Research Article Antitumor Activity of Chinese Propolis in Human...

Research ArticleAntitumor Activity of Chinese Propolis in Human BreastCancer MCF-7 and MDA-MB-231 Cells

Hongzhuan Xuan1 Zhen Li1 Haiyue Yan2 Qing Sang1 Kai Wang3 Qingtao He1

Yuanjun Wang1 and Fuliang Hu3

1 School of Life Science Liaocheng University Liaocheng 252059 China2 School of Life Science Anhui University Hefei 230039 China3 College of Animal Sciences Zhejiang University Hangzhou 310029 China

Correspondence should be addressed to Fuliang Hu flhuzjueducn

Received 10 February 2014 Revised 10 April 2014 Accepted 13 April 2014 Published 22 May 2014

Academic Editor Thomas Efferth

Copyright copy 2014 Hongzhuan Xuan 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

Chinese propolis has been reported to possess various biological activities such as antitumor In present study anticancer activityof ethanol extract of Chinese propolis (EECP) at 25 50 100 and 200 120583gmL was explored by testing the cytotoxicity in MCF-7(human breast cancer ER(+)) and MDA-MB-231 (human breast cancer ER(minus)) cells EECP revealed a dose- and time-dependentcytotoxic effect Furthermore annexin A7 (ANXA7) p53 nuclear factor-120581B p65 (NF-120581B p65) reactive oxygen species (ROS)levels and mitochondrial membrane potential were investigated Our data indicated that treatment of EECP for 24 and 48 hinduced both cells apoptosis obviously Exposure to EECP significantly increased ANXA7 expression and ROS level and NF-120581Bp65 level and mitochondrial membrane potential were depressed by EECP dramatically The effects of EECP on p53 level weredifferent in MCF-7 and MDA-MB-231 cells which indicated that EECP exerted its antitumor effects in MCF-7 and MDA-MB-231cells by inducing apoptosis regulating the levels of ANXA7 p53 and NF-120581B p65 upregulating intracellular ROS and decreasingmitochondrialmembrane potential Interestingly EECPhad little or small cytotoxicity on normal human umbilical vein endothelialcells (HUVECs) These results suggest that EECP is a potential alternative agent on breast cancer treatment

1 Introduction

Propolis a natural product and healthy food raw material iscollected by honeybees from various plants It has been usedsince ancient times for its widely biological activities such asantibacterial antiviral antioxidant anti-inflammatory andantitumor [1 2]

There are different types of propolis according to the loca-tion of its botanical origin and collecting season [3] Basedon Bankova classification there are six main types of propo-lis that is poplar propolis birch propolis Brazilian greenpropolis red propolis pacific propolis andCanarian propolis[4] Chinese propolis is mainly classified as poplar-type andthe predominant chemical compositions are flavonoids andphenolic compounds [5] It exerts various biological activitiesincluding antitumor

Recently antitumor properties of propolis the mostremarkable activity have been widely documented in various

culture cell lines such as human leukemia (HL-60 CI41U937) human colon cancer cells (SW480 HCT116) humancervical cancer (ME180 Hela) human glioblastoma cells(U87MG) human lung carcinoma (A549) human hepatocel-lular carcinoma (HepG2 SNU449) human pancreatic cancer(PANC-1 BxPC-3) cells and mammary carcinoma (MCF-7) [6ndash15] However the molecular mechanisms of Chinesepropolis on antitumor effects have not been fully elucidatedand need to be deeply clarified Here we investigated theanticancer effects of Chinese propolis in MCF-7 (humanbreast cancer ER(+)) and MDA-MB-231 (human breast can-cer ER (minus)) cells and then studied the underlying molecularmechanisms of Chinese propolis on both breast cancer cells

2 Materials and Methods

21 Materials DMEM was obtained from Gibco BRL Co(USA) Fetal bovine serum was from Hyclone Laboratories

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2014 Article ID 280120 11 pageshttpdxdoiorg1011552014280120

2 Evidence-Based Complementary and Alternative Medicine

Inc (USA) Acridine orange (AO) was fromAmresco (USA)Hoechst 33258 2101584071015840-dichlorodihydrofluorescin (DCHF) JC-1 propidium iodide (PI) and sulforhodamine B (SRB) werefrom Sigma Co (USA) The antibodies against p53 ANXA7NF-120581B p65 120573-actin and horseradish peroxidase-conjugatedsecondary antibodies were from Santa Cruz Biotechnology(USA) Secondary antibodies for immunofluorescence don-key anti-rabbit IgG Alexa Fluor-488 were purchased fromLife Technologies (USA) All the other reagents were ofultrapure grade

22 Preparation of Propolis Extracts Propolis used in thepresent study was Chinese propolis and had been used inour previous study [16] The main plant origin was poplar(Populus sp) Samples were maintained at minus20∘C beforeprocessing Propolis sample was extracted with 95 ethanolat room temperature for 24 h The ethanol suspension wasfiltered under reduced pressure The filter liquid was thenconcentrated in a rotary evaporator under reduced pressureat 40∘C until it reached a constant weight and was later redis-solved in ethanol The ethanol-extracted Chinese propolis(EECP) had a brown color The prepared propolis was storedunder a dry condition at 4∘C

To determine the constituents in EECP we used high-performance liquid chromatography (HPLC) analysis witha photodiode array (PDA) EECP was dissolved in ethanol(2mgmL) to the injection of 10 120583L into the HPLC systemThe HPLC system used was Agilent 1100 (Germany) witha C18 column (46 times 250mm id 5 120583m) at 28∘C with adetection wavelength at 280 nmThemobile phases consistedof methanol (A) and 01 phosphoric acid (B) at a flow rateof 10mLmin

23 Cell Cultures The human breast cancer cells MCF-7and MDA-MB-231 were purchased from American TypeCulture Collection (ATCC USA) Human umbilical veinendothelial cells (HUVECs) were gifted by AtherosclerosisResearch Institute of Taishan Medical University of Chinaand purchased from ATCC All cells were respectivelycultured in DMEM medium supplemented with 10 (vv)FBS and 100UmL of penicillin 100 120583gmL streptomycin at37∘C under humidified 95ndash5 (vv) air and CO

2

24 Cell Viability Assay Cells were seeded at the densityof 4 times 104mL into 96-well cell culture plates and weretreated with different concentrations of EECP (25 50 100and 200120583gmL) At 24 and 48 h cells were precipitated for1 h at 4∘C with 100 120583L 10 trichloroacetic acid and stainedwith SRB The optical density was measured at 492 nm afterreconstitution of the dye in 100 120583L 10mM Tris base Theviability () was expressed as (OD of treated groupOD ofcontrol group) times100 The viability of the control cells wasset to 100

25 Nuclear Fragmentation Assay Nuclear fragmentationwas detected by acridine orange staining Briefly at 48 hcells were stained with 5120583gmL AO at room temperature for

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Figure 1The constituents from ethanol-extracted Chinese propolis(EECP) 1 Caffeic acid 2 p-coumaric acid 3 ferulic acid 4pinobanksin 5 7-hydroxy-5-methoxyflavanone 6 kaempferol 7Pinocembrin 8 pinobanksin 3-acetate 9 chrysin 10 caffeic acidphenethyl ester 11 galangin 12 tectochrysin

5min and then were observed under a TE2000S fluorescencemicroscope (Nikon Japan)

26 Hoechst 33258 Staining Hoechst 33258 stainingwas usedto observe apoptotic morphology At 48 h cells in all groupswere stained with 10 120583gmL Hoechst 33258 for 15min Cellswere gentlywashedwith PBS onceNuclear condensation andfragmentation were observed under a TE2000S fluorescencemicroscope (Nikon Japan)

27 Wound-Healing Assay Cells were grown to 80 conflu-ence in a 24-well plate The monolayers were scratched witha plastic tip washed by PBS to remove floating cell debrisand then incubated in medium in the absence or presenceof different concentration of EECP for 48 h Cell migrationinto the wound surface was determined under a TE2000Sinverted microscope (Nikon Japan) Migrated cells acrossthe scratched lines were counted by Image-Pro Plus software(USA)

