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Indonesian Journal of Biotechnology, June, 2016 Vol. 21, No. 1, pp. 29–37

The Synergistic Effect of Doxorubicin and Ethanolic Extracts of Caesalpiniasappan L. Wood and Ficus septica Burm. f. Leaves on Viability, Cell Cycle

Progression, and Apoptosis Induction of MCF­7 Cells

Sari Haryanti1, Suwijiyo Pramono2, Retno Murwanti3, Edy Meiyanto4,*

1Balai Besar Penelitian dan Pengembangan Tanaman Obat dan Obat Tradisional, KementerianKesehatan Republik Indonesia, Tawangmangu

2Departement of Pharmaceutical Biology, Faculty of Pharmacy, UGM, Yogyakarta3Departement of Pharmacology and Clinical Pharmacy, Faculty of Pharmacy, UGM, Yogyakarta4Departement of Pharmaceutical Chemistry, and Cancer Chemoprevention Research Center,Faculty of Pharmacy, UGM, Yogyakarta

AbstractCaesalpinia sappan L. and Ficus septica Burm. f known as a potential plant with wide variety of

medicinal properties, including anticancer. Present study was aimed to explore cytotoxic effect of sappanwood (ECS) and awar­awar leaves (EFS), and its combination with doxorubicin (dox) on MCF­7 cellsfocusing on cell cycle progression and apoptosis induction. The result of MTT assay showed that singletreatment of ECS and dox performed cytotoxic effect with the IC50 value of 32 µg/mL and 6 µM respectively,while EFS performed low cytotoxic effect with the IC50 value of 282 µg/mL. The combination of ECSwith EFS and doxorubicin showed synergistic cytotoxic effect. Flow cytometry analysis revealed thatcombination of ECS (16 µg/mL) with EFS (8 µg/mL) and doxorubicin (2 µM) induced apoptosis, andcell accumulation at sub­G1 and G2/M phases. Immunoblotting assay confirmed the apoptosis inductionof this combination through increasing of cleavage of PARP­1. Based on these results, the synergisticcytotoxic effect of this combination was through G2/M phase accumulation and apoptosis inductionand potentially to be developed as co­chemotherapeutic agent.

Keywords: Sappan wood, Ficus septica leaves, doxorubicin, MCF­7, cell cycle, apoptosis

*Corresponding author:Edy MeiyantoDepartement of Pharmaceutical Chemistry, andCancer Chemoprevention Research Center, Facultyof Pharmacy, UGM, YogyakartaE­mail: meiyan_e@ugm.ac.id

IntroductionCancer is a malignant disease, marked

by uncontrolled cell proliferation andspreading due to the aberration of manyregulatory growth system (Witsch et al., 2010).Continuing proliferation and evadingapoptosis are the main factors of themalignancy and mainly driven by cell cycleprogression with regardless of check pointcontrol (Barnum and O’Connell, 2014). Thereare many molecular event in thesephysiological processes involving growthsignaling through MAP Kinases, cell cycleregulatory proteins, and apoptosis regulation(Santarpia et al., 2012). Most of the signal

regulatory system are the potential pivotaltargets of anti cancer agents. Therefore, thedevelopment of combinatorial anti canceragents should consider to the mainpredominantly molecular event in particularcancers (Kummar et al., 2010).

To date, many molecules have beenidentified to perform cytotoxic activitytargeted on (some) cancer markers such asRTK (receptor tyrosine kinase) inhibitors(Takeuchi and Ito, 2011), CDK (cyclin­dependent kinase) inhibitors (Asghar et al.,2015), NF­κB inhibitors (Hoesel and Schmid,2013) and MMPs inhibitors (Cathcart et al.,2015). Some of these active molecules arenatural plant origin. Meiyanto et al., 2012noted that some citrus flavonoids performcytotoxic activities to some cancer cells throughinhibition of MAPK and NF­κB activation(Meiyanto et al., 2012). Curcumin and othercompounds in turmeric are also reported to

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have anti cancer activity with some moleculartargets in cell cycle, apoptosis and cellmigration (Tuorkey, 2014). Some alkaloidscomponent also known to have cytotoxicactivity to some cancer cells, such as taxanealkaloids and phenanthroindolizidine(Chemler, 2009; Dumontet and Jordan, 2010).Interestingly, the compounds composed inthe potential anticancer plants are believedto contribute in the cytotoxic activity troughdifferent targets and may perform synergisticeffect.

