
620V1-L V1-R
380 770V1-L V1-R
230 872V1-L V1-R
128
Cou
ntCa
9-22
Cou
ntCa
9-22
0 120583gmL 5120583gmL 10120583gmL
15120583gmL 20120583gmL 25120583gmLMMP intensity MMP intensity MMP intensity
1200
1000
500
0
1200
1000
500
0
10110
210
310
410
510
610
7210
110
210
310
410
510
610
7210
110
210
310
410
510
610
72
(a)
500V2-L V2-R
500 440V2-L V2-R
560 455V2-L V2-R
545
744V2-L V2-R
256 868V2-L V2-R
132 964V2-L V2-R
Cou
ntC
ount
0120583gmL 5120583gmL 10120583gmL
15120583gmL 20120583gmL 25120583gmLMMP intensity MMP intensity MMP intensity
1200
1000
500
0
1200
1000
500
0
10110
210
310
410
510
610
7210
110
210
310
410
510
610
7210
110
210
310
410
510
610
72
36
CAL 27
CAL 27
(b)
lowast
lowastlowast
lowastlowast
lowastlowast
120
100
80
60
40
20
0
Relat
ive M
MP-
posit
ive (
)
0 5 10 15 20 25
MECCrt (120583gmL)
(c)
lowastlowastlowastlowast
lowastlowast
lowastlowast
lowastlowast
120
140
100
80
60
40
20
0
Relat
ive M
MP-
posit
ive (
)
0 5 10 15 20 25
MECCrt (120583gmL)
(d)
Figure 5 Depolarization of mitochondrial membrane potential (MMP) of Ca9-22 and CAL 27 oral cancer cell lines was induced byMECCrt Oral cancer Ca9-22 and CAL 27 cell lines were treated with different concentrations (0ndash25120583gmL) of MECCrt for 24 h ((a) (b))RepresentativeMMPprofiles of flow cytometry forMECCrt-treated oral cancer Ca9-22 andCAL 27 cells ((c) and (d))Quantification analysisof relative MMP intensity in (a) and (b) respectively Data means plusmn SDs (119899 = 3) lowast119875 lt 005 and lowastlowast119875 lt 001 against vehicle
8 The Scientific World Journal
The anticancer effects for other Cryptocarya-derivedcompounds from methanolic extracts of nonroot parts havebeen reported earlier For example for murine leukemiaP-388 cells the IC
50values of 2101584041015840-dihydroxy-5101584061015840-
dimethoxychalcone and isodidymocarpin isolated fromtree bark of C costata were 57 and 111 120583M [25] and IC
50
values of the chalcone derivative (desmethylinfectocaryone)and phenolic compound (infectocaryone) isolated fromwood of C konishii were 217 and 08 120583M [26] at 48 hrespectively For compounds from leaves of C chinensis theIC50
values of infectocaryone and cryptocaryanone A wereat the 120583M level for human lung cancer NCI-H460 cells andglioblastoma SF-268 cells [27] These Cryptocarya-derivedcompounds from methanolic extracts of nonroot partsshowed the IC
50values ranging from 08 to 11 120583M This is
close to our preliminary result that the IC50
of the clinicalanticancer drug cisplatin at 24 h treatment in oral cancerCa9-22 cells is 306 120583gmL (102 120583M) (data not shown) Inthe present study the IC
50values of the MECCrt in oral
cancer Ca9-22 and CAL 27 cell lines at 24 h were 1867 and1322 120583gmL respectively Although the IC
50values of the
MECCrt were about 3-4 folds of cisplatin for oral cancercells its crude extract nature has to be concerned Thereforeit is warranted to further investigate the particular bioactivecomponents that are included in the methanolic extracts ofCryptocarya concinnaHance roots
Moreover the anticancer effect for trunk bark ofC infectoria-derived methanol extracts was reported tobe cytotoxic to KB cells [24] KB cells were regardedas oral epidermal carcinoma however it was recentlyvalidated to have marker chromosomes and DNAfinger printings of human cervical cancer HeLacells (httpwwwncbinlmnihgovmeshDb=meshampterm=KB+Cells) [32] Accordingly the anticancer effect of oralcancer by the bioactive compounds from Cryptocarya plantremains unclear Conversely we here demonstrate theantioral cancer effect of methanolic extracts of a Cryptocaryaspecies for the first time using two OSCC cell lines Ca9-22and CAL 27
In several anticancer drugs [6 7 10 11 33ndash36] ROSgeneration is one of the common strategies to inhibit cancercell proliferation ROS plays a vital role in early stagesof apoptosis [37] and leads to MMP depolarization [3839] Escaping apoptosis is demonstrated to be involved inthe drug resistance of cancer cells [40 41] To enhanceapoptotic induction of anticancer drugs may interfere thedrug resistance if there In the present study we observed thatapoptosis was inducible by MECCrt in two OSCC cell linesas it was demonstrated by sub-G1 monitoring and annexinVPI assayWe also found that MECCrt significantly inducedthe ROS level and reduced the MMP level in two oral cancercell lines in dose-responsive ways These findings suggestthat oxidative stress may be involved in theMECCrt-inducedantiproliferative effect in two oral cancer Ca9-22 and CAL 27cell lines However the role of oxidative stress in MECCrtneed to be further examined by the ROS scavenger such asN-acetylcysteine [42] to confirm if raised ROS has played acritical role in the process of apoptosis Furthermore the ROSmay generate nonapoptotic effect like autophagy described
in literature [43 44] Therefore it was warranted to furtherinvestigate the role of autophagy in MECCrt-treated oralcancer cell lines in future
5 Conclusions
We demonstrated the antiproliferative and apoptotic effectsof MECCrt through ROS generation and mitochondrialdepolarization in twoOSCC cell linesTherefore these resultssuggest that MECCrt has anticancer potential for oral cancertherapy
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was supported by funds of the NSC102-2314-B-384-002 MOST103-2320-B-037-008 the 103CM-KMU-09the National Sun Yat-sen University-KMU Joint ResearchProject (NSYSU-KMU 103-p014) the kmtth-102-011 andthe Health and welfare surcharge of tobacco products theMinistry of Health and Welfare Taiwan Republic of China(MOHW103-TD-B-111-05)The authors also acknowledgeDrHans-Uwe Dahms for the English editing
References
[1] C-Y Yen C-H Chen C-H Chang et al ldquoMatrix metallopro-teinases (MMP) 1 andMMP10 but notMMP12 are potential oralcancer markersrdquo Biomarkers vol 14 no 4 pp 244ndash249 2009
[2] C-Y Yen C-Y HuangM-F Hou et al ldquoEvaluating the perfor-mance of fibronectin 1 (FN1) integrin12057241205731 (ITGA4) syndecan-2 (SDC2) and glycoprotein CD44 as the potential biomarkersof oral squamous cell carcinoma (OSCC)rdquo Biomarkers vol 18no 1 pp 63ndash72 2013
[3] H Myoung S-P Hong P-Y Yun J-H Lee and M-J KimldquoAnti-cancer effect of genistein in oral squamous cell carcinomawith respect to angiogenesis and in vitro invasionrdquo CancerScience vol 94 no 2 pp 215ndash220 2003
[4] H K Kim E G Wilson Y H Choi and R VerpoorteldquoMetabolomics a tool for anticancer lead-finding from naturalproductsrdquo Planta Medica vol 76 no 11 pp 1094ndash1102 2010
[5] A D Kinghorn L Pan J N Fletcher and H Chai ldquoThe rele-vance of higher plants in lead compound discovery programsrdquoJournal of Natural Products vol 74 no 6 pp 1539ndash1555 2011
[6] C-C Yeh C-N Tseng J-I Yang et al ldquoAntiproliferation andinduction of apoptosis in Ca9-22 oral cancer cells by ethanolicextract of Gracilaria tenuistipitatardquoMolecules vol 17 no 9 pp10916ndash10927 2012