28 Immunofluorescence Microscopy Assay As described inthe previous report [17] MCF-7 and MDA-MB-231 cellstreated with EECP were fixed in 4 paraformaldehyde (wv)for 15min at room temperature and blocked in 5 donkeyserum (vv) and then incubated with primary antibodiesovernight at 4∘CThen cells were rinsed with 01M PBS threetimes and incubated with corresponding FITC-conjugatedsecondary antibodies 1 h at 37∘C Cells were then rinsedthree times with 01M PBS to eliminate the uncombinedsecondary antibody Nuclei were counterstained with PIA laser scanning confocal microscope (Olympus FV1200Japan) was used for fluorescence detection Analysis wasmade by the Image-Pro Plus software (USA) Images wererepresentative of three independent experiments

29 Western Blot Assay After treatment with EECP cellswere lysed in lysis buffer at ice Thirty micrograms of proteinwere separated by 12 SDS-PAGE and transferred onto

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Figure 2 EECP decreased MCF-7 and MDA-MB-231 cells proliferation but had littlesmall effect on normal HUVECs (a) EECP inhibitedMCF-7 proliferation at 24 and 48 h (b) EECP inhibited MDA-MB-231 cells proliferation at 24 and 48 h (c) EECP had little effect on normalHUVECs viability under the concentration of 100 120583gmL (lowast119875 lt 005 lowastlowast119875 lt 001 versus control 119899 = 3) Data are means plusmn SEM

polyvinylidene difluoride (PVDF) membrane The mem-brane was blocked with 5 (wv) nonfat dry milk in PBS-Tween 20 (PBST 005) for 1 h and was incubated withprimary antibody (1 1000 in PBST) at 4∘C overnight Afterthree washings in PBST the PVDF membrane was incu-bated with appropriate horseradish peroxidase-conjugatedsecondary antibodies (1 5000) for 1 h at room temperatureThe immunoreactive bands were developed with ECL west-ern blotting system The relative quantity of proteins wasanalyzed by use ofQuantityOne software (Bio-RadHerculesCA USA)

210 Determination of Intracellular ROS Levels IntracellularROS level was measured with 2101584071015840-dichlorodihydrofluores-cein (DCHF) which could be rapidly oxidized into the highlyfluorescent 2101584071015840-dichlorofluorescein (DCF) in the presenceof intracellular ROS [18]We treated cells as mentioned abovefor 48 h then washed cells with basal DMEM medium for5min and incubated the cells with DCHF 1mL at 37∘C for30min After washing the cells three times with basal DMEMmedium the fluorescence was monitored with a confocallaser scanning microscope (Olympus FV1200 Japan) usingexcitation and emission wavelengths of 488 nm The amountof ROS was quantified by Image-Pro Plus software (USA)

The images were representative of three independent experi-ments

211 Intracellular Mitochondrial Membrane Potential AssayFluorescence probe of JC-1 was used to test mitochondrialmembrane potential JC-1 exists as a monomer at low mito-chondrial membrane potential and emits green fluorescencebut forms aggregates and emits red fluorescence at highmito-chondrial membrane potential [19] Cells were treated for48 h then washed with basal DMEM medium for 5min andincubatedwith JC-1 1mL at 37∘C for 15min Afterwashing thecells three times with basal DMEMmedium the fluorescencewas monitored with a confocal laser scanning microscope(Olympus FV1200 Japan) using excitation and emissionwavelengths of 488 and 546 nm respectively Results wereshown by ratio of red to green fluorescence as comparedwith the control Image-Pro Plus software (USA) was used toanalysis fluorescence intensity

212 Statistical Analysis Data are fromat least three indepen-dent experiments and expressed as means plusmn SEM Statisticalanalysis involved the paired Student 119905 test and ANOVA withSPSS version 115 Differences were considered statisticallysignificant at 119875 lt 005


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Figure 3 EECP induced apoptosis in MCF-7 and MDA-MB-231 cells (a) Morphological changes of nuclei in MCF-7 cells by staining withacridine orange andHoechst 33258 at 48 h (times200) (b)Morphological changes of nuclei inMDA-MB-231 cells by stainingwith acridine orangeand Hoechst 33258 at 48 h (times200) (c) The levels of procaspase 3 (35KD) were detected by western blot at 24 h (d) The hemiquantification ofprocaspase 3 levels in MCF-7 and MDA-MB-231 cells (lowast119875 lt 005 lowastlowast119875 lt 001 versus control 119899 = 3)

3 Results

31 Constituents of EECP The main constituents identifiedin our sample are caffeic acid p-coumaric acid ferulic acidpinobanksin 7-hydroxy-5-methoxyflavanone kaempferolpinocembrin pinobanksin 3-acetate chrysin caffeic acidphenethyl ester galangin and tectochrysin by HPLC analysis(Figure 1)

32 EECP Decreased MCF-7 and MDA-MB-231 Cells Pro-liferation but Had Little Effect on Normal HUVECs Weinvestigated the sensitivity of MCF-7 and MDA-MB-231 cellsto EECP (25 50 100 and 200120583gmL) using SRB assay at24 and 48 h EECP significantly inhibited MCF-7 and MDA-MB-231 cells proliferation in a dose- and time-dependentmanner Notably the inhibitory effect of EECP on MDA-MB-231 cells was higher than on MCF-7 cells (lowast119875 lt 005lowastlowast119875 lt 001 Figures 2(a) and 2(b))We also investigated the effect of EECP on normal

HUVECs viability and the results showed that there was little

effect on proliferation of normal HUVECs under the con-centrations of 100 120583gmL However EECP at concentrationof 200120583gmL had small cytotoxicity (lowast119875 lt 005 Figure 2(c))

33 EECP Induced Apoptosis in MCF-7 and MDA-MB-231Cells Acridine orange and Hoechst 33258 staining resultsindicated that different concentrations of EECP evidentlyinduced nuclear condensation and fragmentation of MCF-7 and MDA-MB-231 cells in a dose-dependent mannerImportantly nuclei of MDA-MB-231 cells treated with EECP200120583gmL were almost fragmentation (Figures 3(a) and3(b)) In addition we also tested procaspase 3 (35KD) bywestern blot at 24 h The results showed that caspase 3 wasactivated in MCF-7 and MDA-MB-231 cells treated withEECP (Figures 3(c) and 3(d))

34 EECP Inhibited MDA-MB-231 Cells Migration Roughly70 of all patients dying of breast cancer have bone metas-tases [20] Therefore we also performed wounding-healingexperiments to detect the effect of EECP on migration of


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Figure 4 EECP inhibited MDA-MB-231 cells migration (a) Cell migration micrographs obtained under a phase contrast microscope at 024 and 48 h (times100) (b) Relative levels of cell migration (lowastlowast119875 lt 001 versus control 119899 = 3)

MCF-7 andMDA-MB-231 cells EECP significantly inhibitedMDA-MB-231 cells migration in a dose-dependent mannerat 48 h (lowastlowast119875 lt 001 Figure 4) whereas inhibitory effect ofEECP on migration of MCF-7 cells was not significant (datawere not shown)

35 EECP Regulated the Levels of ANXA7 p53 and NF-120581Bp65 in MCF-7 and MDA-MB-231 Cells EECP significantlyupregulated the expression of ANXA7 and downregulatedNF-120581B p65 level in a dose-dependent manner by westernblot and immunofluorescent assay in MCF-7 and MDA-MB-231 cells Furthermore the translocation of NF-120581B p65 fromcytoplasm to nuclei was also inhibited by EECP in bothcells The effect of EECP on p53 level in MCF-7 and inMDA-MB-231 cells was different p53 level was significantlyincreased in MCF-7 cells However in MDA-MB-231 cellsEECP evidently inhibited p53 level (lowast119875 lt 005 lowastlowast119875 lt 001Figures 5 and 6)

36 EECP Increased ROS Level in MCF-7 and MDA-MB-231 Cells EECP at concentration of 25ndash200120583gmL signif-icantly increased ROS level in MCF-7 cells whereas inMDA-MB-231 cells EECP at concentration of 50ndash200120583gmL

evidently increased ROS level (lowast119875 lt 005 lowastlowast119875 lt 001Figure 7)

