Genistein potentiates the effect of 17-beta estradiol on human hepatocellular carcinoma cell line


1 Department of Anatomical Sciences, Medical School, Jahrom University of Medical Sciences, Jahrom, Iran

2 Department of Anatomical Sciences and Molecular Biology, Medical School, Isfahan University of Medical Sciences, Isfahan, Iran

3 Cellular and Molecular Immunology Research Center, Isfahan University of Medical Sciences, Isfahan, Iran


Background: Hepatocellular carcinoma (HCC) is one of the most common malignant tumors. This cancer may be due to a multistep process with an accumulation of epigenetic alterations in tumor suppressor genes (TSGs), leading to hypermethylation of the genes. Hypermethylation of TSGs is associated with silencing and inactivation of them. It is well-known that DNA hypomethylation is the initial epigenetic abnormality recognized in human tumors. Estrogen receptor alpha (ERα) is one of the TSGs which modulates gene transcription and its hypermethylation is because of overactivity of DNA methyltransferases. Fortunately, epigenetic changes especially hypermethylation can be reversed by pharmacological compounds such as genistein (GE) and 17-beta estradiol (E2) which involve in preventing the development of certain cancers by maintaining a protective DNA methylation. The aim of the present study was to analyze the effects of GE on ERα and DNMT1 genes expression and also apoptotic and antiproliferative effects of GE and E2 on HCC.
Materials and Methods: Cells were treated with various concentrations of GE and E2 and the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay was used. Furthermore, cells were treated with single dose of GE and E2 (25 μM) and flow cytometry assay was performed. The expression level of the genes was determined by quantitative real-time reverse transcription polymerase chain reaction.
Results: GE increased ERα and decreased DNMT1 genes expression, GE and E2 inhibited cell viability and induced apoptosis significantly.
Conclusion: GE can epigenetically increase ERα expression by inhibition of DNMT1 expression which in turn increases apoptotic effect of E2. Furthermore, a combination of GE and E2 can induce apoptosis more significantly.