[7] C-C Yeh J-I Yang J-C Lee et al ldquoAnti-proliferative effect ofmethanolic extract of Gracilaria tenuistipitata on oral cancercells involves apoptosis DNA damage and oxidative stressrdquoBMC Complementary and Alternative Medicine vol 12 article142 2012
[8] J S Yang C W Lin C H Hsin M J Hsieh and Y C ChangldquoSelaginella tamariscina attenuates metastasis via Akt pathways
The Scientific World Journal 9
in oral cancer cellsrdquo PLoS ONE vol 8 no 6 Article ID e680352013
[9] B Narotzki A Z Reznick D Aizenbud and Y Levy ldquoGreentea a promising natural product in oral healthrdquoArchives of OralBiology vol 57 no 5 pp 429ndash435 2012
[10] C-Y Yen C-C Chiu R-W Haung et al ldquoAntiprolifera-tive effects of goniothalamin on Ca9-22 oral cancer cellsthrough apoptosis DNAdamage andROS inductionrdquoMutationResearch vol 747 no 2 pp 253ndash258 2012
[11] C-C Chiu J-W Haung F-R Chang et al ldquoGolden berry-derived 4120573-hydroxywithanolide E for selectively killing oralcancer cells by generating ROS DNA damage and apoptoticpathwaysrdquo PLoS ONE vol 8 no 5 Article ID e64739 2013
[12] M T Davies-Coleman andD E A Rivett ldquoNaturally occurring6-substituted 56-dihydro-120572-pyronesrdquo Fortschritte der ChemieOrganischer Naturstoffe vol 55 pp 1ndash35 1989
[13] A Toribio A Bonfils E Delannay et al ldquoNovel seco-dibenzopyrrocoline alkaloid from Cryptocarya oubatchensisrdquoOrganic Letters vol 8 no 17 pp 3825ndash3828 2006
[14] A J Cavalheiro and M Yoshida ldquo6-[120596-arylalkenyl]-56-dihydro-120572-pyrones from Cryptocarya moschata (Lauraceae)rdquoPhytochemistry vol 53 no 7 pp 811ndash819 2000
[15] L D Juliawaty M Kitajima H Takayama S A Achmad andN Aimi ldquoA new type of stilbene-related secondary metaboliteidenburgene from Cryptocarya idenburgensisrdquo Chemical andPharmaceutical Bulletin vol 48 no 11 pp 1726ndash1728 2000
[16] P-M Allard E T H Dau C Eydoux et al ldquoAlkylatedflavanones from the bark of Cryptocarya chartacea as denguevirus NS5 polymerase inhibitorsrdquo Journal of Natural Productsvol 74 no 11 pp 2446ndash2453 2011
[17] T-S Wu C-R Su and K-H Lee ldquoCytotoxic and anti-HIVphenanthroindolizidine alkaloids from Cryptocarya chinensisrdquoNatural Product Communications vol 7 no 6 pp 725ndash727 2012
[18] T-H Chou J-J Chen C-F Peng M-J Cheng and I-S ChenldquoNew flavanones from the leaves of Cryptocarya chinensis andtheir antituberculosis activityrdquo Chemistry and Biodiversity vol8 no 11 pp 2015ndash2024 2011
[19] A A Nasrullah A Zahari J Mohamad and K AwangldquoAntiplasmodial alkaloids from the bark of Cryptocarya nigra(Lauraceae)rdquoMolecules vol 18 no 7 pp 8009ndash8017 2013
[20] R A Davis O Demirkiran M L Sykes et al ldquo7101584081015840-Dihydroobolactone a typanocidal 120572-pyrone from the rainforesttree Cryptocarya obovatardquo Bioorganic andMedicinal ChemistryLetters vol 20 no 14 pp 4057ndash4059 2010
[21] R Feng Z K Guo C M Yan E G Li R X Tan and H MGe ldquoAnti-inflammatory flavonoids from Cryptocarya chingiirdquoPhytochemistry vol 76 pp 98ndash105 2012
[22] V Dumontet N Van Hung M-T Adeline et al ldquoCytotoxicflavonoids and 120572-pyrones from Cryptocarya obovatardquo Journalof Natural Products vol 67 no 5 pp 858ndash862 2004
[23] C Y Ong S K Ling R M Ali et al ldquoSystematic analysis of invitro photo-cytotoxic activity in extracts from terrestrial plantsin Peninsula Malaysia for photodynamic therapyrdquo Journal ofPhotochemistry and Photobiology B vol 96 no 3 pp 216ndash2222009
[24] V Dumontet C Gaspard N van Hung et al ldquoNew cytotoxicflavonoids fromCryptocarya infectoriardquoTetrahedron vol 57 no29 pp 6189ndash6196 2001
[25] H Usman E H Hakim T Harlim et al ldquoCytotoxic chalconesand flavanones from the tree bark of Cryptocarya costatardquoZeitschrift fur Naturforschung C vol 61 no 3-4 pp 184ndash1882006
[26] F Kurniadewi L D Juliawaty Y M Syah et al ldquoPhenolic com-pounds from Cryptocarya konishii their cytotoxic and tyrosinekinase inhibitory propertiesrdquo Journal of Natural Medicines vol64 no 2 pp 121ndash125 2010
[27] T-H Chou J-J Chen S-J Lee M Y Chiang C-W Yang andI-S Chen ldquoCytotoxic flavonoids from the leaves ofCryptocaryachinensisrdquo Journal of Natural Products vol 73 no 9 pp 1470ndash1475 2010
[28] J C Liao ldquoLauraceae in flora of Taiwanrdquo in Editorial Committeeof the Flora of Taiwan vol 2 pp 448ndash451 Taipei Taiwan 1996
[29] B H Chen H W Chang H M Huang et al ldquo(-)-anonaineinduces DNA damage and inhibits growth and migration ofhuman lung carcinomaH1299 cellsrdquo Journal of Agricultural andFood Chemistry vol 59 no 6 pp 2284ndash2290 2011
[30] M-Y Lin Y-R Lee S-Y Chiang et al ldquoCortexMoutan inducesbladder cancer cell death via apoptosis and retards tumorgrowth inmouse bladdersrdquoEvidence-based Complementary andAlternative Medicine vol 2013 Article ID 207279 8 pages 2013
[31] C-C Chiu P-L Liu K-J Huang et al ldquoGoniothalamininhibits growth of human lung cancer cells through DNAdamage apoptosis and reduced migration abilityrdquo Journal ofAgricultural and Food Chemistry vol 59 no 8 pp 4288ndash42932011
[32] J Masters ldquoFalse cell linesrdquoCarcinogenesis vol 23 no 2 p 3712002
[33] H Ding C Han D Guo et al ldquoSelective induction of apoptosisof human oral cancer cell lines by avocado extracts via a ROS-mediated mechanismrdquo Nutrition and Cancer vol 61 no 3 pp348ndash356 2009
[34] J-C Lee M-F Hou H-W Huang et al ldquoMarine algal natu-ral products with anti-oxidative anti-inflammatory and anti-cancer propertiesrdquoCancer Cell International vol 13 no 1 article55 2013
[35] C Gorrini I S Harris and TWMak ldquoModulation of oxidativestress as an anticancer strategyrdquoNature Reviews DrugDiscoveryvol 12 no 12 pp 931ndash947 2013
[36] D Trachootham J Alexandre and P Huang ldquoTargeting can-cer cells by ROS-mediated mechanisms a radical therapeuticapproachrdquo Nature Reviews Drug Discovery vol 8 no 7 pp579ndash591 2009
[37] A K Samhan-Arias F J Martın-Romero and C Gutierrez-Merino ldquoKaempferol blocks oxidative stress in cerebellar gran-ule cells and reveals a key role for reactive oxygen speciesproduction at the plasma membrane in the commitment toapoptosisrdquo Free Radical Biology and Medicine vol 37 no 1 pp48ndash61 2004
[38] S-H Oh and S-C Lim ldquoA rapid and transient ROS generationby cadmium triggers apoptosis via caspase-dependent pathwayin HepG2 cells and this is inhibited through N-acetylcysteine-mediated catalase upregulationrdquo Toxicology and Applied Phar-macology vol 212 no 3 pp 212ndash223 2006
[39] R A Ehlers A Hernandez L S Bloemendal R T EthridgeB Farrow and B M Evers ldquoMitochondrial DNA damage andaltered membrane potential (Δ120595) in pancreatic acinar cellsinduced by reactive oxygen speciesrdquo Surgery vol 126 no 2 pp148ndash155 1999