37 EECP Reduced Mitochondrial Membrane Potential inMCF-7 and MDA-MB-231 Cells The mitochondrial mem-brane potential sensor JC-1 was used to determine the mito-chondrial function As shown in Figure 7 EECP significantlydecreased mitochondrial membrane potential in a dose-dependent manner inMCF-7 andMDA-MB-231 cells at 48 hNote that the decreased level of mitochondrial membranepotential inMDA-MB-231 cells was higher than that inMCF-7 cells (lowastlowast119875 lt 001 Figure 8)

4 Discussion

In propolis there is usually a variety of chemical compoundssuch as polyphenols terpenoids steroids and amino acidPropolis samples obtained from different plants are com-posed of different chemical compounds Chinese propolisis mainly classified as poplar-type and the predominantchemical constituents are flavonoids and phenolic com-pounds and their percentage ranges from 35 to 50[21] Our results from HPLC also indicate that the major


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Figure 5 EECP regulated the levels of ANXA7 p53 and NF-120581B p65 in MCF-7 cells (a) Fluorescent micrographs obtained at 48 h (times400)Nuclei were counterstained with PI (b) and (c)The relative fluorescence intensity of NF-120581B p65 and p53 inMCF-7 cells (d)The levels of p53ANXA7 and NF-120581B p65 were detected by western blot at 48 h (e) (f) and (g)The hemiquantification of p53 ANXA7 and NF-120581B p65 levelsin MCF-7 cells (lowast119875 lt 005 lowastlowast119875 lt 001 versus control 119899 = 3)


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Figure 6 EECP regulated the levels of ANXA7 p53 and NF-120581B p65 in MDA-MB-231 cells (a) Fluorescent micrographs obtained at 48 h(times400) (b) and (c)The relative fluorescence intensity of NF-120581B p65 and p53 in MDA-MB-231 cells (d)The levels of ANXA7 and NF-120581B p65were detected by western blot at 48 h the level of p53 was detected by western blot at 24 h (e) (f) and (g) The hemiquantification of p53ANXA7 and NF-120581B p65 levels in MDA-MB-231 cells (lowast119875 lt 005 lowastlowast119875 lt 001 versus control 119899 = 3)


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Figure 7 EECP increased ROS level in MCF-7 andMDA-MB-231 cells (a) Fluorescent micrographs of MCF-7 cells obtained at 48 h (times200)(b) Relative quantity of ROS inMCF-7 cells (c) Fluorescentmicrographs ofMDA-MB-231 cells obtained at 48 h (times200) (d) Relative quantity ofROS inMDA-MB-231 cells Values represent the relative fluorescent intensity determined by laser scanning confocal microscopy (lowast119875 lt 005lowastlowast119875 lt 001 versus control 119899 = 3)

chemical constituents of EECP are polyphenolicflavonoidsAnd caffeic acid phenethyl ester caffeic acid galanginchrysin kaempferol pinobanksin and pinocembrin are themajor compounds Furthermore accumulating evidence hasindicated that polyphenolicflavonoids may serve as a potentadjunct to chemotherapy and radiotherapy in the treatmentof cancers [22ndash24]

Breast cancer ranks among the most common malignanttumors afflicting women worldwide [25] In this study weinvestigated the antitumor activities of EECP in MCF-7and MDA-MB-231 cells Our results showed that EECPpotentially exerted its antitumor effect by inhibiting cellproliferation inducing apoptosis inhibiting cell migrationregulating ANXA7 and p53 levels downregulating NF-120581Bp65 level and inhibiting its translocation from cytoplasmto nuclei and increasing intracellular ROS level decreasingmitochondrial membrane potential Besides these EECP hadlittle or small effect on normalHUVECs InterestinglyMDA-MB-231 cells were more sensitive to EECP than MCF-7 cells

The vascular endothelium cells play a critical role in thephysiological and pathological progress for their locationbetween the intravascular compartment and extravasculartissues [17] Endothelial cells are the primary target for manychemical agents Many anticancer chemical agents cannot beused in clinic for their cytotoxicity on endothelial cells Herewe found that EECP under concentration 100 120583gmL hadgood antitumor activity but had little cytotoxicity on normalHUVECs and concentration of EECP 200 120583gmL had somesmall toxicity on HUVECs which indicated the low toxicityof EECP when used as anticancer agent

Cancer metastasis is the leading cause of mortality inpatients with breast cancer Metastasis is multistep processMDA-MB-231 breast cancer cells a highly metastatic humanbreast carcinoma cell line are widely used as a model tostudy breast cancer cell metastasis Here we found thatEECP 25ndash200120583gmL remarkably inhibited MDA-MB-231cells migration which indicated the good ability of EECP oninhibiting breast cancer cells metastasis

ANXA7 a member of the annexin family of calcium-dependent phospholipid binding proteins codes for Ca2+dependent GTPase which involves several different roles inautophagy exocytosis carcinogenesis and tumor suppres-sion [26ndash28] Recently it is described as a candidate tumorsuppressor gene for prostate cancer [29] HumanANXA7 hasbeen mapped to tumor susceptibility locus 10q21 with 35loss of heterozygosity in prostate and breast cancer indicatingits possible tumor suppressive function [30] Srivastava et al(2001) indicated that ANXA7 could be a biomarker in theprogression of breast cancer [31] In present study we foundthat EECP significantly upregulated ANXA7 level in MCF-7and MDA-MB-231 cells This is the first time indicating theeffect of propolis on ANXA7 in breast cancer cells whichmight be a new target of propolis on antitumor study andtreatment

p53 another tumor suppressor protein is a central targetof inactivation in human cancer and a key regulator ofgenotoxic stress-induced growth arrest or apoptosis [32] Wepreviously reported that both Chinese propolis and Braziliangreen propolis affected p53 level in HUVECs with nutritiondeprivation [16 33] MCF-7 has a wild-type p53 here we


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Figure 8 EECP reduced mitochondrial membrane potential in MCF-7 and MDA-MB-231 cells (a) Fluorescent micrographs of MCF-7 cellsobtained at 48 h (times400) (b) Relative quantity of mitochondrial membrane potential in MCF-7 cells (c) Fluorescent micrographs of MDA-MB-231 cells obtained at 48 h (times400) (d) Relative quantity of mitochondrial membrane potential in MDA-MB-231 cells Values represent therelative fluorescent intensity determined by laser scanning confocal microscopy (lowastlowast119875 lt 001 versus control 119899 = 3)

found that EECP higher 25 120583gmL significantly upregulatedp53 level to induce apoptosis However MDA-MB-231 hasa high level of a mutant p53 which contributes to thesuppression of apoptosis in human breast cancer cells Incurrent study we found that the effect of EECP on mutant-type p53 was complex and was dose-dependent p53 levelwas inhibited with the increase of concentration by westernblot assay However these changes could not be tested byimmunofluorescencemicroscopy assay at 24 h for there was ahigh level of mutant p53-expressing in MDA-MB-231 Takentogether EECP exerts its antitumor effect through regulatingp53 level

The NF-120581B signal transduction pathway is deregulatedin a variety of human cancers [34 35] In most types ofcancer cells NF-120581B is constitutively active Blocking NF-120581Bhas been shown to stop tumor cells from proliferating todie or to become more sensitive to the action of antitumoragents especially antioxidants [36]Therefore agents capableof downmodulating the activation of NF-120581B have a potentialfor use in therapeutic interventions [37] Here we found thatEECP downregulated the activation of NF-120581B p65 a subunitof NF-120581B and inhibited its translocation from cytoplasmto nuclear to activate in MCF-7 and MDA-MB-231 cellswhich indicated that EECP could become a useful antitumoragent

Accumulating evidence has demonstrated that ROS areimportant signals in the regulation of diverse cellular func-tions High levels of ROS induce oxidative stress leading to anumber of different diseases including cancer [38] Howeverrecent studies indicated that high levels ROS induce apoptosisby triggering proapoptotic signaling molecules to antitumor

