J BIOCHEM MOLECULAR TOXICOLOGYVolume 28, Number 9, 2014

Investigation of Gallic Acid Induced Anticancer Effectin Human Breast Carcinoma MCF-7 CellsKe Wang,1 Xue Zhu,1 Kai Zhang,1 Ling Zhu,2 and Fanfan Zhou3

1Key Laboratory of Nuclear Medicine, Ministry of Health, Jiangsu Key Laboratory of Molecular Nuclear Medicine, Jiangsu Institute of NuclearMedicine, Wuxi 214063, Jiangsu Province, People’s Republic of China; E-mail: wangke@jsinm.org2Save Sight Institute, University of Sydney, NSW 2000, Australia3Faculty of Pharmacy, University of Sydney, NSW 2006, Australia

Received 19 January 2014; revised 8 April 2014; accepted 30 April 2014

ABSTRACT: Gallic acid (GA), a polyhydroxylpheno-lic compound abundantly distributed in plants, fruits,and foods, has been reported to have various biolog-ical activities including an anticancer effect. In thisstudy, we extensively investigated the anticancer ef-fect of GA in human breast carcinoma MCF-7 cells.Our study indicated that treatment with GA resultedin inhibition of proliferation and induction of apopto-sis in MCF-7 cells. Then, the molecular mechanism ofGA’s apoptotic action in MCF-7 cells was further inves-tigated. The results revealed that GA induced apopto-sis by triggering the extrinsic or Fas/FasL pathway aswell as the intrinsic or mitochondrial pathway. Further-more, the apoptotic signaling induced by GA was am-plified by cross-link between the two pathways. Takentogether, our findings may be useful for understandingthe mechanism of action of GA on breast cancer cellsand provide new insights into the possible applicationof such compound and its derivatives in breast can-cer therapy. C© 2014 Wiley Periodicals, Inc. J. Biochem.Mol. Toxicol. 28:387–393, 2014; View this article online atwileyonlinelibrary.com. DOI 10.1002/jbt.21575

KEYWORDS: Gallic Acid; MCF-7; Cell Apoptosis;Fas/FasL Pathway; Mitochondrial Pathway

Correspondence to: Ke Wang.Contract Grant Sponsor: National Natural Science Foundation.Contract Grant Number: 81300787.Contract Grant Sponsor: Natural Science Foundation of Jiangsu

Province.Contract Grant Numbers: BK2011168 and BK2012105.Contract Grant Sponsor: Youth Foundation of Jiangsu Institute

of Nuclear Medicine.Contract Grant Number: QN201111.

C© 2014 Wiley Periodicals, Inc.

INTRODUCTION

Breast cancer is the most common malignancy inwomen worldwide [1]. Incidence of global breast can-cer is increasing at a speed of 3.1% annually over thelast three decades and it is still the leading cause ofcancer deaths among women in developed and devel-oping countries [2–4]. In China, the incidence of breastcancer is also increasing rapidly and about 1.1 millionnew cases appeared every year [5]. Although progresshas been made in the management of breast cancer,the mechanism underlying this heterogeneous diseasedevelopment still remains unclear. The conventionaltreatment of breast cancer includes surgery combinedwith radiotherapy and/or chemotherapy; however, thetherapy is hampered by the problem of drug resistanceand toxicity [6]. Therefore, it is of critical importance tofurther continue the search for the effective drugs forthe treatment of breast cancer.

In recent decades, natural plants, fruits, or foodshave been recognized as a valuable resource in thedevelopment of novel and advanced cancer therapies[7, 8]. For example, plant-derived anticancer drugssuch as paclitaxel, vinorelbine, or vincristine have cur-rently been proved to be effective in clinical can-cer trails [9–11]. Gallic acid (GA; 3,4,5-triphydroxyl-benzoic acid) is a polyhydroxylphenolic compound,which is widely distributed in various plants, fruits,and foods [12]. GA was demonstrated to have variousbiological activities including antibacterial, antiviral,and anti-inflammatory, in which the antitumor activ-ity is most striking [13, 14]. Until now, the activities ofGA on various cancer cells such as leukemia, glioma,prostate, and lung cancer have been reported [15–18].The anticancer effect of GA has also been investigatedon human breast carcinoma MCF-7 cells, which focusedon the mechanisms of cell cycle arrest; however, the

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anticancer effect of GA on this cell line is still far fromclear [19].