Caesalpinia sappan L., commonlyknown as sappan wood or secang in Indonesia,is a plant of Leguminosae family, which hasbeen used widely as traditional medicine inAsia (Nirmal et al., 2015). Chemicalinvestigation resulted that brazilin/brazileinis the major compound of sappan wood(Mekala and Radha, 2015). Brazilin is easilyoxidized to produce brazilein by air and light.Both brazilin and brazilein responsible forvarious biological activities of sappan wood,including anticancer (Nirmal et al., 2015).Ficus septica Burm. f. (awar­awar) ethanolicleaves extract induced apoptosis anddownregulated Bcl2 expression on MCF­7breast cancer cell lines (Sekti et al., 2010). Then­hexane insoluble fraction and ethyl acetatesoluble fraction from leaves ethanolic extractexhibited cytotoxic activity on T47D breastcancer cell lines (Nugroho et al., 2011). Thehexane fraction combined with doxorubicinshowed synergistic activity on T47D (Nugrohoet al., 2013). In our previous study(unpublished data) ethanolic extract of F.septica leaves showed strong antimigrationactivity on 4T1 cells by wound healing assay,while sappan wood did not show the effect.Therefore, we suggest the combination ofboth extract might support each other withdifferent mechanism and resulting asynergistic effect with lower dose ofdoxorubicin. Thus, the combination mightbe able to combat cancer through inhibitionof proliferation and also metastasis. Thispresent study was aimed to explore cytotoxiceffect of extract in MCF­7, the human breastcancer cell line, and its combination effectwith doxorubicin. Furthermore, we also

investigated the possible combinatorial effectof doxorubicin and the extract in cell cycleprogression and apoptosis induction.

Materials and Methods

Materials and extractionSappan wood (Caesalpinia sappan L.)

and awar (Ficus septica Burm. f.) leavesobtained from Medicinal Plant and TraditionalMedicine Research and Development Center,Tawangmangu, Central Java, Indonesia. Theywere sliced, dried in 40°C, pulverized, andextracted with ethanol 96% by macerationmethod. The dried extract of sappan wood(ECS) and ficus leaves (EFS), and alsodoxorubicin (dox) were dissolved in DMSO(Sigma),andfreshlydilutedinculturemediumin several concentration before used.

Cell cultureMCF­7 cell lines were obtained from

ATCC HTB­22 and maintained in laboratoryof Molecular Biology, Medicinal Plant andTraditional Medicine Research andDevelopment Centre, Ministry of Health,Tawangmangu, Jawa Tengah, Indonesia.Cells were cultured in Minimum EssentialMedium (MEM Gibco) containing 10% fetalbovine serum/FBS (Gibco), 1% penicillin­streptomycin (Gibco), and incubated in CO2incubator 5% at 37°C.

Cytotoxic MTT assayApproximately 1x104 MCF­7 cells/well

were seeded in 96­well plates and incubatedfor 48 hours. Cells were treated with increasingconcentration of the extract or doxorubicineither alone and in combination for 24 hours.Cultured medium was removed and cellswere washed with PBS (Sigma). MTT 0.5mg/ml in medium were added into each welland incubated for 3–4 hours. MTT reactionwas stopped by the addition of 10% SDS in0.01 N HCl, and incubated overnight in thedark room. The absorbance was measuredusing ELISA reader at λ 595 nm (Biorad).Each treatment were carried out in triplicate,and the absorbance data are provided aspercent viability compared to control cells(untreated).

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Cell cycle and apoptosis induction by flowcytometry assay

Approximately 5x105 MCF­7 cells/wellwere cultured in 6­well plate and incubatedfor 48 hours. Cells were then treated with theextract and doxorubicin, either alone orcombination for 24 hours. Cells were harvestedwith trypsin EDTA, washed with phosphate­buffered saline (PBS), and centrifuged 500rpm for 5 minutes. For apoptosis induction,cells then incubated with annexin­V­FITCand propidium iodide (BD Pharmingen) for15 min in the dark, and analyzed by BD AccuriC6 Flow cytometer. To determine cell cycledistribution, cells were fixed with cold ethanol70% for 30 minutes, washed with PBS, andcentrifuged 500 rpm for 5 minutes. Cells werethen resuspended in PBS containing 40 μg/mlpropidium iodide (Sigma), 20 μg/mL RNAse(Roche) and 0.1% TritonX­114 (Sigma) for 15minutes in the dark, and then subjected toBD Accuri C6 flow cytometer.