[39] And we previously found that a high concentration ofBrazilian propolis extract induced HUVECs apoptosis withROS level increase and at a low concentration propolis pro-tected HUVECs by decreasing ROS level [33] These findingstaken together it appears that propolis plays a dual role onROS depending on concentrations at high concentrationit exerts a prooxidant effect at low concentration it canalso act as an antioxidant by scavenging free radicals Inpresent study EECP induced MCF-7 andMDA-MB-231 cellsapoptosis with ROS increase in a dose-dependent mannerEECP may therefore exert prooxidant effect in breast cancercells

The decrease of mitochondrial membrane potentialwould lead to the release of cytochrome c to activate caspaseto initiate apoptotic signaling pathway [40] Mitochondriaare the most important intracellular source of ROS andelevated ROS levels can also decrease mitochondrial mem-brane potential [33] Here we found that EECP decreasedmitochondrial membrane potential in MCF-7 and MDA-MB-231 cells From this viewpoint we deduced that EECPinduced apoptosis in MCF-7 and MDA-MB-231 cells wereROS-dependent mitochondrial pathway

In conclusion our results suggest that EECP and itspolyphenolicflavonoid components exert antitumor effectsmainly through inducing apoptosis of breast cancer cellsTheinvolved mechanisms commonly contain ANXA7 and p53proteins regulating NF-120581B inhibition and regulation of ROSand mitochondrial membrane potential Attractively EECPhas non-low toxicity to normal cells because of its selectivetoxicities to tumor cells So it is believed that propolis maybecome an attractive and promising agent for breast cancer


treatment However further research is needed to clarifyprecise targets of propolis in breast cancer cells

Conflict of Interests

The authors declare no conflict of interest regarding thepublication of this paper

Authorsrsquo Contribution

Hongzhuan Xuan and Zhen Li contributed equally to thiswork

Acknowledgments

This work was supported by the Grants from the NationalNatural Science Foundation of China (nos 31201860 and31272512) Shandong Provincial Natural Science Foundationof China (no ZR2012CQ003) the Modern AgroindustryTechnology Research System from the Ministry of Agricul-ture of China (CARS-45) and theCollege Student InnovationFoundation of Liaocheng University China (nos SF2013279SF2013282 and SF2013292)

References

[1] J M Sforcin and V Bankova ldquoPropolis is there a potential forthe development of new drugsrdquo Journal of Ethnopharmacologyvol 133 no 2 pp 253ndash260 2011

[2] V C Toreti H H Sato G M Pastore and Y K Park ldquoRecentprogress of propolis for its biological and chemical composi-tions and its botanical originrdquo Evidence-Based Complementaryand Alternative Medicine vol 2013 Article ID 697390 13 pages2013

[3] V Bankova ldquoChemical diversity of propolis and the problem ofstandardizationrdquo Journal of Ethnopharmacology vol 100 no 1-2 pp 114ndash117 2005

[4] V Bankova ldquoRecent trends and important developments inpropolis researchrdquo Evidence-Based Complementary and Alter-native Medicine vol 2 no 1 pp 29ndash32 2005

[5] L P Sun A L Chen H C Hung et al ldquoChrysin a histonedeacetylase 8 inhibitor with anticancer activity and a suitablecandidate for the standardization of Chinese propolisrdquo Journalof Agricultural and Food Chemistry vol 60 pp 11748ndash117582012

[6] K Aso S Kanno T Tadano S Satoh and M IshikawaldquoInhibitory effect of propolis on the growth of human leukemiaU937rdquo Biological and Pharmaceutical Bulletin vol 27 no 5 pp727ndash730 2004

[7] S Mishima Y Narita S Chikamatsu et al ldquoEffects of propolison cell growth and gene expression in HL-60 cellsrdquo Journal ofEthnopharmacology vol 99 no 1 pp 5ndash11 2005

[8] S M Alencar T L C Oldoni M L Castro et al ldquoChemicalcomposition and biological activity of a new type of Brazilianpropolis red propolisrdquo Journal of Ethnopharmacology vol 113no 2 pp 278ndash283 2007

[9] S Awale F Li H Onozuka H Esumi Y Tezuka and S KadotaldquoConstituents of Brazilian red propolis and their preferentialcytotoxic activity against human pancreatic PANC-1 cancer cell

line in nutrient-deprived conditionrdquo Bioorganic and MedicinalChemistry vol 16 no 1 pp 181ndash189 2008

[10] S M Messerli M Ahn K Kunimasa et al ldquoArtepillin C (ARC)in Brazilian green propolis selectively blocks oncogenic PAK1signaling and suppresses the growth of NF tumors in micerdquoPhytotherapy Research vol 23 no 3 pp 423ndash427 2009

[11] C N Chen C J Hsiao S S Lee et al ldquoChemical modificationand anticancer effect of prenylated flavanones from Taiwanesepropolisrdquo Natural Product Research vol 26 pp 116ndash124 2012

[12] G C Chan K W Cheung and D M Sze ldquoThe immunomod-ulatory and anticancer properties of propolisrdquo Clinical Reviewsin Allergy amp Immunology vol 44 pp 262ndash273 2013

[13] T K Ha M E Kim J H Yoon S J Bae J Yeom and J SLee ldquoGalangin induces human colon cancer cell death via themitochondrial dysfunction and caspase-dependent pathwayrdquoExperimental Biology and Medicine vol 238 pp 1047ndash10542013

[14] R Markiewicz-Zukowska M H Borawska A FiedorowiczS K Naliwajko D Sawicka and H Car ldquoPropolis changesthe anticancer activity of temozolomide in U87MG humanglioblastoma cell linerdquo BMC Complementary and AlternativeMedicine vol 13 article 50 2013

[15] F Yang H Jin J Pi et al ldquoAnti-tumor activity evaluation ofnovel chrysin-organogermanium(IV) complex inMCF-7 cellsrdquoBioorganic amp Medicinal Chemistry Letters vol 23 pp 5544ndash5551 2013

[16] H Xuan R Zhu Y Li and F Hu ldquoInhibitory effect ofchinese propolis on phosphatidylcholine-specific phospholi-pase C activity in vascular endothelial cellsrdquo Evidence-BasedComplementary and Alternative Medicine vol 2011 Article ID985278 8 pages 2011

[17] N Meng L Wu J Gao et al ldquoLipopolysaccharide inducesautophagy through BIRC2 in human umbilical vein endothelialcellsrdquo Journal of Cellular Physiology vol 225 no 1 pp 174ndash1792010

[18] N SuematsuH Tsutsui JWen et al ldquoOxidative stressmediatestumor necrosis factor-120572-induced mitochondrial DNA damageand dysfunction in cardiac myocytesrdquo Circulation vol 107 no10 pp 1418ndash1423 2003

[19] H Izuta M Shimazawa S Tazawa Y Araki S Mishimaand H Hara ldquoProtective effects of Chinese propolis and itscomponent chrysin against neuronal cell death via inhibitionof mitochondrial apoptosis pathway in SH-SY5Y cellsrdquo Journalof Agricultural and Food Chemistry vol 56 no 19 pp 8944ndash8953 2008

[20] A Jemal R Siegel E Ward Y Hao J Xu and M J ThunldquoCancer statistics 2009rdquo CA Cancer Journal for Clinicians vol59 no 4 pp 225ndash249 2009

[21] N Orsolic and I Basic ldquoCancer chemoprevention by propolisand its polyphenolic compounds in experimental animalsrdquoRecent Progress in Medicinal Plants vol 17 pp 55ndash113 2007

[22] D Lamoral-Theys L Pottier F Dufrasne et al ldquoNaturalpolyphenols that display anticancer properties through inhibi-tion of kinase activityrdquo Current Medicinal Chemistry vol 17 no9 pp 812ndash825 2010

[23] N Orsolic A Horvat Knezevic L Sver S Terzic and I BasicldquoImmunomodulatory and antimetastatic action of propolis andrelated polyphenolic compoundsrdquo Journal of Ethnopharmacol-ogy vol 94 pp 307ndash315 2004