[40] S McKenzie and N Kyprianou ldquoApoptosis evasion the role ofsurvival pathways in prostate cancer progression and therapeu-tic resistancerdquo Journal of Cellular Biochemistry vol 97 no 1 pp18ndash32 2006
10 The Scientific World Journal
[41] S Fulda ldquoEvasion of apoptosis as a cellular stress response incancerrdquo International Journal of Cell Biology vol 2010 ArticleID 370835 6 pages 2010
[42] H Lin X-B Liu J-J Yu F Hua and Z-W Hu ldquoAntioxidantN-acetylcysteine attenuates hepatocarcinogenesis by inhibitingROSER stress in TLR2 deficient mouserdquo PLoS ONE vol 8 no10 Article ID e74130 2013
[43] R Scherz-Shouval and Z Elazar ldquoROS mitochondria and theregulation of autophagyrdquoTrends in Cell Biology vol 17 no 9 pp422ndash427 2007
[44] R Scherz-Shouval and Z Elazar ldquoRegulation of autophagy byROS physiology and pathologyrdquoTrends in Biochemical Sciencesvol 36 no 1 pp 30ndash38 2011
Submit your manuscripts athttpwwwhindawicom
PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom
Volume 2014
ToxinsJournal of
VaccinesJournal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
AntibioticsInternational Journal of
ToxicologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Drug DeliveryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in Pharmacological Sciences
Tropical MedicineJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
AddictionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Autoimmune Diseases
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anesthesiology Research and Practice
ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Pharmaceutics
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
The Scientific World Journal 7
500V1-L V1-R
500 517V1-L V1-R
483 539V1-L V1-R
461
620V1-L V1-R
380 770V1-L V1-R
230 872V1-L V1-R
128
Cou
ntCa
9-22
Cou
ntCa
9-22
0 120583gmL 5120583gmL 10120583gmL
15120583gmL 20120583gmL 25120583gmLMMP intensity MMP intensity MMP intensity
1200
1000
500
0
1200
1000
500
0
10110
210
310
410
510
610
7210
110
210
310
410
510
610
7210
110
210
310
410
510
610
72
(a)
500V2-L V2-R
500 440V2-L V2-R
560 455V2-L V2-R
545
744V2-L V2-R
256 868V2-L V2-R
132 964V2-L V2-R
Cou
ntC
ount
0120583gmL 5120583gmL 10120583gmL
15120583gmL 20120583gmL 25120583gmLMMP intensity MMP intensity MMP intensity
1200
1000
500
0
1200
1000
500
0
10110
210
310
410
510
610
7210
110
210
310
410
510
610
7210
110
210
310
410
510
610
72
36
CAL 27
CAL 27
(b)
lowast
lowastlowast
lowastlowast
lowastlowast
120
100
80
60
40
20
0
Relat
ive M
MP-
posit
ive (
)
0 5 10 15 20 25
MECCrt (120583gmL)
(c)
lowastlowastlowastlowast
lowastlowast
lowastlowast
lowastlowast
120
140
100
80
60
40
20
0
Relat
ive M
MP-
posit
ive (
)
0 5 10 15 20 25
MECCrt (120583gmL)
(d)
Figure 5 Depolarization of mitochondrial membrane potential (MMP) of Ca9-22 and CAL 27 oral cancer cell lines was induced byMECCrt Oral cancer Ca9-22 and CAL 27 cell lines were treated with different concentrations (0ndash25120583gmL) of MECCrt for 24 h ((a) (b))RepresentativeMMPprofiles of flow cytometry forMECCrt-treated oral cancer Ca9-22 andCAL 27 cells ((c) and (d))Quantification analysisof relative MMP intensity in (a) and (b) respectively Data means plusmn SDs (119899 = 3) lowast119875 lt 005 and lowastlowast119875 lt 001 against vehicle
8 The Scientific World Journal
The anticancer effects for other Cryptocarya-derivedcompounds from methanolic extracts of nonroot parts havebeen reported earlier For example for murine leukemiaP-388 cells the IC
50values of 2101584041015840-dihydroxy-5101584061015840-
dimethoxychalcone and isodidymocarpin isolated fromtree bark of C costata were 57 and 111 120583M [25] and IC
50
values of the chalcone derivative (desmethylinfectocaryone)and phenolic compound (infectocaryone) isolated fromwood of C konishii were 217 and 08 120583M [26] at 48 hrespectively For compounds from leaves of C chinensis theIC50
values of infectocaryone and cryptocaryanone A wereat the 120583M level for human lung cancer NCI-H460 cells andglioblastoma SF-268 cells [27] These Cryptocarya-derivedcompounds from methanolic extracts of nonroot partsshowed the IC
50values ranging from 08 to 11 120583M This is
close to our preliminary result that the IC50
of the clinicalanticancer drug cisplatin at 24 h treatment in oral cancerCa9-22 cells is 306 120583gmL (102 120583M) (data not shown) Inthe present study the IC
50values of the MECCrt in oral
cancer Ca9-22 and CAL 27 cell lines at 24 h were 1867 and1322 120583gmL respectively Although the IC
50values of the
MECCrt were about 3-4 folds of cisplatin for oral cancercells its crude extract nature has to be concerned Thereforeit is warranted to further investigate the particular bioactivecomponents that are included in the methanolic extracts ofCryptocarya concinnaHance roots
Moreover the anticancer effect for trunk bark ofC infectoria-derived methanol extracts was reported tobe cytotoxic to KB cells [24] KB cells were regardedas oral epidermal carcinoma however it was recentlyvalidated to have marker chromosomes and DNAfinger printings of human cervical cancer HeLacells (httpwwwncbinlmnihgovmeshDb=meshampterm=KB+Cells) [32] Accordingly the anticancer effect of oralcancer by the bioactive compounds from Cryptocarya plantremains unclear Conversely we here demonstrate theantioral cancer effect of methanolic extracts of a Cryptocaryaspecies for the first time using two OSCC cell lines Ca9-22and CAL 27
In several anticancer drugs [6 7 10 11 33ndash36] ROSgeneration is one of the common strategies to inhibit cancercell proliferation ROS plays a vital role in early stagesof apoptosis [37] and leads to MMP depolarization [3839] Escaping apoptosis is demonstrated to be involved inthe drug resistance of cancer cells [40 41] To enhanceapoptotic induction of anticancer drugs may interfere thedrug resistance if there In the present study we observed thatapoptosis was inducible by MECCrt in two OSCC cell linesas it was demonstrated by sub-G1 monitoring and annexinVPI assayWe also found that MECCrt significantly inducedthe ROS level and reduced the MMP level in two oral cancercell lines in dose-responsive ways These findings suggestthat oxidative stress may be involved in theMECCrt-inducedantiproliferative effect in two oral cancer Ca9-22 and CAL 27cell lines However the role of oxidative stress in MECCrtneed to be further examined by the ROS scavenger such asN-acetylcysteine [42] to confirm if raised ROS has played acritical role in the process of apoptosis Furthermore the ROSmay generate nonapoptotic effect like autophagy described
in literature [43 44] Therefore it was warranted to furtherinvestigate the role of autophagy in MECCrt-treated oralcancer cell lines in future
5 Conclusions
We demonstrated the antiproliferative and apoptotic effectsof MECCrt through ROS generation and mitochondrialdepolarization in twoOSCC cell linesTherefore these resultssuggest that MECCrt has anticancer potential for oral cancertherapy