Western BlotApproximately 106 MCF­7 cells were

seeded in 10 cm tissue culture dish, andincubated for 24 hours. Cell were treated withthe extract and doxorubicin, either alone orcombination for 24 hours. Protein wasextracted using Pro­prep (IntronBiotechnology), then separated in 14%acrylamide gel by SDS­PAGE electrophoresis.After transferring to polyvinylidene fluoride(PVDF) membrane, the membrane wasincubated overnight at 4°C with either therabbit monoclonal antibody of PARP­1 (CellSignaling D64E10) or mouse monoclonalantibody of β­actin (Santa Cruz sc­47778).After incubation with secondary antibodyanti­rabbit (Santa Cruz sc­2357) and anti­mouse (Santa Cruz sc­516102) for 1 hour, theprotein bands were visualized using ECL(Amersham) and detected by Luminograph.The relative protein levels were calculated inreference to the amount of β­actin protein.

Data analysisTo calculate the IC50 value from single

cytotoxicity assay, we plotted linear regression

of concentration and % cells viability usingExcel MS Office 2013. Combination treatmentwas evaluated by calculating the CombinationIndex (CI) value with Compusyn software.The data obtained from flow cytometer wasanalyzed using BD Accuri C6 software.

Results and Discussion

Results

Cell growth inhibitory effect by single treatmentof ECS, EFS, and dox

Our previous report noted that ethanolicextract of ficus leaves and sappan woodperformed cytotoxic effect on T47D breastcancer cells through cell cycle arrest andapoptosis induction (Nugroho et al., 2015;Nurzijah et al., 2012). In this report, we useMCF7 breast cancer cells, a type of cancer cellwith caspase­3 mutation (Ghavami et al., 2009),to explore the response of the cells to theagents in single or in combination. The MCF­7 cells treated with single ECS, EFS, and doxshowed cytotoxic effect in dose dependentmanner with the IC50 value of 32 µg/mL, 282µg/mL, and 6 µM respectively (Figure 1).Based on these results, we were then assessedthe combination effect of the extract anddoxorubicin in some series concentration onMCF­7 cells.

Combinatorial effect of ECS, EFS, and dox onMCF­7 cells viability

To evaluate the combinatorial effectof these agents, we set the treatment withthe series concentration of each agent underIC50 values. The result showed that theincreasing concentration of ECS 4, 5, 8, and16 µg/ml and EFS 2, 3, 4, and 8 µg/ml incombination was not followed by decreasingcell viability, and resulted combination index(CI) value 1.0–4.1. These CI values indicatedthat combinational treatment of this extractexhibited an additive and antagonisticinhibitory effect on MCF­7 cells. Additiveeffect was only achieved at the highest leavesconcentration used in this experiment.However, when those extract combined withdoxorubicin 1.0, 1.5, and 2 µM, it resultedsynergistic effect. The only antagonistic effect

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was showed at the lowest concentration ofdoxorubicin (0.5 µM) (Figure 2 and Table 1).

Cell cycle progression and apoptosis inductionThe combination effect of dox and the

extract in cell growth inhibition could be asthe result of the modulation in physiologicalprocess of the cells. To investigate the effectof the combinatorial treatment in particularphysiological process of the cells we furtherexplored MCF­7 cell cycle progression andcell death using flow cytometry. We wereselected one combination, ECS­EFS­doxorubicin with the concentration of16:8:2. Cell cycle histogram of the treatmentwas presented in Figure 3. Single treatmentof dox 2 μM slightly induced G2/M arrested(27.4%), while 16 μg/ml of ECS stronglyinduced G2/M (43.1%) compared to cellcontrol (21.2%). Whereas, EFS did not affected

cell cycle profile. Interestingly, thecombination of ECS and EFS induced G2/Marrested but lower than sappan alone (41.5%).Moreover, the treatment of ECS, EFS and doxcombination increased cell population insubG1 (7.1%) compared to untreated cells(2.3%) and any single treatment. The G2/Mwas still arrested with higher population(32.8%) than that of doxorubicin treatment,but lower than sappan alone (Figure 3).

To understand the cell deathmechanism in subG1 population, whethersynergistic combination was mediatedthrough apoptosis, we stained treated cellswith propidium iodide – annexin V andsubjected to flow cytometry (Figure 4). Theresults showed the apoptotic population wassignificantly increased in MCF­7 cells treatedby combination of ECS­EFS­dox (37.0%)compared to untreated cells (2.7%) and single

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Figure 1. Cytotoxic effect of ECS, EFS, and dox on MCF­7 cells. Cells were seeded in 96­well plate and treatedwith ECS (A), EFS (B) or dox (C) for 24 hours with the series of concentration as indicated. Cells viability weredetermined by MTT assay as described in methods, performed in triplicate, and represented as mean ± SD of %cell viability. IC50 value was calculated using linier regression.