In this study, to establish the precise mechanism ofGA on MCF-7 cells, the induction of GA on cell apopto-sis was further investigated in regard to the extrinsic ordeath receptor pathway and the intrinsic or mitochon-drial pathway.

MATERIALS AND METHODS

Reagents

GA (purity > 98%) was purchased from NationalInstitute for the Control of Pharmaceutical and Bio-logical Products (Beijing, People’s Republic of China)and dissolved in dimethyl sulfoxide (DMSO). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bro-mide (MTT), sodiumditolyl-4,4-bis(2-azo-8-amino-1-naphthol-3,6-disulfonate) (trypan blue), rhodamine123(Rh123), and DMSO were purchased from Sigma (St.Louis, MO). Annexin V-FITC and PI double staining kitwas purchased from BD Biosciences (San Jose, CA). Theantibodies to Bax, Bcl-2, cytochrome c, Bid, and β-actinwere obtained from Santa Cruz Biotechnology (SantaCruz, CA). Caspase fluorometric assay kit, caspase-8 in-hibitor Z-IETD-FMK, and caspase-9 inhibitor Z-LEHD-FMK were obtained from BioVision (Milpitas, CA). Allother chemicals and reagents were of analytical grade.

Cell Culture

The human breast cancer cell line MCF-7 was ob-tained from Shanghai Institute of Cell Biology, ChineseAcademy of Sciences, Shanghai, People’s Republic ofChina. MCF-7 cells were cultured in Deulbecco’s mod-ified eagle medium supplemented with 10% fetal calfserum, 1% penicillin–streptomycin, 0.25 µg/mL am-photericin, and 5 µg/mL insulin.

Cell Proliferation Assay

Inhibition of cell proliferation was measured bythe MTT assay and the trypan blue exclusion assay asmentioned before [20]. Cells (1 × 104 cells/well) wereplated in a 96-well plate. After 24-h incubation, cellswere treated with GA (0, 25, 50, 100, 200, 400 µM)for 24 h and 10 µL MTT stock solution (5 mg/mL)was added to each well for incubation for another 4 hat 37°C. Then, the culture medium was removed and100 µL DMSO was added to dissolve the formazancrystals. Absorbance was measured at 570 nm with anenzyme-linked immunosorbent assay (ELISA) reader(Bio-Rad, Hercules, CA). For the trypan blue exclu-

sion assay, cells were washed with phosphate buffersaline (PBS) twice at the end of the incubation periodand trypsinized. Then, the cells were resuspended andcounted. Cell viability was expressed as a percentageof the value against the nontreated control group.

Measurement of Cell Apoptosis

Apoptosis was determined by double stain-ing cells with the annexin V-FITC and PI. Af-ter treatment with the indicated concentrations ofGA, cells were washed twice with cold PBS, resus-pended in binding buffer (10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid/NaOH (pH 7.4),140 mM NaCl, 2.5 mM CaCl2) and stained with annexinV-FITC and PI at room temperature for 15 min in thedark. Cells were then analyzed using flow cytometry(Becton-Dickinson, San Jose, CA) within 1 h after stain-ing. Apoptotic cells were defined as annexin V+/PI−

cells and the percentage of which was calculated byCellQuest software (Becton-Dickinson).

Measurement of Fas/APO-1 and FasL Levels

The levels of Fas/APO-1 and Fas ligand (FasL)including membrane FasL (mFasL) of cell lysate andsoluble FasL (sFasL) of cell culture supernatant wereassessed by ELISA kit (Calbiochem, Billerica, MA). Thesamples were incubated in a 96-well plate with anti-bodies, then the unbound materials were removed andhorseradish peroxidase (HRP) conjugated streptavidinwas added to bind antibodies. After catalyzing withchromogenic substrate, absorbance was measured at450 nm with an ELISA reader. The concentrations ofFas/APO-1 and FasL were determined by interpolat-ing from standard curves.

Measurement of Mitochondrial MembranePotential

Mitochondrial membrane potential was measuredby uptake of Rh123 into mitochondria. Cells (1 × 105

cells/well) treated with GA were incubated with Rh123(5 µM) for 30 min. After collection, excess Rh123 wasremoved by washing and the cells were incubated at37°C for the indicated lengths of time. Then cellularlevels of Rh123 were measured by flow cytometry.