[24] N Orsolic ldquoA review of propolis antitumor action in vivo andin vitrordquo Journal of ApiProduct and ApiMedical Science vol 2no 1 pp 1ndash20 2010


[25] L Wang Z Dong B Huang et al ldquoDistinct patterns of auto-phagy evoked by two benzoxazine derivatives in vascular endo-thelia cellsrdquo Autophagy vol 6 no 8 pp 1115ndash1124 2010

[26] H Li S Huang S Wang et al ldquoRelationship between annexinA7 and integrin 1205734 in autophagyrdquo The International Journal ofBiochemistry amp Cell Biology vol 45 no 11 pp 2605ndash2611 2013

[27] H Li S Huang S Wang et al ldquoTargeting annexin A7 by asmall molecule suppressed the activity of phosphatidylcholine-specific phospholipase C in vascular endothelial cells andinhibited atherosclerosis in apolipoprotein E(minus)(minus) icerdquo CellDeath amp Disease vol 4 article e806 2013

[28] H Li N Liu S Wang et al ldquoIdentification of a small moleculetargeting annexin A7rdquo Biochimica et Biophysica Acta vol 1833pp 2092ndash2099 2013

[29] M Srivastava L Bubendorf V Srikantan et al ldquoAnx7 a candi-date tumor suppressor gene for prostate cancerrdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 98 no 8 pp 4575ndash4580 2001

[30] M Srivastava C Montagna X Leighton et al ldquoHaploinsuffi-ciency of Anx7 tumor suppressor gene and consequent genomicinstability promotes tumorigenesis in the Anx7(+minus) mouserdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 100 no 2 pp 14287ndash14292 2003

[31] M Srivastava L Bubendorf L Nolan et al ldquoANX7 as a bio-marker in prostate and breast cancer progressionrdquo DiseaseMarkers vol 17 no 2 pp 115ndash120 2001

[32] O Karni-Schmidt A Zupnick M Castillo et al ldquop53 islocalized to a sub-nucleolar compartment after proteasomalinhibition in an energy-dependent mannerrdquo Journal of CellScience vol 121 no 24 pp 4098ndash4105 2008

[33] H Xuan J Zhao J Miao Y Li Y Chu and F Hu ldquoEffectof Brazilian propolis on human umbilical vein endothelial cellapoptosisrdquo Food and Chemical Toxicology vol 49 no 1 pp 78ndash85 2011

[34] T Lu andG R Stark ldquoCytokine overexpression and constitutiveNF120581B in cancerrdquo Cell Cycle vol 3 no 9 pp 1114ndash1117 2004

[35] X Zhang B Jin and C Huang ldquoThe PI3KAkt pathway andits downstream transcriptional factors as targets for chemopre-ventionrdquo Current Cancer Drug Targets vol 7 no 4 pp 305ndash3162007

[36] R O Escarcega S Fuentes-Alexandro M Garcıa-Carrasco AGatica andA Zamora ldquoThe transcription factor nuclear factor-kappaB and cancerrdquoClinical Oncology vol 19 no 2 pp 154ndash1612007

[37] Y K Choi S G Cho S M Woo et al ldquoSaussurea lappa clarke-derived costunolide prevents TNF alpha-induced breast cancercell migration and invasion by inhibiting NF-kappa B activityrdquoEvidence-Based Complementary and Alternative Medicine vol2013 Article ID 936257 10 pages 2013

[38] P Storz ldquoReactive oxygen species in tumor progressionrdquo Fron-tiers in Bioscience vol 10 no 2 pp 1881ndash1896 2005

[39] YWang Q Tang S Jiang M Li and XWang ldquoAnti-colorectalcancer activity of macrostemonoside A mediated by reactiveoxygen speciesrdquo Biochemical and Biophysical Research Com-munications vol 441 no 4 pp 825ndash830 2013

[40] R Singh N K Avliyakulov M Braga et al ldquoProteomic iden-tification of mitochondrial targets of arginase in human breastcancerrdquo PLoS ONE vol 8 Article ID e79242 2013

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


Inc (USA) Acridine orange (AO) was fromAmresco (USA)Hoechst 33258 2101584071015840-dichlorodihydrofluorescin (DCHF) JC-1 propidium iodide (PI) and sulforhodamine B (SRB) werefrom Sigma Co (USA) The antibodies against p53 ANXA7NF-120581B p65 120573-actin and horseradish peroxidase-conjugatedsecondary antibodies were from Santa Cruz Biotechnology(USA) Secondary antibodies for immunofluorescence don-key anti-rabbit IgG Alexa Fluor-488 were purchased fromLife Technologies (USA) All the other reagents were ofultrapure grade

22 Preparation of Propolis Extracts Propolis used in thepresent study was Chinese propolis and had been used inour previous study [16] The main plant origin was poplar(Populus sp) Samples were maintained at minus20∘C beforeprocessing Propolis sample was extracted with 95 ethanolat room temperature for 24 h The ethanol suspension wasfiltered under reduced pressure The filter liquid was thenconcentrated in a rotary evaporator under reduced pressureat 40∘C until it reached a constant weight and was later redis-solved in ethanol The ethanol-extracted Chinese propolis(EECP) had a brown color The prepared propolis was storedunder a dry condition at 4∘C

To determine the constituents in EECP we used high-performance liquid chromatography (HPLC) analysis witha photodiode array (PDA) EECP was dissolved in ethanol(2mgmL) to the injection of 10 120583L into the HPLC systemThe HPLC system used was Agilent 1100 (Germany) witha C18 column (46 times 250mm id 5 120583m) at 28∘C with adetection wavelength at 280 nmThemobile phases consistedof methanol (A) and 01 phosphoric acid (B) at a flow rateof 10mLmin

23 Cell Cultures The human breast cancer cells MCF-7and MDA-MB-231 were purchased from American TypeCulture Collection (ATCC USA) Human umbilical veinendothelial cells (HUVECs) were gifted by AtherosclerosisResearch Institute of Taishan Medical University of Chinaand purchased from ATCC All cells were respectivelycultured in DMEM medium supplemented with 10 (vv)FBS and 100UmL of penicillin 100 120583gmL streptomycin at37∘C under humidified 95ndash5 (vv) air and CO

2

24 Cell Viability Assay Cells were seeded at the densityof 4 times 104mL into 96-well cell culture plates and weretreated with different concentrations of EECP (25 50 100and 200120583gmL) At 24 and 48 h cells were precipitated for1 h at 4∘C with 100 120583L 10 trichloroacetic acid and stainedwith SRB The optical density was measured at 492 nm afterreconstitution of the dye in 100 120583L 10mM Tris base Theviability () was expressed as (OD of treated groupOD ofcontrol group) times100 The viability of the control cells wasset to 100

25 Nuclear Fragmentation Assay Nuclear fragmentationwas detected by acridine orange staining Briefly at 48 hcells were stained with 5120583gmL AO at room temperature for

12

3

4

5

6

78

9

1011

12

0060

0050

0040

0030

0020

0010

0000

minus0010

000 500 1000 1500 2000 2500 3000 3500 4000 4500 5000

Time (min)

AU

Figure 1The constituents from ethanol-extracted Chinese propolis(EECP) 1 Caffeic acid 2 p-coumaric acid 3 ferulic acid 4pinobanksin 5 7-hydroxy-5-methoxyflavanone 6 kaempferol 7Pinocembrin 8 pinobanksin 3-acetate 9 chrysin 10 caffeic acidphenethyl ester 11 galangin 12 tectochrysin

5min and then were observed under a TE2000S fluorescencemicroscope (Nikon Japan)

26 Hoechst 33258 Staining Hoechst 33258 stainingwas usedto observe apoptotic morphology At 48 h cells in all groupswere stained with 10 120583gmL Hoechst 33258 for 15min Cellswere gentlywashedwith PBS onceNuclear condensation andfragmentation were observed under a TE2000S fluorescencemicroscope (Nikon Japan)

27 Wound-Healing Assay Cells were grown to 80 conflu-ence in a 24-well plate The monolayers were scratched witha plastic tip washed by PBS to remove floating cell debrisand then incubated in medium in the absence or presenceof different concentration of EECP for 48 h Cell migrationinto the wound surface was determined under a TE2000Sinverted microscope (Nikon Japan) Migrated cells acrossthe scratched lines were counted by Image-Pro Plus software(USA)