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was supported by funds of the NSC102-2314-B-384-002 MOST103-2320-B-037-008 the 103CM-KMU-09the National Sun Yat-sen University-KMU Joint ResearchProject (NSYSU-KMU 103-p014) the kmtth-102-011 andthe Health and welfare surcharge of tobacco products theMinistry of Health and Welfare Taiwan Republic of China(MOHW103-TD-B-111-05)The authors also acknowledgeDrHans-Uwe Dahms for the English editing
References
[1] C-Y Yen C-H Chen C-H Chang et al ldquoMatrix metallopro-teinases (MMP) 1 andMMP10 but notMMP12 are potential oralcancer markersrdquo Biomarkers vol 14 no 4 pp 244ndash249 2009
[2] C-Y Yen C-Y HuangM-F Hou et al ldquoEvaluating the perfor-mance of fibronectin 1 (FN1) integrin12057241205731 (ITGA4) syndecan-2 (SDC2) and glycoprotein CD44 as the potential biomarkersof oral squamous cell carcinoma (OSCC)rdquo Biomarkers vol 18no 1 pp 63ndash72 2013
[3] H Myoung S-P Hong P-Y Yun J-H Lee and M-J KimldquoAnti-cancer effect of genistein in oral squamous cell carcinomawith respect to angiogenesis and in vitro invasionrdquo CancerScience vol 94 no 2 pp 215ndash220 2003
[4] H K Kim E G Wilson Y H Choi and R VerpoorteldquoMetabolomics a tool for anticancer lead-finding from naturalproductsrdquo Planta Medica vol 76 no 11 pp 1094ndash1102 2010
[5] A D Kinghorn L Pan J N Fletcher and H Chai ldquoThe rele-vance of higher plants in lead compound discovery programsrdquoJournal of Natural Products vol 74 no 6 pp 1539ndash1555 2011
[6] C-C Yeh C-N Tseng J-I Yang et al ldquoAntiproliferation andinduction of apoptosis in Ca9-22 oral cancer cells by ethanolicextract of Gracilaria tenuistipitatardquoMolecules vol 17 no 9 pp10916ndash10927 2012
[7] C-C Yeh J-I Yang J-C Lee et al ldquoAnti-proliferative effect ofmethanolic extract of Gracilaria tenuistipitata on oral cancercells involves apoptosis DNA damage and oxidative stressrdquoBMC Complementary and Alternative Medicine vol 12 article142 2012
[8] J S Yang C W Lin C H Hsin M J Hsieh and Y C ChangldquoSelaginella tamariscina attenuates metastasis via Akt pathways
The Scientific World Journal 9
in oral cancer cellsrdquo PLoS ONE vol 8 no 6 Article ID e680352013
[9] B Narotzki A Z Reznick D Aizenbud and Y Levy ldquoGreentea a promising natural product in oral healthrdquoArchives of OralBiology vol 57 no 5 pp 429ndash435 2012
[10] C-Y Yen C-C Chiu R-W Haung et al ldquoAntiprolifera-tive effects of goniothalamin on Ca9-22 oral cancer cellsthrough apoptosis DNAdamage andROS inductionrdquoMutationResearch vol 747 no 2 pp 253ndash258 2012
[11] C-C Chiu J-W Haung F-R Chang et al ldquoGolden berry-derived 4120573-hydroxywithanolide E for selectively killing oralcancer cells by generating ROS DNA damage and apoptoticpathwaysrdquo PLoS ONE vol 8 no 5 Article ID e64739 2013
[12] M T Davies-Coleman andD E A Rivett ldquoNaturally occurring6-substituted 56-dihydro-120572-pyronesrdquo Fortschritte der ChemieOrganischer Naturstoffe vol 55 pp 1ndash35 1989
[13] A Toribio A Bonfils E Delannay et al ldquoNovel seco-dibenzopyrrocoline alkaloid from Cryptocarya oubatchensisrdquoOrganic Letters vol 8 no 17 pp 3825ndash3828 2006
[14] A J Cavalheiro and M Yoshida ldquo6-[120596-arylalkenyl]-56-dihydro-120572-pyrones from Cryptocarya moschata (Lauraceae)rdquoPhytochemistry vol 53 no 7 pp 811ndash819 2000
[15] L D Juliawaty M Kitajima H Takayama S A Achmad andN Aimi ldquoA new type of stilbene-related secondary metaboliteidenburgene from Cryptocarya idenburgensisrdquo Chemical andPharmaceutical Bulletin vol 48 no 11 pp 1726ndash1728 2000
[16] P-M Allard E T H Dau C Eydoux et al ldquoAlkylatedflavanones from the bark of Cryptocarya chartacea as denguevirus NS5 polymerase inhibitorsrdquo Journal of Natural Productsvol 74 no 11 pp 2446ndash2453 2011
[17] T-S Wu C-R Su and K-H Lee ldquoCytotoxic and anti-HIVphenanthroindolizidine alkaloids from Cryptocarya chinensisrdquoNatural Product Communications vol 7 no 6 pp 725ndash727 2012
[18] T-H Chou J-J Chen C-F Peng M-J Cheng and I-S ChenldquoNew flavanones from the leaves of Cryptocarya chinensis andtheir antituberculosis activityrdquo Chemistry and Biodiversity vol8 no 11 pp 2015ndash2024 2011
[19] A A Nasrullah A Zahari J Mohamad and K AwangldquoAntiplasmodial alkaloids from the bark of Cryptocarya nigra(Lauraceae)rdquoMolecules vol 18 no 7 pp 8009ndash8017 2013
[20] R A Davis O Demirkiran M L Sykes et al ldquo7101584081015840-Dihydroobolactone a typanocidal 120572-pyrone from the rainforesttree Cryptocarya obovatardquo Bioorganic andMedicinal ChemistryLetters vol 20 no 14 pp 4057ndash4059 2010
[21] R Feng Z K Guo C M Yan E G Li R X Tan and H MGe ldquoAnti-inflammatory flavonoids from Cryptocarya chingiirdquoPhytochemistry vol 76 pp 98ndash105 2012
[22] V Dumontet N Van Hung M-T Adeline et al ldquoCytotoxicflavonoids and 120572-pyrones from Cryptocarya obovatardquo Journalof Natural Products vol 67 no 5 pp 858ndash862 2004
[23] C Y Ong S K Ling R M Ali et al ldquoSystematic analysis of invitro photo-cytotoxic activity in extracts from terrestrial plantsin Peninsula Malaysia for photodynamic therapyrdquo Journal ofPhotochemistry and Photobiology B vol 96 no 3 pp 216ndash2222009
[24] V Dumontet C Gaspard N van Hung et al ldquoNew cytotoxicflavonoids fromCryptocarya infectoriardquoTetrahedron vol 57 no29 pp 6189ndash6196 2001
[25] H Usman E H Hakim T Harlim et al ldquoCytotoxic chalconesand flavanones from the tree bark of Cryptocarya costatardquoZeitschrift fur Naturforschung C vol 61 no 3-4 pp 184ndash1882006
[26] F Kurniadewi L D Juliawaty Y M Syah et al ldquoPhenolic com-pounds from Cryptocarya konishii their cytotoxic and tyrosinekinase inhibitory propertiesrdquo Journal of Natural Medicines vol64 no 2 pp 121ndash125 2010
[27] T-H Chou J-J Chen S-J Lee M Y Chiang C-W Yang andI-S Chen ldquoCytotoxic flavonoids from the leaves ofCryptocaryachinensisrdquo Journal of Natural Products vol 73 no 9 pp 1470ndash1475 2010
[28] J C Liao ldquoLauraceae in flora of Taiwanrdquo in Editorial Committeeof the Flora of Taiwan vol 2 pp 448ndash451 Taipei Taiwan 1996
[29] B H Chen H W Chang H M Huang et al ldquo(-)-anonaineinduces DNA damage and inhibits growth and migration ofhuman lung carcinomaH1299 cellsrdquo Journal of Agricultural andFood Chemistry vol 59 no 6 pp 2284ndash2290 2011
[30] M-Y Lin Y-R Lee S-Y Chiang et al ldquoCortexMoutan inducesbladder cancer cell death via apoptosis and retards tumorgrowth inmouse bladdersrdquoEvidence-based Complementary andAlternative Medicine vol 2013 Article ID 207279 8 pages 2013
[31] C-C Chiu P-L Liu K-J Huang et al ldquoGoniothalamininhibits growth of human lung cancer cells through DNAdamage apoptosis and reduced migration abilityrdquo Journal ofAgricultural and Food Chemistry vol 59 no 8 pp 4288ndash42932011