Western Blotting Analysis

Total cell exacts were prepared in lysis buffer(50 mM Tris-HCl, 150 mM NaCl, 1 mM ethylene gly-col tetraacetic acid, 1 mM ethylene diamine tetraacetic

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FIGURE 1. Effect of GA on cell proliferation in MCF-7 cells. Cells were treated with various concentrations of GA (0, 25, 50, 100, 200, 400 µM)for 24 h and cell viability was evaluated by (A) the MTT assay and (B) the trypan blue exclusion assay. The results were shown as mean ± SEMof three experiments and each included triplicate sets.

acid, 20 mM NaF, 100 mM Na3VO4, 1% NonidetP-40, 1% Triton X-100, 1 mM phenylmethylsulfonyl flu-oride, 10 mg/mL aprotinin, and 10 mg/mL leupeptin)as described previously [21]. Equivalent amounts ofsamples were resolved by SDS-PAGE (10%) and elec-trophoretically transferred onto polyvinylidene fluo-ride membranes. After getting blocked with 5% bovineserum albumin in Tris-buffer saline (TBST) at 37°C for2 h, the membranes were incubated with the desiredprimary antibody at 4°C for overnight. After wash-ing with TBST for three times, the membranes wereincubated with HRP-conjugated secondary antibodyat 37°C for 2 h. The detection of protein bands wasperformed using the enhanced chemiluminescence de-tection kit (Beyotime, Nantong, People’s Republic ofChina).

Measurement of Caspase Activity

Caspase activity was assessed by the fluorometricassay. Cells (2 × 105 cells/well) treated with the in-dicated concentrations of GA were harvested in lysisbuffer and protein was separated by centrifugation. Anequivalent amount of protein from each sample wasincubated with caspase substrate at 37°C for 30 min.The activity was quantified by a spectrofluorometer(Molecular Devices, Sunnyvale, CA) with an excitationwavelength at 400 nm and an emission wavelength at505 nm.

Statistical Analysis

Biostatistical analyses were conducted with theGraphPad Prism 5.0 (GraphPad, La Jolla, CA) andSPSS 16.0 software package (Chicago, IL). All exper-iments were repeated three times. Results of multi-ple experiments were expressed as mean ± SEM. AP value of less than 0.05 was accepted as statisticallysignificant.

RESULTS

Effect of GA on Cell Proliferation

The inhibitory effect of GA on cell proliferationwas evaluated through the MTT assay and the try-pan blue exclusion assay. As shown in Figure 1A, GAtreatment remarkably induced proliferation inhibitionin MCF-7 cells. The inhibition of cell growth was dosedependent, with the IC50 value of 80.5 µM at 24 h.The cytotoxic effect of GA was also verified by the try-pan blue exclusion assay, which was consistent withthe results of the MTT assay (Figure 1B). Our resultsindicated that GA had an inhibitory effect on breastcarcinoma MCF-7 cells in a dose-dependent manner.

Effect of GA on Cell Apoptosis

Cell apoptosis induced by GA was assessed byflow cytometric analysis using annexin V-PI dual-staining assay. The results indicated that percentageof apoptotic cells were significantly increased by treat-ment with GA at 50 µM (14.29 ± 3.18%), 100 µM (26.35 ±4.48%), and 200 µM (45.58%±7.69%) for 24 h in compar-ison with the control (1.27 ± 0.48%, P < 0.05) (Figure 2).Our data suggested that GA could inhibit the growthof MCF-7 cells by inducing apoptosis.

Effect of GA on Fas/FasL Apoptotic Pathway

To determine whether the Fas/FasL apoptoticpathway was activated, the expression levels ofFas/FasL (mFasL and sFasL) were measured by ELISA.As shown in Figure 3A, treatment with GA resultedin the enhancement of both Fas/APO1 and FasL pro-tein levels, whose effect was dose dependent. In ad-dition, the level of mFasL was higher than sFasL atall points. Then, the activity of initiator caspase-8 of theFas/FasL system was assessed. The results showed that

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FIGURE 2. Effect of GA on cell apoptosis in MCF-7 cells. Cells were treated with various concentrations of GA (0, 50, 100, 200 µM) for 24 h andcell apoptosis was assessed by flow cytometric analysis. (A) Flow cytometry analysis of cell apoptosis using annexin V-FITC/PI dual staining.(B) The bar chart describes the percentage distribution of apoptotic cells. The results were shown as mean ± SEM of three experiments and eachincluded triplicate sets. *P < 0.05, **P < 0.01 versus control.

caspase-8 activity was increased in GA-treated MCF-7cells (P < 0.05) (Figure 3B).