28 Immunofluorescence Microscopy Assay As described inthe previous report [17] MCF-7 and MDA-MB-231 cellstreated with EECP were fixed in 4 paraformaldehyde (wv)for 15min at room temperature and blocked in 5 donkeyserum (vv) and then incubated with primary antibodiesovernight at 4∘CThen cells were rinsed with 01M PBS threetimes and incubated with corresponding FITC-conjugatedsecondary antibodies 1 h at 37∘C Cells were then rinsedthree times with 01M PBS to eliminate the uncombinedsecondary antibody Nuclei were counterstained with PIA laser scanning confocal microscope (Olympus FV1200Japan) was used for fluorescence detection Analysis wasmade by the Image-Pro Plus software (USA) Images wererepresentative of three independent experiments

29 Western Blot Assay After treatment with EECP cellswere lysed in lysis buffer at ice Thirty micrograms of proteinwere separated by 12 SDS-PAGE and transferred onto


020406080

100120

Control 25 50 100 200

MCF-7

24h48h


lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast


lowast

Cel

l via

bilit

y (

of c

ontro

l)

(a)

020406080

100120

25 50 100 200

MDA-MB-231

Control

24h48h


lowast

lowastlowast


lowastlowast

lowastlowast

lowastlowast

Cel

l via

bilit

y (

of c

ontro

l)

(b)

020406080

100120

25 50 100 200Control

24h48h


lowast

lowastlowast

HUVEC

Cel

l via

bilit

y (

of c

ontro

l)

(c)








25 50 100 200Control

MCF-7

48h

48h

(120583gmL)

(a)

25 50 100 200Control

MDA-MB-231

48h

48h

(120583gmL)

(b)

25 50 100 200Control


Procaspase 3

120573-Actin

120573-Actin

MCF-7(120583gmL)

(c)

002040608

112

25 50 100 200Control(120583gmL)

The r

elat

ive l

evel

of

proc

aspa

se3

120573-a

ctin

002040608

112

25 50 100 200Control(120583gmL)

The r

elat

ive l

evel

of

proc

aspa

se3

120573-a

ctin

MCF-7

lowastlowast


lowastlowastlowast

MDA-MB-231

(d)


3 Results








48h

24h

0h

25 50 100 200(120583gmL)

Control

(a)

0010203040506070809

The r

elat

ive l

evel

of c

ell

mig

ratio

n

25 50 100 200Control

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(120583gmL)

(b)







4 Discussion



25 50 100 200(120583gmL)

Control

Nuclei

Merge

p53

NF-120581B p65

MCF-7

(a)

005

115

225

335

4

25 50 100 200

Relat

ive fl

uore

scen

t in

tens

ity o

f p53

(120583gmL)Control

lowastlowast


lowastlowast

MCF-7

(b)

0

05

1

15

2

25

25 50 100 200(120583gmL)

Control



Relat

ive fl

uore

scen

tin

tens

ity o

f NF-120581

B p6

5MCF-7

(c)

p53

120573-Actin

ANXA7

25 50 100 200(120583gmL)

Control

NF-120581B p65

(d)

005

115

225

25 50 100 200(120583gmL)

Control


lowast

The r

elat

ive l

evel

of

p53

120573-a

ctin

(e)

0

05

1

15

2

25 50 100 200(120583gmL)

Control


The r

elat

ive l

evel

of

AN

XA7

120573-a

ctin

(f)

002040608

112

25 50 100 200(120583gmL)

Control


lowastlowast

The r

elat

ive l

evel

of

NF-120581

B p6

5120573

-act

in

(g)



Nuclei

Merge

p53

NF-120581B p65

25 50 100 200(120583gmL)

Control

MDA-MB-231

(a)

0123456789

10

25 50 100 200

Relat

ive fl

uore

scen

t in

tens

ity o

f p53

(120583gmL)Control

MDA-MB-231

(b)

0

05

1

15

2

25

25 50 100 200(120583gmL)

Control

Relat

ive fl

uore

scen

tin

tens

ity N

F-120581

B p6

5



MDA-MB-231

(c)

p53

120573-Actin

120573-Actin

ANXA7

NF-120581B p65

25 50 100 200(120583gmL)

Control

(d)

002040608

112

25 50 100 200(120583gmL)

Control

lowastlowastlowast

The r

elat

ive l

evel

of

p53

120573-a

ctin

(e)

002040608

112141618

2

25 50 100 200(120583gmL)

Control

lowastlowast

lowastlowast

lowastlowast

The r

elat

ive l

evel

of

AN

XA7

120573-a

ctin

(f)

002040608

112

25 50 100 200(120583gmL)

Control

lowastlowast

lowast

lowast

The r

elat

ive l

evel

of

NF-120581

B p6

5120573

-act

in

(g)



25 50 100 200Control(120583gmL)

48h

MCF-7

(a)

02468

10121416

25 50 100 200Control

MCF-7

lowast lowast

lowastlowast

lowastlowast

The r

elat

ive l

evel

of R

OS

(120583gmL)

(b)

25 50 100 200Control

48h

(120583gmL)MDA-MB-231

(c)

0123456

25 50 100 200

The r

elat

ive l

evel

of R

OS

Control


lowast

(120583gmL)

MDA-MB-231

(d)









25 50 100 200(120583gmL)

Control

48h

MCF-7

(a)

020

040608

1121416

25 50 100 200

The r

elat

ive l

evel

of

Control

mito

chon

dria

mem

bran

e pot

entia

l MCF-7




(b)

48h

25 50 100 200(120583gmL)

Control

MDA-MB-231

(c)

005

115

225

3

25 50 100 200Control


The r

elat

ive l

evel

of

mito

chon

dria

mem

bran

e pot

entia

l MDA-MB-231


(d)














Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















020406080

100120

Control 25 50 100 200

MCF-7

24h48h


lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast


lowast

Cel

l via

bilit

y (

of c

ontro

l)

(a)

020406080

100120

25 50 100 200

MDA-MB-231

Control

24h48h


lowast

lowastlowast


lowastlowast

lowastlowast

lowastlowast

Cel

l via

bilit

y (

of c

ontro

l)

(b)

020406080

100120

25 50 100 200Control

24h48h


lowast

lowastlowast

HUVEC

Cel

l via

bilit

y (

of c

ontro

l)

(c)








25 50 100 200Control

MCF-7

48h

48h

(120583gmL)

(a)

25 50 100 200Control

MDA-MB-231

48h

48h

(120583gmL)

(b)

25 50 100 200Control


Procaspase 3

120573-Actin

120573-Actin

MCF-7(120583gmL)

(c)

002040608

112

25 50 100 200Control(120583gmL)

The r

elat

ive l

evel

of

proc

aspa

se3

120573-a

ctin

002040608

112

25 50 100 200Control(120583gmL)

The r

elat

ive l

evel

of

proc

aspa

se3

120573-a

ctin

MCF-7

lowastlowast


lowastlowastlowast

MDA-MB-231

(d)


3 Results








48h

24h

0h

25 50 100 200(120583gmL)

Control

(a)

0010203040506070809

The r

elat

ive l

evel

of c

ell

mig

ratio

n

25 50 100 200Control

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(120583gmL)

(b)







4 Discussion



25 50 100 200(120583gmL)

Control

Nuclei

Merge

p53

NF-120581B p65

MCF-7

(a)

005

115

225

335

4

25 50 100 200

Relat

ive fl

uore

scen

t in

tens

ity o

f p53

(120583gmL)Control

lowastlowast


lowastlowast

MCF-7

(b)

0

05

1

15

2

25

25 50 100 200(120583gmL)

Control



Relat

ive fl

uore

scen

tin

tens

ity o

f NF-120581

B p6

5MCF-7

(c)

p53

120573-Actin

ANXA7

25 50 100 200(120583gmL)

Control

NF-120581B p65

(d)

005

115

225

25 50 100 200(120583gmL)