[32] J Masters ldquoFalse cell linesrdquoCarcinogenesis vol 23 no 2 p 3712002
[33] H Ding C Han D Guo et al ldquoSelective induction of apoptosisof human oral cancer cell lines by avocado extracts via a ROS-mediated mechanismrdquo Nutrition and Cancer vol 61 no 3 pp348ndash356 2009
[34] J-C Lee M-F Hou H-W Huang et al ldquoMarine algal natu-ral products with anti-oxidative anti-inflammatory and anti-cancer propertiesrdquoCancer Cell International vol 13 no 1 article55 2013
[35] C Gorrini I S Harris and TWMak ldquoModulation of oxidativestress as an anticancer strategyrdquoNature Reviews DrugDiscoveryvol 12 no 12 pp 931ndash947 2013
[36] D Trachootham J Alexandre and P Huang ldquoTargeting can-cer cells by ROS-mediated mechanisms a radical therapeuticapproachrdquo Nature Reviews Drug Discovery vol 8 no 7 pp579ndash591 2009
[37] A K Samhan-Arias F J Martın-Romero and C Gutierrez-Merino ldquoKaempferol blocks oxidative stress in cerebellar gran-ule cells and reveals a key role for reactive oxygen speciesproduction at the plasma membrane in the commitment toapoptosisrdquo Free Radical Biology and Medicine vol 37 no 1 pp48ndash61 2004
[38] S-H Oh and S-C Lim ldquoA rapid and transient ROS generationby cadmium triggers apoptosis via caspase-dependent pathwayin HepG2 cells and this is inhibited through N-acetylcysteine-mediated catalase upregulationrdquo Toxicology and Applied Phar-macology vol 212 no 3 pp 212ndash223 2006
[39] R A Ehlers A Hernandez L S Bloemendal R T EthridgeB Farrow and B M Evers ldquoMitochondrial DNA damage andaltered membrane potential (Δ120595) in pancreatic acinar cellsinduced by reactive oxygen speciesrdquo Surgery vol 126 no 2 pp148ndash155 1999
[40] S McKenzie and N Kyprianou ldquoApoptosis evasion the role ofsurvival pathways in prostate cancer progression and therapeu-tic resistancerdquo Journal of Cellular Biochemistry vol 97 no 1 pp18ndash32 2006
10 The Scientific World Journal
[41] S Fulda ldquoEvasion of apoptosis as a cellular stress response incancerrdquo International Journal of Cell Biology vol 2010 ArticleID 370835 6 pages 2010
[42] H Lin X-B Liu J-J Yu F Hua and Z-W Hu ldquoAntioxidantN-acetylcysteine attenuates hepatocarcinogenesis by inhibitingROSER stress in TLR2 deficient mouserdquo PLoS ONE vol 8 no10 Article ID e74130 2013
[43] R Scherz-Shouval and Z Elazar ldquoROS mitochondria and theregulation of autophagyrdquoTrends in Cell Biology vol 17 no 9 pp422ndash427 2007
[44] R Scherz-Shouval and Z Elazar ldquoRegulation of autophagy byROS physiology and pathologyrdquoTrends in Biochemical Sciencesvol 36 no 1 pp 30ndash38 2011
Submit your manuscripts athttpwwwhindawicom
PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom
Volume 2014
ToxinsJournal of
VaccinesJournal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
AntibioticsInternational Journal of
ToxicologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Drug DeliveryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in Pharmacological Sciences
Tropical MedicineJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
AddictionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Autoimmune Diseases
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anesthesiology Research and Practice
ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Pharmaceutics
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
8 The Scientific World Journal
The anticancer effects for other Cryptocarya-derivedcompounds from methanolic extracts of nonroot parts havebeen reported earlier For example for murine leukemiaP-388 cells the IC
50values of 2101584041015840-dihydroxy-5101584061015840-
dimethoxychalcone and isodidymocarpin isolated fromtree bark of C costata were 57 and 111 120583M [25] and IC
50
values of the chalcone derivative (desmethylinfectocaryone)and phenolic compound (infectocaryone) isolated fromwood of C konishii were 217 and 08 120583M [26] at 48 hrespectively For compounds from leaves of C chinensis theIC50
values of infectocaryone and cryptocaryanone A wereat the 120583M level for human lung cancer NCI-H460 cells andglioblastoma SF-268 cells [27] These Cryptocarya-derivedcompounds from methanolic extracts of nonroot partsshowed the IC
50values ranging from 08 to 11 120583M This is
close to our preliminary result that the IC50
of the clinicalanticancer drug cisplatin at 24 h treatment in oral cancerCa9-22 cells is 306 120583gmL (102 120583M) (data not shown) Inthe present study the IC
50values of the MECCrt in oral
cancer Ca9-22 and CAL 27 cell lines at 24 h were 1867 and1322 120583gmL respectively Although the IC
50values of the
MECCrt were about 3-4 folds of cisplatin for oral cancercells its crude extract nature has to be concerned Thereforeit is warranted to further investigate the particular bioactivecomponents that are included in the methanolic extracts ofCryptocarya concinnaHance roots
Moreover the anticancer effect for trunk bark ofC infectoria-derived methanol extracts was reported tobe cytotoxic to KB cells [24] KB cells were regardedas oral epidermal carcinoma however it was recentlyvalidated to have marker chromosomes and DNAfinger printings of human cervical cancer HeLacells (httpwwwncbinlmnihgovmeshDb=meshampterm=KB+Cells) [32] Accordingly the anticancer effect of oralcancer by the bioactive compounds from Cryptocarya plantremains unclear Conversely we here demonstrate theantioral cancer effect of methanolic extracts of a Cryptocaryaspecies for the first time using two OSCC cell lines Ca9-22and CAL 27
In several anticancer drugs [6 7 10 11 33ndash36] ROSgeneration is one of the common strategies to inhibit cancercell proliferation ROS plays a vital role in early stagesof apoptosis [37] and leads to MMP depolarization [3839] Escaping apoptosis is demonstrated to be involved inthe drug resistance of cancer cells [40 41] To enhanceapoptotic induction of anticancer drugs may interfere thedrug resistance if there In the present study we observed thatapoptosis was inducible by MECCrt in two OSCC cell linesas it was demonstrated by sub-G1 monitoring and annexinVPI assayWe also found that MECCrt significantly inducedthe ROS level and reduced the MMP level in two oral cancercell lines in dose-responsive ways These findings suggestthat oxidative stress may be involved in theMECCrt-inducedantiproliferative effect in two oral cancer Ca9-22 and CAL 27cell lines However the role of oxidative stress in MECCrtneed to be further examined by the ROS scavenger such asN-acetylcysteine [42] to confirm if raised ROS has played acritical role in the process of apoptosis Furthermore the ROSmay generate nonapoptotic effect like autophagy described
in literature [43 44] Therefore it was warranted to furtherinvestigate the role of autophagy in MECCrt-treated oralcancer cell lines in future
5 Conclusions