Effect of GA on Mitochondrial ApoptoticPathway

To investigate whether the mitochondrial pathwaywas involved in GA-induced apoptosis, the associatedevents were analyzed. Mitochondrial dysfunction in-cudes the change of mitochondrial membrane potentialas well as Bax/Bcl-2 ratio, cytochrome c release into cy-tosol, and activation of caspase-9. As shown in Figure 4,treatment with GA (200 µM) markedly decreased theproportion of Rh123 positive cells from 94.25 ± 3.82%to 29.51 ± 4.27%, increased the ratio of Bax/Bcl-2 to ap-proximately 8.92-fold compared to that of the control,and enhanced cytochrome c levels in cytosol. Then, theactivity of initiator caspase-9 of mitochondrial apop-totic pathway was assessed. The results showed that

caspase-9 activity was dose dependently increased inGA-treated cells (P < 0.05).

Effect of Caspase-8-Mediated Bid Cleavageon the Activation of MitochondrialApoptotic Pathway

Since both Fas/FasL and mitochondrial signalingpathways were involved in GA-mediated apoptosis,it was possible that GA could activate the mitochon-drial apoptotic pathway through caspase-8-mediatedBid cleavage, which then resulted in cytochrome crelease and caspase-9 activation. To test this idea, thestatus of Bid protein was checked. As shown in Figure 5,a full-size Bid protein was cleaved to yield a fragmentafter treatment of cells with GA. Furthermore, we in-vestigated the effects of various caspase inhibitors onGA-induced Bid cleavage. When cells were cotreatedwith the caspase-8 inhibitor, the level of Bid was recov-ered and cleavage of which was not appeared, whereas

FIGURE 3. Effect of GA on the Fas/FasL apoptotic pathway in MCF-7 cells. Cells were treated with various concentrations of GA (0, 50, 100,200 µM) for 24 h, then the expression levels of Fas/APO1 and FasL were measured by ELISA (A) and the activity of caspase-8 was assessed bythe fluorometric assay (B). The results were shown as mean ± SEM of three experiments and each included triplicate sets. *P < 0.05, **P < 0.01versus control.

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FIGURE 4. Effect of GA on the mitochondrial apoptotic pathway in MCF-7 cells. Cells were treated with various concentrations of GA (0,50, 100, 200 µM) for 24 h and for the determination of associated events. (A) Analysis of disturbances of mitochondrial membrane potentialusing fluorescence probe Rh123. MFI: mean fluorescence intensity. (B) Assessment of Bax and Bcl-2 protein levels by Western blot analysis.(C) Determination of the levels of mitochondrial cytochrome c (Mito Cyto c) and cytosolic cytochrome c (Cyto Cyto c) by Western blot analysis.(D) Assessment of the activity of caspase-9 by the fluorometric assay. The results were shown as mean ± SEM of three experiments and eachincluded triplicate sets. *P <0.05, **P < 0.01 versus control.

cotreating with the caspase-9 inhibitor did not affectGA-induced cleavage of Bid. The results of which in-dicated that activation of caspase-9 might be a down-stream event of caspase-8-mediated Bid cleavage.

DISCUSSION

GA is one of the major components of natural prod-ucts such as green tea, grapes, strawberries, bananas,and many other fruits [12]. Previous studies have re-ported that GA plays an important role in the preven-tion of malignant transformation and cancer develop-ment [15]. Consistently, the cytotoxicity effect of GA inhuman breast carcinoma MCF-7 cells was estimated in

our research, which was likely mediated via inducingcell apoptosis. However, little was done on research ofthe molecular mechanisms underneath the effect of GAand which was extensively characterized in the currentstudy.

To gain insight into the molecular mechanismof GA’s action on cell apoptosis, the involvement ofapoptosis-related signaling pathways has been inves-tigated in MCF-7 cells, which include the extrinsic andintrinsic pathways. The Fas/FasL system is recognizedas an extrinsic pathway for the induction of apopto-sis [22]. Fas is expressed at low level on the surface ofresting cells; however, the expression of which is en-hanced after cell activation [23]. In contrast with Fas,the expression of FasL is reported to be much more

FIGURE 5. Effect of GA on Bid cleavage in MCF-7 cells. (A) Cells were treated with various concentrations of GA (0, 50, 100, 200 µM) for 24 hand then Bid cleavage was determined. (B) The effect of caspase-8 and caspase-9 inhibitors on GA-mediated (100 µM) Bid cleavage.