Table 1. Combination index (CI) and dose reduction index (DRI) values of doxorubicin in combination. CI < 1indicates synergistic effect, CI = 1 indicates additive effects and CI > 1 indicates antagonistic effect (Chou, 2010).

­

8

CI value

8

­

2.9

Combo 3­3

Treatment

Concentration

% viability DRI (doxo)ECS EFS Dox

(µg/ml) (µg/ml) (µM)

Combo 2­1 4 2 ­ 84.4 + 9.2 1.10

Combo 2­2 5 3 ­ 88.1 + 6.5 4.10 ­Combo 2­3 4 ­ 83.9 + 5.7 1.90

Combo 2­4 16 ­ 73.4 + 6.5 1.00

Combo 3­1 4 88.4 + 1.8 3.20 5.2

Combo 3­2 78.4 + 1.9 0.88

4 1.5 56.1 + 2.2 0.62

Combo 3­4 8 2 37.7 + 1.9 0.83

3.3

3.0

­

16

8

5

2

3 1

0.5

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treatment of dox (7.7%), ECS (17%), and EFS(4.2%). The combination of ECS and EFS hadlower apoptotic cells (14.2%), but showed thehighest population on necrotic cells (4.6%).To confirm apoptosis induction of the singleand combination treatment, we analyzed theexpression level of protein poly (ADP­ribose)polymerase (PARP) by Western Blot.

Cleavage PARP­1, as indicator of apoptosis,were shown in cells treated with singledoxorubicin and its combination with ECSand EFS (Figure 4­H).

DiscussionDoxorubicin is one of the most active

chemotherapeutic agent and widely used for

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Figure 2. Combinational cytotoxic effect of ECS, EFS and dox on MCF­7 cells. Cells were seeded in 96­well plate andtreated with ECS, EFS, or doxorubicin, either in single and combination. The concentration of ECS was 4, 5, 8, 16µg/mL, EFS was 2, 3, 4, 8 µg/mL, and dox 0.5, 1.0, 1.5, 2.0 µM. The combination concentration of ECS and EFS was4:2, 5:3, 8:4, and 16:8 µg/mL, while combination of ECS, EFS, and dox was 4:2:0.5, 5:3:1.0, 8:4:1.5, and 16:8:2.0. Cellsviability were performed in triplicate, and represented as mean ± SD.

DNA content

Cou

nt

Figure 3. The effect of ECS, EFS, dox and their combination on cell cycle distribution. Cells were treated for 24 hoursand stained with PI reagent, each sample was subjected to flow cytometer. (A) Cell control, (B) dox 2 μM, (C) ECS16 μg/ml, (D) EFS 8 μg/mL, (E) combination of ECS and EFS (F) combination of ECS, EFS, and dox.

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breast cancer treatment (Anampa et al., 2015).Its application in chemotherapy is oftenlimited due to cardio toxicity risk andresistance progression (Vejpongsa and Yeh,2014). The development of doxorubicinresistance in breast cancer is multifactorialprocesses, mainly associated with wide anddiverse expression of drug­resistance genes,and many other changes in genes responsiblefor cell cycle, apoptosis and DNA repair(AbuHammad and Zihlif, 2013). Thecombination with a natural chemopreventiveagents is one of the promising strategy toimprove doxorubicin anticancer effectivenessand reduce its toxicity (Ko and Moon, 2015).Therefore, we investigated the modulatoryeffect of ECS and EFS combined withdoxorubicin on cytotoxicity, cell cycleprogression and apoptosis induction in MCF­7 human breast cancer cell line.

In this study, the MTT assay revealedthat MCF­7 cells viability was stronglyinhibited by single ECS, and also doxorubicin,while the EFS exhibited low activity. We

further demonstrated MTT assay combinationof the extract and doxorubicin to obtain CIvalue. The CI is widely accepted as the simplestpossible way to express pharmacologic druginteraction for quantifying synergism orantagonism. Synergism interaction will bevery useful to treat the dreadful diseases,such as cancer. The main gains are theachievement of synergistic therapeutic effect,reduction of dose and toxicity, and alsominimize or delay drug resistance (Chou,2010).