We demonstrated the antiproliferative and apoptotic effectsof MECCrt through ROS generation and mitochondrialdepolarization in twoOSCC cell linesTherefore these resultssuggest that MECCrt has anticancer potential for oral cancertherapy
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was supported by funds of the NSC102-2314-B-384-002 MOST103-2320-B-037-008 the 103CM-KMU-09the National Sun Yat-sen University-KMU Joint ResearchProject (NSYSU-KMU 103-p014) the kmtth-102-011 andthe Health and welfare surcharge of tobacco products theMinistry of Health and Welfare Taiwan Republic of China(MOHW103-TD-B-111-05)The authors also acknowledgeDrHans-Uwe Dahms for the English editing
References
[1] C-Y Yen C-H Chen C-H Chang et al ldquoMatrix metallopro-teinases (MMP) 1 andMMP10 but notMMP12 are potential oralcancer markersrdquo Biomarkers vol 14 no 4 pp 244ndash249 2009
[2] C-Y Yen C-Y HuangM-F Hou et al ldquoEvaluating the perfor-mance of fibronectin 1 (FN1) integrin12057241205731 (ITGA4) syndecan-2 (SDC2) and glycoprotein CD44 as the potential biomarkersof oral squamous cell carcinoma (OSCC)rdquo Biomarkers vol 18no 1 pp 63ndash72 2013
[3] H Myoung S-P Hong P-Y Yun J-H Lee and M-J KimldquoAnti-cancer effect of genistein in oral squamous cell carcinomawith respect to angiogenesis and in vitro invasionrdquo CancerScience vol 94 no 2 pp 215ndash220 2003
[4] H K Kim E G Wilson Y H Choi and R VerpoorteldquoMetabolomics a tool for anticancer lead-finding from naturalproductsrdquo Planta Medica vol 76 no 11 pp 1094ndash1102 2010
[5] A D Kinghorn L Pan J N Fletcher and H Chai ldquoThe rele-vance of higher plants in lead compound discovery programsrdquoJournal of Natural Products vol 74 no 6 pp 1539ndash1555 2011
[6] C-C Yeh C-N Tseng J-I Yang et al ldquoAntiproliferation andinduction of apoptosis in Ca9-22 oral cancer cells by ethanolicextract of Gracilaria tenuistipitatardquoMolecules vol 17 no 9 pp10916ndash10927 2012
[7] C-C Yeh J-I Yang J-C Lee et al ldquoAnti-proliferative effect ofmethanolic extract of Gracilaria tenuistipitata on oral cancercells involves apoptosis DNA damage and oxidative stressrdquoBMC Complementary and Alternative Medicine vol 12 article142 2012
[8] J S Yang C W Lin C H Hsin M J Hsieh and Y C ChangldquoSelaginella tamariscina attenuates metastasis via Akt pathways
The Scientific World Journal 9
in oral cancer cellsrdquo PLoS ONE vol 8 no 6 Article ID e680352013
[9] B Narotzki A Z Reznick D Aizenbud and Y Levy ldquoGreentea a promising natural product in oral healthrdquoArchives of OralBiology vol 57 no 5 pp 429ndash435 2012
[10] C-Y Yen C-C Chiu R-W Haung et al ldquoAntiprolifera-tive effects of goniothalamin on Ca9-22 oral cancer cellsthrough apoptosis DNAdamage andROS inductionrdquoMutationResearch vol 747 no 2 pp 253ndash258 2012
[11] C-C Chiu J-W Haung F-R Chang et al ldquoGolden berry-derived 4120573-hydroxywithanolide E for selectively killing oralcancer cells by generating ROS DNA damage and apoptoticpathwaysrdquo PLoS ONE vol 8 no 5 Article ID e64739 2013
[12] M T Davies-Coleman andD E A Rivett ldquoNaturally occurring6-substituted 56-dihydro-120572-pyronesrdquo Fortschritte der ChemieOrganischer Naturstoffe vol 55 pp 1ndash35 1989
[13] A Toribio A Bonfils E Delannay et al ldquoNovel seco-dibenzopyrrocoline alkaloid from Cryptocarya oubatchensisrdquoOrganic Letters vol 8 no 17 pp 3825ndash3828 2006
[14] A J Cavalheiro and M Yoshida ldquo6-[120596-arylalkenyl]-56-dihydro-120572-pyrones from Cryptocarya moschata (Lauraceae)rdquoPhytochemistry vol 53 no 7 pp 811ndash819 2000
[15] L D Juliawaty M Kitajima H Takayama S A Achmad andN Aimi ldquoA new type of stilbene-related secondary metaboliteidenburgene from Cryptocarya idenburgensisrdquo Chemical andPharmaceutical Bulletin vol 48 no 11 pp 1726ndash1728 2000
[16] P-M Allard E T H Dau C Eydoux et al ldquoAlkylatedflavanones from the bark of Cryptocarya chartacea as denguevirus NS5 polymerase inhibitorsrdquo Journal of Natural Productsvol 74 no 11 pp 2446ndash2453 2011
[17] T-S Wu C-R Su and K-H Lee ldquoCytotoxic and anti-HIVphenanthroindolizidine alkaloids from Cryptocarya chinensisrdquoNatural Product Communications vol 7 no 6 pp 725ndash727 2012
[18] T-H Chou J-J Chen C-F Peng M-J Cheng and I-S ChenldquoNew flavanones from the leaves of Cryptocarya chinensis andtheir antituberculosis activityrdquo Chemistry and Biodiversity vol8 no 11 pp 2015ndash2024 2011
[19] A A Nasrullah A Zahari J Mohamad and K AwangldquoAntiplasmodial alkaloids from the bark of Cryptocarya nigra(Lauraceae)rdquoMolecules vol 18 no 7 pp 8009ndash8017 2013
[20] R A Davis O Demirkiran M L Sykes et al ldquo7101584081015840-Dihydroobolactone a typanocidal 120572-pyrone from the rainforesttree Cryptocarya obovatardquo Bioorganic andMedicinal ChemistryLetters vol 20 no 14 pp 4057ndash4059 2010
[21] R Feng Z K Guo C M Yan E G Li R X Tan and H MGe ldquoAnti-inflammatory flavonoids from Cryptocarya chingiirdquoPhytochemistry vol 76 pp 98ndash105 2012
[22] V Dumontet N Van Hung M-T Adeline et al ldquoCytotoxicflavonoids and 120572-pyrones from Cryptocarya obovatardquo Journalof Natural Products vol 67 no 5 pp 858ndash862 2004
[23] C Y Ong S K Ling R M Ali et al ldquoSystematic analysis of invitro photo-cytotoxic activity in extracts from terrestrial plantsin Peninsula Malaysia for photodynamic therapyrdquo Journal ofPhotochemistry and Photobiology B vol 96 no 3 pp 216ndash2222009
[24] V Dumontet C Gaspard N van Hung et al ldquoNew cytotoxicflavonoids fromCryptocarya infectoriardquoTetrahedron vol 57 no29 pp 6189ndash6196 2001
[25] H Usman E H Hakim T Harlim et al ldquoCytotoxic chalconesand flavanones from the tree bark of Cryptocarya costatardquoZeitschrift fur Naturforschung C vol 61 no 3-4 pp 184ndash1882006
[26] F Kurniadewi L D Juliawaty Y M Syah et al ldquoPhenolic com-pounds from Cryptocarya konishii their cytotoxic and tyrosinekinase inhibitory propertiesrdquo Journal of Natural Medicines vol64 no 2 pp 121ndash125 2010
[27] T-H Chou J-J Chen S-J Lee M Y Chiang C-W Yang andI-S Chen ldquoCytotoxic flavonoids from the leaves ofCryptocaryachinensisrdquo Journal of Natural Products vol 73 no 9 pp 1470ndash1475 2010
[28] J C Liao ldquoLauraceae in flora of Taiwanrdquo in Editorial Committeeof the Flora of Taiwan vol 2 pp 448ndash451 Taipei Taiwan 1996
[29] B H Chen H W Chang H M Huang et al ldquo(-)-anonaineinduces DNA damage and inhibits growth and migration ofhuman lung carcinomaH1299 cellsrdquo Journal of Agricultural andFood Chemistry vol 59 no 6 pp 2284ndash2290 2011
[30] M-Y Lin Y-R Lee S-Y Chiang et al ldquoCortexMoutan inducesbladder cancer cell death via apoptosis and retards tumorgrowth inmouse bladdersrdquoEvidence-based Complementary andAlternative Medicine vol 2013 Article ID 207279 8 pages 2013
[31] C-C Chiu P-L Liu K-J Huang et al ldquoGoniothalamininhibits growth of human lung cancer cells through DNAdamage apoptosis and reduced migration abilityrdquo Journal ofAgricultural and Food Chemistry vol 59 no 8 pp 4288ndash42932011