Control


lowast

The r

elat

ive l

evel

of

p53

120573-a

ctin

(e)

0

05

1

15

2

25 50 100 200(120583gmL)

Control


The r

elat

ive l

evel

of

AN

XA7

120573-a

ctin

(f)

002040608

112

25 50 100 200(120583gmL)

Control


lowastlowast

The r

elat

ive l

evel

of

NF-120581

B p6

5120573

-act

in

(g)



Nuclei

Merge

p53

NF-120581B p65

25 50 100 200(120583gmL)

Control

MDA-MB-231

(a)

0123456789

10

25 50 100 200

Relat

ive fl

uore

scen

t in

tens

ity o

f p53

(120583gmL)Control

MDA-MB-231

(b)

0

05

1

15

2

25

25 50 100 200(120583gmL)

Control

Relat

ive fl

uore

scen

tin

tens

ity N

F-120581

B p6

5



MDA-MB-231

(c)

p53

120573-Actin

120573-Actin

ANXA7

NF-120581B p65

25 50 100 200(120583gmL)

Control

(d)

002040608

112

25 50 100 200(120583gmL)

Control

lowastlowastlowast

The r

elat

ive l

evel

of

p53

120573-a

ctin

(e)

002040608

112141618

2

25 50 100 200(120583gmL)

Control

lowastlowast

lowastlowast

lowastlowast

The r

elat

ive l

evel

of

AN

XA7

120573-a

ctin

(f)

002040608

112

25 50 100 200(120583gmL)

Control

lowastlowast

lowast

lowast

The r

elat

ive l

evel

of

NF-120581

B p6

5120573

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in

(g)



25 50 100 200Control(120583gmL)

48h

MCF-7

(a)

02468

10121416

25 50 100 200Control

MCF-7

lowast lowast

lowastlowast

lowastlowast

The r

elat

ive l

evel

of R

OS

(120583gmL)

(b)

25 50 100 200Control

48h

(120583gmL)MDA-MB-231

(c)

0123456

25 50 100 200

The r

elat

ive l

evel

of R

OS

Control


lowast

(120583gmL)

MDA-MB-231

(d)









25 50 100 200(120583gmL)

Control

48h

MCF-7

(a)

020

040608

1121416

25 50 100 200

The r

elat

ive l

evel

of

Control

mito

chon

dria

mem

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(b)

48h

25 50 100 200(120583gmL)

Control

MDA-MB-231

(c)

005

115

225

3

25 50 100 200Control


The r

elat

ive l

evel

of

mito

chon

dria

mem

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entia

l MDA-MB-231


(d)














Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















25 50 100 200Control

MCF-7

48h

48h

(120583gmL)

(a)

25 50 100 200Control

MDA-MB-231

48h

48h

(120583gmL)

(b)

25 50 100 200Control


Procaspase 3

120573-Actin

120573-Actin

MCF-7(120583gmL)

(c)

002040608

112

25 50 100 200Control(120583gmL)

The r

elat

ive l

evel

of

proc

aspa

se3

120573-a

ctin

002040608

112

25 50 100 200Control(120583gmL)

The r

elat

ive l

evel

of

proc

aspa

se3

120573-a

ctin

MCF-7

lowastlowast


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MDA-MB-231

(d)


3 Results








48h

24h

0h

25 50 100 200(120583gmL)

Control

(a)

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The r

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25 50 100 200Control

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(120583gmL)

(b)







4 Discussion



25 50 100 200(120583gmL)

Control

Nuclei

Merge

p53

NF-120581B p65

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(a)

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115

225

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4

25 50 100 200

Relat

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f p53

(120583gmL)Control

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(b)

0

05

1

15

2

25

25 50 100 200(120583gmL)

Control



Relat

ive fl

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tin

tens

ity o

f NF-120581

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(c)

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25 50 100 200(120583gmL)

Control

NF-120581B p65

(d)

005

115

225

25 50 100 200(120583gmL)

Control


lowast

The r

elat

ive l

evel

of

p53

120573-a

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(e)

0

05

1

15

2

25 50 100 200(120583gmL)

Control


The r

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ive l

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of

AN

XA7

120573-a

ctin

(f)

002040608

112

25 50 100 200(120583gmL)

Control


lowastlowast

The r

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of

NF-120581

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5120573

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in

(g)



Nuclei

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p53

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25 50 100 200(120583gmL)

Control

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(a)

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25 50 100 200

Relat

ive fl

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t in

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f p53

(120583gmL)Control

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(b)

0

05

1

15

2

25

25 50 100 200(120583gmL)

Control

Relat

ive fl

uore

scen

tin

tens

ity N

F-120581

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5



MDA-MB-231

(c)

p53

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120573-Actin

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25 50 100 200(120583gmL)

Control

(d)

002040608

112

25 50 100 200(120583gmL)

Control

lowastlowastlowast

The r

elat

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evel

of

p53

120573-a

ctin

(e)

002040608

112141618

2

25 50 100 200(120583gmL)

Control

lowastlowast

lowastlowast

lowastlowast

The r

elat

ive l

evel

of

AN

XA7

120573-a

ctin

(f)

002040608

112

25 50 100 200(120583gmL)

Control

lowastlowast

lowast

lowast

The r

elat

ive l

evel

of

NF-120581

B p6

5120573

-act

in

(g)



25 50 100 200Control(120583gmL)

48h

MCF-7

(a)

02468

10121416

25 50 100 200Control

MCF-7

lowast lowast

lowastlowast

lowastlowast

The r

elat

ive l

evel

of R

OS

(120583gmL)

(b)

25 50 100 200Control

48h

(120583gmL)MDA-MB-231

(c)

0123456

25 50 100 200

The r

elat

ive l

evel

of R

OS

Control


lowast

(120583gmL)

MDA-MB-231

(d)









25 50 100 200(120583gmL)

Control

48h

MCF-7

(a)

020

040608

1121416

25 50 100 200

The r

elat

ive l

evel

of

Control

mito

chon

dria

mem

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e pot

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l MCF-7




(b)

48h

25 50 100 200(120583gmL)

Control

MDA-MB-231

(c)

005

115

225

3

25 50 100 200Control


The r

elat

ive l

evel

of

mito

chon

dria

mem

bran

e pot

entia

l MDA-MB-231


(d)














Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















48h

24h

0h

25 50 100 200(120583gmL)

Control

(a)

0010203040506070809

The r

elat

ive l

evel

of c

ell

mig

ratio

n

25 50 100 200Control

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(120583gmL)

(b)







4 Discussion



25 50 100 200(120583gmL)

Control

Nuclei

Merge

p53

NF-120581B p65

MCF-7

(a)

005

115

225

335

4

25 50 100 200

Relat

ive fl

uore

scen

t in

tens

ity o

f p53

(120583gmL)Control

lowastlowast


lowastlowast

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(b)

0

05

1

15

2

25

25 50 100 200(120583gmL)

Control



Relat

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tin

tens

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f NF-120581

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25 50 100 200(120583gmL)

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(d)

005

115

225

25 50 100 200(120583gmL)

Control


lowast

The r

elat

ive l

evel

of

p53

120573-a

ctin

(e)

0

05

1

15

2

25 50 100 200(120583gmL)

Control


The r

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of

AN

XA7

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ctin

(f)

002040608

112

25 50 100 200(120583gmL)

Control


lowastlowast

The r

elat

ive l

evel

of

NF-120581

B p6

5120573

-act

in

(g)



Nuclei

Merge

p53

NF-120581B p65

25 50 100 200(120583gmL)

Control

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(a)

0123456789

10

25 50 100 200

Relat

ive fl

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scen

t in

tens

ity o

f p53

(120583gmL)Control

MDA-MB-231

(b)

0

05

1

15

2

25

25 50 100 200(120583gmL)

Control

Relat

ive fl

uore

scen

tin

tens

ity N

F-120581

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5



MDA-MB-231

(c)

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25 50 100 200(120583gmL)

Control

(d)

002040608

112

25 50 100 200(120583gmL)

Control

lowastlowastlowast

The r

elat

ive l

evel

of

p53

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(e)