The synergistic inhibitory effectachieved with the combination of doxorubicin1.0, 1.5, and 2.0 µM (equal to 1/6, ¼ and 1/3of IC50 respectively), with ECS (5, 8, 16µg/mL) and EFS (3, 4, 8 µg/mL). The biggestconcentration of EFS was approximately 1/35of IC50, based on its antimigration activity on4T1 cells by wound healing method (data notshown). Therefore, the combined extract maylead to reduce of doxorubicin dose therapyand furthermore minimizing its cardiotoxicity risk.

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Figure 4. The effect of ECS, EFS, dox and their combination on cell apoptosis. Cells were treated for 24 hours andstained with PI reagent, each sample was subjected to flow cytometer. (A) Cell control, (B) dox 2 μM, (C) ECS 16μg/ml, (D) EFS 8 μg/mL, (E) combination of ECS­EFS, (F) combination of ECS­EFS­dox. The diagram A­F dividedinto 4 area, which showed distribution profiles of living cells (bottom­left), early apoptotic (bottom­right), lateapoptosis (upper­right), necrosis (upper­left) in various indicated treatment. Graphic (G) showed analysis resultsfor apoptosis induction for each treatment. (H) The effect of ECS, EFS, doxorubicin and their combination on cellularexpression levels of PARP­1 by Western blot. Protein β­actin was used as loading control.

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Cell cycle regulation and apoptosisinduction play a critical role in cytotoxicactivity. The cell cycle is a tidy and tightlyregulated mechanism by which cell divide,involving four phases namely G1, S(synthesis), G2 and M (mitosis) (Deep andAgarwal, 2008). The abrogation of cell cyclecheckpoints at critical time is expected totarget the errors of cell cycle regulation toattain cancer cells specific cytotoxicity and tomake the tumor cell susceptible to apoptosis.Currently, these agents are combined withconventional chemotherapeutic agent toovercome cell cycle mediated drug resistanceand to enhance cytotoxic efficacy (Kelley etal., 2014). Doxorubicin modulates cell cyclethrough G1 and G2 phases arrest, as theresult of its interaction with topoisomeraseII mediated DNA damage (Lal et al., 2010).Our result confirmed that single treatmentof dox 2 µM and ECS 16 µg/ml induced cellaccumulation at G2/M phase. However, theEFS 6 µg/ml alone did not show significantchanges in cell cycle profile. Thecombination of dox with both ECS and EFSenhanced cell accumulation at subG1,compared to the untreated cells and eachsingle treatment. These results suggestedthat EFS contributed to enhance the cytotoxiceffect of dox and ECS leading to cell death.

The cell cycle arrest depicted asurvival mechanism for the cancer cell torepair its own damaged DNA. Thedisruption of cell cycle checkpoints byspecific agent before completing DNArepair, can activate the apoptotic pathwayleading to cell death (Carrassa, 2013). In ourstudy, the combination of doxorubicin withboth of the extract increased apoptotic andnecrotic cell induction, compared tountreated cell and each single treatment.Apoptosis induction was confirmed byWestern Blot analysis that showed PARP­1cleavage in the treatment of singledoxorubicin, and its combination with ECSand EFS.

PARP­1 can be cleaved by activatedcaspases. The cleavage of PARP­1 willinhibit uncleaved PARP­1 activity, thuspromote DNA damage response in multiple

pathway (Weaver and Yang, 2013). Theeffect of PARP­1 inhibitors in apoptosisinduction, providing it as a promising targetin cancer therapy (Wang et al., 2012).Apoptosis is one of cell death design,showed an ordered and orchestratedcellular process that occurs in physiologicalcondition and pathological of manydiseases. Apoptosis evasion plays a crucialrole in carcinogenesis, therefore it isbecoming as a popular target for cancertreatment strategy (Wong, 2011). Also, theaberrant cell cycle progression plays acrucial role in cancer cell growth, thustargeting the cell cycle have been regardedas an ideal cancer treatment (Deep andAgarwal, 2008).

ConclusionsBased on these findings, progressive

cytotoxicity effect of doxorubicin combinedwith ECS and EFS extract inhibited MCF­7cells growth through apoptosis inductionand cell cycle perturbation (G2/M arrest).Therefore, the extract combination performspotential natural sources to be developedfurther as co­chemotherapeutic agent.

AcknowledgementsThis research was supported by

Cancer Chemoprevention Research Center,Faculty of Pharmacy, Universitas GadjahMada, and Medicinal Plant and TraditionalMedicine Research and DevelopmentCentre, National Institute of HealthResearch and Development, Ministry ofHealth, Indonesia.

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