[32] J Masters ldquoFalse cell linesrdquoCarcinogenesis vol 23 no 2 p 3712002
[33] H Ding C Han D Guo et al ldquoSelective induction of apoptosisof human oral cancer cell lines by avocado extracts via a ROS-mediated mechanismrdquo Nutrition and Cancer vol 61 no 3 pp348ndash356 2009
[34] J-C Lee M-F Hou H-W Huang et al ldquoMarine algal natu-ral products with anti-oxidative anti-inflammatory and anti-cancer propertiesrdquoCancer Cell International vol 13 no 1 article55 2013
[35] C Gorrini I S Harris and TWMak ldquoModulation of oxidativestress as an anticancer strategyrdquoNature Reviews DrugDiscoveryvol 12 no 12 pp 931ndash947 2013
[36] D Trachootham J Alexandre and P Huang ldquoTargeting can-cer cells by ROS-mediated mechanisms a radical therapeuticapproachrdquo Nature Reviews Drug Discovery vol 8 no 7 pp579ndash591 2009
[37] A K Samhan-Arias F J Martın-Romero and C Gutierrez-Merino ldquoKaempferol blocks oxidative stress in cerebellar gran-ule cells and reveals a key role for reactive oxygen speciesproduction at the plasma membrane in the commitment toapoptosisrdquo Free Radical Biology and Medicine vol 37 no 1 pp48ndash61 2004
[38] S-H Oh and S-C Lim ldquoA rapid and transient ROS generationby cadmium triggers apoptosis via caspase-dependent pathwayin HepG2 cells and this is inhibited through N-acetylcysteine-mediated catalase upregulationrdquo Toxicology and Applied Phar-macology vol 212 no 3 pp 212ndash223 2006
[39] R A Ehlers A Hernandez L S Bloemendal R T EthridgeB Farrow and B M Evers ldquoMitochondrial DNA damage andaltered membrane potential (Δ120595) in pancreatic acinar cellsinduced by reactive oxygen speciesrdquo Surgery vol 126 no 2 pp148ndash155 1999
[40] S McKenzie and N Kyprianou ldquoApoptosis evasion the role ofsurvival pathways in prostate cancer progression and therapeu-tic resistancerdquo Journal of Cellular Biochemistry vol 97 no 1 pp18ndash32 2006
10 The Scientific World Journal
[41] S Fulda ldquoEvasion of apoptosis as a cellular stress response incancerrdquo International Journal of Cell Biology vol 2010 ArticleID 370835 6 pages 2010
[42] H Lin X-B Liu J-J Yu F Hua and Z-W Hu ldquoAntioxidantN-acetylcysteine attenuates hepatocarcinogenesis by inhibitingROSER stress in TLR2 deficient mouserdquo PLoS ONE vol 8 no10 Article ID e74130 2013
[43] R Scherz-Shouval and Z Elazar ldquoROS mitochondria and theregulation of autophagyrdquoTrends in Cell Biology vol 17 no 9 pp422ndash427 2007
[44] R Scherz-Shouval and Z Elazar ldquoRegulation of autophagy byROS physiology and pathologyrdquoTrends in Biochemical Sciencesvol 36 no 1 pp 30ndash38 2011
Submit your manuscripts athttpwwwhindawicom
PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom
Volume 2014
ToxinsJournal of
VaccinesJournal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
AntibioticsInternational Journal of
ToxicologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Drug DeliveryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in Pharmacological Sciences
Tropical MedicineJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
AddictionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Autoimmune Diseases
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anesthesiology Research and Practice
ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Pharmaceutics
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
The Scientific World Journal 9
in oral cancer cellsrdquo PLoS ONE vol 8 no 6 Article ID e680352013
[9] B Narotzki A Z Reznick D Aizenbud and Y Levy ldquoGreentea a promising natural product in oral healthrdquoArchives of OralBiology vol 57 no 5 pp 429ndash435 2012
[10] C-Y Yen C-C Chiu R-W Haung et al ldquoAntiprolifera-tive effects of goniothalamin on Ca9-22 oral cancer cellsthrough apoptosis DNAdamage andROS inductionrdquoMutationResearch vol 747 no 2 pp 253ndash258 2012
[11] C-C Chiu J-W Haung F-R Chang et al ldquoGolden berry-derived 4120573-hydroxywithanolide E for selectively killing oralcancer cells by generating ROS DNA damage and apoptoticpathwaysrdquo PLoS ONE vol 8 no 5 Article ID e64739 2013
[12] M T Davies-Coleman andD E A Rivett ldquoNaturally occurring6-substituted 56-dihydro-120572-pyronesrdquo Fortschritte der ChemieOrganischer Naturstoffe vol 55 pp 1ndash35 1989
[13] A Toribio A Bonfils E Delannay et al ldquoNovel seco-dibenzopyrrocoline alkaloid from Cryptocarya oubatchensisrdquoOrganic Letters vol 8 no 17 pp 3825ndash3828 2006
[14] A J Cavalheiro and M Yoshida ldquo6-[120596-arylalkenyl]-56-dihydro-120572-pyrones from Cryptocarya moschata (Lauraceae)rdquoPhytochemistry vol 53 no 7 pp 811ndash819 2000
[15] L D Juliawaty M Kitajima H Takayama S A Achmad andN Aimi ldquoA new type of stilbene-related secondary metaboliteidenburgene from Cryptocarya idenburgensisrdquo Chemical andPharmaceutical Bulletin vol 48 no 11 pp 1726ndash1728 2000
[16] P-M Allard E T H Dau C Eydoux et al ldquoAlkylatedflavanones from the bark of Cryptocarya chartacea as denguevirus NS5 polymerase inhibitorsrdquo Journal of Natural Productsvol 74 no 11 pp 2446ndash2453 2011
[17] T-S Wu C-R Su and K-H Lee ldquoCytotoxic and anti-HIVphenanthroindolizidine alkaloids from Cryptocarya chinensisrdquoNatural Product Communications vol 7 no 6 pp 725ndash727 2012
[18] T-H Chou J-J Chen C-F Peng M-J Cheng and I-S ChenldquoNew flavanones from the leaves of Cryptocarya chinensis andtheir antituberculosis activityrdquo Chemistry and Biodiversity vol8 no 11 pp 2015ndash2024 2011
[19] A A Nasrullah A Zahari J Mohamad and K AwangldquoAntiplasmodial alkaloids from the bark of Cryptocarya nigra(Lauraceae)rdquoMolecules vol 18 no 7 pp 8009ndash8017 2013
[20] R A Davis O Demirkiran M L Sykes et al ldquo7101584081015840-Dihydroobolactone a typanocidal 120572-pyrone from the rainforesttree Cryptocarya obovatardquo Bioorganic andMedicinal ChemistryLetters vol 20 no 14 pp 4057ndash4059 2010
[21] R Feng Z K Guo C M Yan E G Li R X Tan and H MGe ldquoAnti-inflammatory flavonoids from Cryptocarya chingiirdquoPhytochemistry vol 76 pp 98ndash105 2012
[22] V Dumontet N Van Hung M-T Adeline et al ldquoCytotoxicflavonoids and 120572-pyrones from Cryptocarya obovatardquo Journalof Natural Products vol 67 no 5 pp 858ndash862 2004
[23] C Y Ong S K Ling R M Ali et al ldquoSystematic analysis of invitro photo-cytotoxic activity in extracts from terrestrial plantsin Peninsula Malaysia for photodynamic therapyrdquo Journal ofPhotochemistry and Photobiology B vol 96 no 3 pp 216ndash2222009
[24] V Dumontet C Gaspard N van Hung et al ldquoNew cytotoxicflavonoids fromCryptocarya infectoriardquoTetrahedron vol 57 no29 pp 6189ndash6196 2001
[25] H Usman E H Hakim T Harlim et al ldquoCytotoxic chalconesand flavanones from the tree bark of Cryptocarya costatardquoZeitschrift fur Naturforschung C vol 61 no 3-4 pp 184ndash1882006