002040608

112141618

2

25 50 100 200(120583gmL)

Control

lowastlowast

lowastlowast

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The r

elat

ive l

evel

of

AN

XA7

120573-a

ctin

(f)

002040608

112

25 50 100 200(120583gmL)

Control

lowastlowast

lowast

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The r

elat

ive l

evel

of

NF-120581

B p6

5120573

-act

in

(g)



25 50 100 200Control(120583gmL)

48h

MCF-7

(a)

02468

10121416

25 50 100 200Control

MCF-7

lowast lowast

lowastlowast

lowastlowast

The r

elat

ive l

evel

of R

OS

(120583gmL)

(b)

25 50 100 200Control

48h

(120583gmL)MDA-MB-231

(c)

0123456

25 50 100 200

The r

elat

ive l

evel

of R

OS

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(120583gmL)

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(d)









25 50 100 200(120583gmL)

Control

48h

MCF-7

(a)

020

040608

1121416

25 50 100 200

The r

elat

ive l

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of

Control

mito

chon

dria

mem

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(b)

48h

25 50 100 200(120583gmL)

Control

MDA-MB-231

(c)

005

115

225

3

25 50 100 200Control


The r

elat

ive l

evel

of

mito

chon

dria

mem

bran

e pot

entia

l MDA-MB-231


(d)














Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















25 50 100 200(120583gmL)

Control

Nuclei

Merge

p53

NF-120581B p65

MCF-7

(a)

005

115

225

335

4

25 50 100 200

Relat

ive fl

uore

scen

t in

tens

ity o

f p53

(120583gmL)Control

lowastlowast


lowastlowast

MCF-7

(b)

0

05

1

15

2

25

25 50 100 200(120583gmL)

Control



Relat

ive fl

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scen

tin

tens

ity o

f NF-120581

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Control

NF-120581B p65

(d)

005

115

225

25 50 100 200(120583gmL)

Control


lowast

The r

elat

ive l

evel

of

p53

120573-a

ctin

(e)

0

05

1

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2

25 50 100 200(120583gmL)

Control


The r

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AN

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120573-a

ctin

(f)

002040608

112

25 50 100 200(120583gmL)

Control


lowastlowast

The r

elat

ive l

evel

of

NF-120581

B p6

5120573

-act

in

(g)



Nuclei

Merge

p53

NF-120581B p65

25 50 100 200(120583gmL)

Control

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(a)

0123456789

10

25 50 100 200

Relat

ive fl

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scen

t in

tens

ity o

f p53

(120583gmL)Control

MDA-MB-231

(b)

0

05

1

15

2

25

25 50 100 200(120583gmL)

Control

Relat

ive fl

uore

scen

tin

tens

ity N

F-120581

B p6

5



MDA-MB-231

(c)

p53

120573-Actin

120573-Actin

ANXA7

NF-120581B p65

25 50 100 200(120583gmL)

Control

(d)

002040608

112

25 50 100 200(120583gmL)

Control

lowastlowastlowast

The r

elat

ive l

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of

p53

120573-a

ctin

(e)

002040608

112141618

2

25 50 100 200(120583gmL)

Control

lowastlowast

lowastlowast

lowastlowast

The r

elat

ive l

evel

of

AN

XA7

120573-a

ctin

(f)

002040608

112

25 50 100 200(120583gmL)

Control

lowastlowast

lowast

lowast

The r

elat

ive l

evel

of

NF-120581

B p6

5120573

-act

in

(g)



25 50 100 200Control(120583gmL)

48h

MCF-7

(a)

02468

10121416

25 50 100 200Control

MCF-7

lowast lowast

lowastlowast

lowastlowast

The r

elat

ive l

evel

of R

OS

(120583gmL)

(b)

25 50 100 200Control

48h

(120583gmL)MDA-MB-231

(c)

0123456

25 50 100 200

The r

elat

ive l

evel

of R

OS

Control


lowast

(120583gmL)

MDA-MB-231

(d)









25 50 100 200(120583gmL)

Control

48h

MCF-7

(a)

020

040608

1121416

25 50 100 200

The r

elat

ive l

evel

of

Control

mito

chon

dria

mem

bran

e pot

entia

l MCF-7




(b)

48h

25 50 100 200(120583gmL)

Control

MDA-MB-231

(c)

005

115

225

3

25 50 100 200Control


The r

elat

ive l

evel

of

mito

chon

dria

mem

bran

e pot

entia

l MDA-MB-231


(d)














Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Nuclei

Merge

p53

NF-120581B p65

25 50 100 200(120583gmL)

Control

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(a)

0123456789

10

25 50 100 200

Relat

ive fl

uore

scen

t in

tens

ity o

f p53

(120583gmL)Control

MDA-MB-231

(b)

0

05

1

15

2

25

25 50 100 200(120583gmL)

Control

Relat

ive fl

uore

scen

tin

tens

ity N

F-120581

B p6

5



MDA-MB-231

(c)

p53

120573-Actin

120573-Actin

ANXA7

NF-120581B p65

25 50 100 200(120583gmL)

Control

(d)

002040608

112

25 50 100 200(120583gmL)

Control

lowastlowastlowast

The r

elat

ive l

evel

of

p53

120573-a

ctin

(e)

002040608

112141618

2

25 50 100 200(120583gmL)

Control

lowastlowast

lowastlowast

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The r

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of

AN

XA7

120573-a

ctin

(f)

002040608

112

25 50 100 200(120583gmL)

Control

lowastlowast

lowast

lowast

The r

elat

ive l

evel

of

NF-120581

B p6

5120573

-act

in

(g)



25 50 100 200Control(120583gmL)

48h

MCF-7

(a)

02468

10121416

25 50 100 200Control

MCF-7

lowast lowast

lowastlowast

lowastlowast

The r

elat

ive l

evel

of R

OS

(120583gmL)

(b)

25 50 100 200Control

48h

(120583gmL)MDA-MB-231

(c)

0123456

25 50 100 200

The r

elat

ive l

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of R

OS

Control


lowast

(120583gmL)

MDA-MB-231

(d)









25 50 100 200(120583gmL)

Control

48h

MCF-7

(a)

020

040608

1121416

25 50 100 200

The r

elat

ive l

evel

of

Control

mito

chon

dria

mem

bran

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entia

l MCF-7




(b)

48h

25 50 100 200(120583gmL)

Control

MDA-MB-231

(c)

005

115

225

3

25 50 100 200Control


The r

elat

ive l

evel

of

mito

chon

dria

mem

bran

e pot

entia

l MDA-MB-231


(d)














Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















25 50 100 200Control(120583gmL)

48h

MCF-7

(a)

02468

10121416

25 50 100 200Control

MCF-7

lowast lowast

lowastlowast

lowastlowast

The r

elat

ive l

evel

of R

OS

(120583gmL)

(b)

25 50 100 200Control

48h

(120583gmL)MDA-MB-231

(c)

0123456

25 50 100 200

The r

elat

ive l

evel

of R

OS

Control


lowast

(120583gmL)

MDA-MB-231

(d)









25 50 100 200(120583gmL)

Control

48h

MCF-7

(a)

020

040608

1121416

25 50 100 200

The r

elat

ive l

evel

of

Control

mito

chon

dria

mem

bran

e pot

entia

l MCF-7




(b)

48h

25 50 100 200(120583gmL)

Control

MDA-MB-231

(c)

005

115

225

3

25 50 100 200Control


The r

elat

ive l

evel

of

mito

chon

dria

mem

bran

e pot

entia

l MDA-MB-231


(d)














Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















25 50 100 200(120583gmL)

Control

48h

MCF-7

(a)

020

040608

1121416

25 50 100 200

The r

elat

ive l

evel

of

Control

mito

chon

dria

mem

bran

e pot

entia

l MCF-7




(b)

48h

25 50 100 200(120583gmL)

Control

MDA-MB-231

(c)

005

115

225

3

25 50 100 200Control


The r

elat

ive l

evel

of

mito

chon

dria

mem

bran

e pot

entia

l MDA-MB-231


(d)














Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Research Article Antitumor Activity of Chinese Propolis in Human...

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