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FIGURE 6. The proposed molecular mechanisms of cell apoptosisinduced by GA in breast carcinoma MCF-7 cells.

restricted and often requires cell activation [24]. Theinteraction of FasL with Fas on a target cell stimulatesa series of intracellular caspase cascade that leads tothe induction of apoptosis [22]. Our study indicatedthat both Fas/FasL protein levels and the activity ofinitiator caspase-8 were enhanced in MCF-7 cells af-ter treatment with GA. Thus, we demonstrated that theFas/FasL apoptotic system participated in GA-inducedcell apoptosis. Mitochondria appear as the central ex-ecutioner of apoptosis; in the mitochondrial pathway,apoptosis-related members of the Bcl-2 family causemitochondrial outer membrane permeabilization, al-lowing the release of cytochrome c, which interactswith Apaf-1 to trigger caspase activation and apoptosis[25, 26]. Our study found that treatment with GA re-sulted in mitochondrial dysfunction via decreasing mi-tochondrial membrane potential, increasing Bax/Bcl-2ratio, cytochrome c release into cytosol, as well as acti-vation of caspase-9. Furthermore, we had also demon-strated that the two pathways were linked and thatmolecules in one pathway could influence the otherby caspase-8-mediated Bid cleavage, which was con-sistent with the previous study reported by Kook et al.[27]. The cleavage of Bid by caspase-8 supported the hy-pothesis that the proteolysis event of Bid suppressed bycotreatment with caspase-8 inhibitor, but not caspase-9inhibitor.

In conclusion, we demonstrated the signalingpathways of GA-induced apoptosis in breast carcinomaMCF-7 cells (Figure 6). This study will contribute inunderstanding the mechanism of action of such com-pound in breast cancer cells; however, much more an-imal and human studies are needed for the possibilityof GA and its derivatives on treatment of breast cancer.

REFERENCES

1. DeSantis C, Siegel R, Bandi P, Jemal A. Breast cancerstatistics, 2011. CA Cancer J Clin 2011;61(6):409–418.

2. Bleyer A, Welch HG. Effect of three decades of screeningmammography on breast-cancer incidence. N Engl J Med2012;367(21):1998–2005.

3. Merlo D, Ceppi M, Filiberti R, Bocchini V, Znaor A,Gamulin M, Primic-Zakelj M, Bruzzi P, Bouchardy C, Fu-cic A. Breast cancer incidence trends in European womenaged 20–39 years at diagnosis. Breast Cancer Res Treat2012;134(1):363–370.

4. Palmer JR, Boggs DA, Wise LA, Adams-CampbellLL, Rosenberg L. Individual and neighborhood so-cioeconomic status in relation to breast cancer inci-dence in African-American women. Am J Epidemiol2012;176(12):1141–1146.

5. Zheng S, Bai JQ, Li J, Fan JH, Pang Y, Song QK, HuangR, Yang HJ, Xu F, Lu N. The pathologic characteristicsof breast cancer in China and its shift during 1999–2008:a national-wide multicenter cross-sectional image over10 years. Int J Cancer 2012;131(11):2622–2631.

6. Maughan KL, Lutterbie MA, Ham PS. Treatment of breastcancer. Am Fam Physician 2010;81(11):1339–1346.

7. HemaIswarya S, Doble M. Potential synergism of nat-ural products in the treatment of cancer. Phytother Res2006;20(4):239–249.

8. Cragg GM, Grothaus PG, Newman DJ. Impact of naturalproducts on developing new anti-cancer agents. ChemRev 2009;109(7):3012–3043.

9. Miller K, Wang M, Gralow J, Dickler M, Cobleigh M,Perez EA, Shenkier T, Cella D, Davidson NE. Paclitaxelplus bevacizumab versus paclitaxel alone for metastaticbreast cancer. N Engl J Med 2007;357(26):2666–2676.

10. Joensuu H, Kellokumpu-Lehtinen P-L, Bono P, AlankoT, Kataja V, Asola R, Utriainen T, Kokko R, HemminkiA, Tarkkanen M. Adjuvant docetaxel or vinorelbine withor without trastuzumab for breast cancer. N Engl J Med2006;354(8):809–820.