[26] F Kurniadewi L D Juliawaty Y M Syah et al ldquoPhenolic com-pounds from Cryptocarya konishii their cytotoxic and tyrosinekinase inhibitory propertiesrdquo Journal of Natural Medicines vol64 no 2 pp 121ndash125 2010
[27] T-H Chou J-J Chen S-J Lee M Y Chiang C-W Yang andI-S Chen ldquoCytotoxic flavonoids from the leaves ofCryptocaryachinensisrdquo Journal of Natural Products vol 73 no 9 pp 1470ndash1475 2010
[28] J C Liao ldquoLauraceae in flora of Taiwanrdquo in Editorial Committeeof the Flora of Taiwan vol 2 pp 448ndash451 Taipei Taiwan 1996
[29] B H Chen H W Chang H M Huang et al ldquo(-)-anonaineinduces DNA damage and inhibits growth and migration ofhuman lung carcinomaH1299 cellsrdquo Journal of Agricultural andFood Chemistry vol 59 no 6 pp 2284ndash2290 2011
[30] M-Y Lin Y-R Lee S-Y Chiang et al ldquoCortexMoutan inducesbladder cancer cell death via apoptosis and retards tumorgrowth inmouse bladdersrdquoEvidence-based Complementary andAlternative Medicine vol 2013 Article ID 207279 8 pages 2013
[31] C-C Chiu P-L Liu K-J Huang et al ldquoGoniothalamininhibits growth of human lung cancer cells through DNAdamage apoptosis and reduced migration abilityrdquo Journal ofAgricultural and Food Chemistry vol 59 no 8 pp 4288ndash42932011
[32] J Masters ldquoFalse cell linesrdquoCarcinogenesis vol 23 no 2 p 3712002
[33] H Ding C Han D Guo et al ldquoSelective induction of apoptosisof human oral cancer cell lines by avocado extracts via a ROS-mediated mechanismrdquo Nutrition and Cancer vol 61 no 3 pp348ndash356 2009
[34] J-C Lee M-F Hou H-W Huang et al ldquoMarine algal natu-ral products with anti-oxidative anti-inflammatory and anti-cancer propertiesrdquoCancer Cell International vol 13 no 1 article55 2013
[35] C Gorrini I S Harris and TWMak ldquoModulation of oxidativestress as an anticancer strategyrdquoNature Reviews DrugDiscoveryvol 12 no 12 pp 931ndash947 2013
[36] D Trachootham J Alexandre and P Huang ldquoTargeting can-cer cells by ROS-mediated mechanisms a radical therapeuticapproachrdquo Nature Reviews Drug Discovery vol 8 no 7 pp579ndash591 2009
[37] A K Samhan-Arias F J Martın-Romero and C Gutierrez-Merino ldquoKaempferol blocks oxidative stress in cerebellar gran-ule cells and reveals a key role for reactive oxygen speciesproduction at the plasma membrane in the commitment toapoptosisrdquo Free Radical Biology and Medicine vol 37 no 1 pp48ndash61 2004
[38] S-H Oh and S-C Lim ldquoA rapid and transient ROS generationby cadmium triggers apoptosis via caspase-dependent pathwayin HepG2 cells and this is inhibited through N-acetylcysteine-mediated catalase upregulationrdquo Toxicology and Applied Phar-macology vol 212 no 3 pp 212ndash223 2006
[39] R A Ehlers A Hernandez L S Bloemendal R T EthridgeB Farrow and B M Evers ldquoMitochondrial DNA damage andaltered membrane potential (Δ120595) in pancreatic acinar cellsinduced by reactive oxygen speciesrdquo Surgery vol 126 no 2 pp148ndash155 1999
[40] S McKenzie and N Kyprianou ldquoApoptosis evasion the role ofsurvival pathways in prostate cancer progression and therapeu-tic resistancerdquo Journal of Cellular Biochemistry vol 97 no 1 pp18ndash32 2006
10 The Scientific World Journal
[41] S Fulda ldquoEvasion of apoptosis as a cellular stress response incancerrdquo International Journal of Cell Biology vol 2010 ArticleID 370835 6 pages 2010
[42] H Lin X-B Liu J-J Yu F Hua and Z-W Hu ldquoAntioxidantN-acetylcysteine attenuates hepatocarcinogenesis by inhibitingROSER stress in TLR2 deficient mouserdquo PLoS ONE vol 8 no10 Article ID e74130 2013
[43] R Scherz-Shouval and Z Elazar ldquoROS mitochondria and theregulation of autophagyrdquoTrends in Cell Biology vol 17 no 9 pp422ndash427 2007
[44] R Scherz-Shouval and Z Elazar ldquoRegulation of autophagy byROS physiology and pathologyrdquoTrends in Biochemical Sciencesvol 36 no 1 pp 30ndash38 2011
Submit your manuscripts athttpwwwhindawicom
PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom
Volume 2014
ToxinsJournal of
VaccinesJournal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
AntibioticsInternational Journal of
ToxicologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Drug DeliveryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in Pharmacological Sciences
Tropical MedicineJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
AddictionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Autoimmune Diseases
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anesthesiology Research and Practice
ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Pharmaceutics
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
10 The Scientific World Journal
[41] S Fulda ldquoEvasion of apoptosis as a cellular stress response incancerrdquo International Journal of Cell Biology vol 2010 ArticleID 370835 6 pages 2010
[42] H Lin X-B Liu J-J Yu F Hua and Z-W Hu ldquoAntioxidantN-acetylcysteine attenuates hepatocarcinogenesis by inhibitingROSER stress in TLR2 deficient mouserdquo PLoS ONE vol 8 no10 Article ID e74130 2013
[43] R Scherz-Shouval and Z Elazar ldquoROS mitochondria and theregulation of autophagyrdquoTrends in Cell Biology vol 17 no 9 pp422ndash427 2007
[44] R Scherz-Shouval and Z Elazar ldquoRegulation of autophagy byROS physiology and pathologyrdquoTrends in Biochemical Sciencesvol 36 no 1 pp 30ndash38 2011
Submit your manuscripts athttpwwwhindawicom
PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom
Volume 2014
ToxinsJournal of
VaccinesJournal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
AntibioticsInternational Journal of
ToxicologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Drug DeliveryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in Pharmacological Sciences
Tropical MedicineJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
AddictionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Autoimmune Diseases
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anesthesiology Research and Practice
ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Pharmaceutics
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Submit your manuscripts athttpwwwhindawicom
PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom
Volume 2014
ToxinsJournal of
VaccinesJournal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
AntibioticsInternational Journal of
ToxicologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Drug DeliveryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in Pharmacological Sciences
Tropical MedicineJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
AddictionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Autoimmune Diseases
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anesthesiology Research and Practice
ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Pharmaceutics
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Top Related