11. Stege EM, Kros JM, de Bruin HG, Enting R, van HeuvelI, Looijenga LH, van der Rijt CD, Smitt PA, van den BentMJ. Successful treatment of low-grade oligodendroglialtumors with a chemotherapy regimen of procarbazine,lomustine, and vincristine. Cancer 2005;103(4):802–809.

12. Fiuza S, Gomes C, Teixeira L, Girao da Cruz M,Cordeiro M, Milhazes N, Borges F, Marques M. Pheno-lic acid derivatives with potential anticancer properties-a structure-activity relationship study. Part 1: methyl,propyl and octyl esters of caffeic and gallic acids. BioorgMed Chem 2004;12(13):3581–3589.

13. Alberto MR, Farıas ME, Manca de Nadra MC. Effect ofgallic acid and catechin on Lactobacillus hilgardii 5wgrowth and metabolism of organic compounds. J AgricFood Chem 2001;49(9):4359–4363.

14. Lapidot T, Walker MD, Kanner J. Antioxidant and proox-idant effects of phenolics on pancreatic β-cells in vitro. JAgric Food Chem 2002;50(25):7220–7225.

15. Faried A, Kurnia D, Faried L, Usman N, Miyazaki T, KatoH, Kuwano H. Anticancer effects of gallic acid isolatedfrom Indonesian herbal medicine, Phaleria macrocarpa(Scheff.) Boerl, on human cancer cell lines. Int J Oncol2007;30(3):605–613.

16. Ji BC, Hsu WH, Yang JS, Hsia TC, Lu CC, Chi-ang JH, Yang JL, Lin CH, Lin JJ, Suen LJW, GibsonWood W, Chung JG. Gallic acid induces apoptosis via

J Biochem Molecular Toxicology DOI 10.1002/jbt

Volume 28, Number 9, 2014 ANTICANCER EFFECT OF GALLIC ACID IN MCF-7 CELLS 393

caspase-3 and mitochondrion-dependent pathways invitro and suppresses lung xenograft tumor growth invivo. J Agric Food Chem 2009;57(16):7596–7604.

17. Lo C, Lai TY, Yang JH, Yang JS, Ma YS, Weng SW,Chen YY, Lin JG, Chung JG. Gallic acid inducesapoptosis in A375.S2 human melanoma cells throughcaspase-dependent and-independent pathways. Int J On-col 2010;37(2):377–385.

18. Lu Y, Jiang F, Jiang H, Wu K, Zheng X, Cai Y, KatakowskiM, Chopp M, To S-S. Gallic acid suppresses cell viabil-ity, proliferation, invasion and angiogenesis in humanglioma cells. Eur J Pharmacol 2010;64(2–3):102–107.

19. Hsu JD, Kao SH, Ou TT, Chen YJ, Li YJ, Wang CJ. Gal-lic acid induces G2/M phase arrest of breast cancer cellMCF-7 through stabilization of p27Kip1 attributed to dis-ruption of p27Kip1/Skp2 complex. J Agric Food Chem2011;59(5):1996–2003.

20. van Meerloo J, Kaspers GJ, Cloos J. Cell sensitivity assays:the MTT assay. Methods Mol Biol 2011;731:237–245.

21. Hammond JB, Kruger NJ. The Bradford method for pro-tein quantitation. Methods Mol Biol 1988;3:25–32.

22. Reichmann E. The biological role of the Fas/FasL systemduring tumor formation and progression. Semin CancerBiol 2002;12(4):309–315.

23. Siegel RM, Frederiksen JK, Zacharias DA, Chan FK-M, Johnson M, Lynch D, Tsien RY, Lenardo MJ. Faspreassociation required for apoptosis signaling anddominant inhibition by pathogenic mutations. Science2000;288(5475):2354–2357.

24. Selam B, Kayisli UA, Mulayim N, Arici A. Regulationof Fas ligand expression by estradiol and progesteronein human endometrium. Biol Reprod 2001;65(4):979–985.

25. Wang X. The expanding role of mitochondria in apopto-sis. Genes Dev 2001;15(22):2922–2933.

26. Desagher S, Martinou JC. Mitochondria as the cen-tral control point of apoptosis. Trends Cell Biol2000;10(9):369–377.

27. Kook S, Zhan X, Cleghorn WM, Benovic JL, Gurevich VV,Gurevich EV. Caspase-cleaved arrestin-2 and BID coop-eratively facilitate cytochrome C release and cell death.Cell Death Differ 2014;21(1):172–184.

J Biochem Molecular Toxicology DOI 10.1002/jbt