Bosch FX, Ribes J, Borràs J. Epidemiology of primary liver cancer. Semin Liver Dis 1999;19:271-85.  Back to cited text no. 1
Michielsen PP, Francque SM, van Dongen JL. Viral hepatitis and hepatocellular carcinoma. World J Surg Oncol 2005;3:27.  Back to cited text no. 2
Bruix J, Sherman M, Llovet JM, Beaugrand M, Lencioni R, Burroughs AK, et al. Clinical management of hepatocellular carcinoma. Conclusions of the Barcelona-2000 EASL conference. European Association for the Study of the Liver. J Hepatol 2001;35:421-30.  Back to cited text no. 3
Tischoff I, Tannapfel A. DNA methylation in hepatocellular carcinoma. World J Gastroenterol 2008;14:1741-8.  Back to cited text no. 4
Beasley RP, Hwang LY, Lin CC, Chien CS. Hepatocellular carcinoma and hepatitis B virus. A prospective study of 22 707 men in Taiwan. Lancet 1981;2:1129-33.  Back to cited text no. 5
Parkin DM, Bray F, Ferlay J, Pisani P. Estimating the world cancer burden: Globocan 2000. Int J Cancer 2001;94:153-6.  Back to cited text no. 6
Riggs AD, Pfeifer GP. X-chromosome inactivation and cell memory. Trends Genet 1992;8:169-74.  Back to cited text no. 7
Razin A, Cedar H. DNA methylation and genomic imprinting. Cell 1994;77:473-6.  Back to cited text no. 8
De Zhu J. The altered DNA methylation pattern and its implications in liver cancer. Cell Res 2005;15:272-80.  Back to cited text no. 9
Cheng P, Schmutte C, Cofer KF, Felix JC, Yu MC, Dubeau L. Alterations in DNA methylation are early, but not initial, events in ovarian tumorigenesis. Br J Cancer 1997;75:396-402.  Back to cited text no. 10
Ehrlich M, Woods CB, Yu MC, Dubeau L, Yang F, Campan M, et al. Quantitative analysis of associations between DNA hypermethylation, hypomethylation, and DNMT RNA levels in ovarian tumors. Oncogene 2006;25:2636-45.  Back to cited text no. 11
Bedford MT, van Helden PD. Hypomethylation of DNA in pathological conditions of the human prostate. Cancer Res 1987;47:5274-6.  Back to cited text no. 12
Lin CH, Hsieh SY, Sheen IS, Lee WC, Chen TC, Shyu WC, et al. Genome-wide hypomethylation in hepatocellular carcinogenesis. Cancer Res 2001;61:4238-43.  Back to cited text no. 13
Ehrlich M, Jiang G, Fiala E, Dome JS, Yu MC, Long TI, et al. Hypomethylation and hypermethylation of DNA in Wilms tumors. Oncogene 2002;21:6694-702.  Back to cited text no. 14
Vo AT, Millis RM. Epigenetics and breast cancers. Obstet Gynecol Int 2012;17:10-7.  Back to cited text no. 15
Mann M, Cortez V, Vadlamudi RK. Epigenetics of estrogen receptor signaling: Role in hormonal cancer progression and therapy. Cancers (Basel) 2011;3:1691-707.  Back to cited text no. 16
Teng J, Wang ZY, Jarrard DF, Bjorling DE. Roles of estrogen receptor alpha and beta in modulating urothelial cell proliferation. Endocr Relat Cancer 2008;15:351-64.  Back to cited text no. 17
Danel L, Cordier G, Revillard JP, Saez S. Presence of estrogen binding sites and growth-stimulating effect of estradiol in the human myelogenous cell line HL60. Cancer Res 1982;42:4701-5.  Back to cited text no. 18
Hatfill SJ, Brusnicky J, Fester E. Immunocytochemical identification of nuclear estrogen-receptors in human acute myeloid leukemia. Leuk Res 1991;15:315-20.  Back to cited text no. 19
Holst F, Stahl PR, Ruiz C, Hellwinkel O, Jehan Z, Wendland M, et al. Estrogen receptor alpha (ESR1) gene amplification is frequent in breast cancer. Nat Genet 2007;39:655-60.  Back to cited text no. 20
Pancholi S, Lykkesfeldt AE, Hilmi C, Banerjee S, Leary A, Drury S, et al. ERBB2 influences the subcellular localization of the estrogen receptor in tamoxifen-resistant MCF-7 cells leading to the activation of AKT and RPS6KA2. Endocr Relat Cancer 2008;15:985-1002.  Back to cited text no. 21
Boix L, Bruix J, Castells A, Fuster J, Bru C, Visa J, et al. Sex hormone receptors in hepatocellular carcinoma. Is there a rationale for hormonal treatment? J Hepatol 1993;17:187-91.  Back to cited text no. 22
van 't Veer LJ, Dai H, van de Vijver MJ, He YD, Hart AA, Mao M, et al. Gene expression profiling predicts clinical outcome of breast cancer. Nature 2002;415:530-6.  Back to cited text no. 23
van 't Veer LJ, Dai H, van de Vijver MJ, He YD, Hart AA, Bernards R, et al. Expression profiling predicts outcome in breast cancer. Breast Cancer Res 2003;5:57-8.  Back to cited text no. 24
Archer KJ, Mas VR, Maluf DG, Fisher RA. High-throughput assessment of CpG site methylation for distinguishing between HCV-cirrhosis and HCV-associated hepatocellular carcinoma. Mol Genet Genomics 2010;283:341-9.  Back to cited text no. 25
Hishida M, Nomoto S, Inokawa Y, Hayashi M, Kanda M, Okamura Y, et al. Estrogen receptor 1 gene as a tumor suppressor gene in hepatocellular carcinoma detected by triple-combination array analysis. Int J Oncol 2013;43:88-94.  Back to cited text no. 26
Day JK, Bauer AM, DesBordes C, Zhuang Y, Kim BE, Newton LG, et al. Genistein alters methylation patterns in mice. J Nutr 2002;132:2419S-23.  Back to cited text no. 27
Nikbakht Dastjerdi M, Kavoosi F. Inhibitory effect of 17-beta estradiol on hepatocellular carcinoma cell line. Indian J Appl Res 2015;5:435-40.  Back to cited text no. 28
Brownson DM, Azios NG, Fuqua BK, Dharmawardhane SF, Mabry TJ. Flavonoid effects relevant to cancer. Int Res Conf Food Nutr Cancer J Nutr 2002;132:3482-9.  Back to cited text no. 29
Kyselova Z. Toxicological aspects of the use of phenolic compounds in disease prevention. Interdiscip Toxicol 2011;4:173-83.  Back to cited text no. 30
Patisaul HB, Jefferson W. The pros and cons of phytoestrogens. Front Neuroendocrinol 2010;31:400-19.  Back to cited text no. 31
Ziegler RG. Phytoestrogens and breast cancer. Am J Clin Nutr 2004;79:183-4.  Back to cited text no. 32
Breithofer A, Graumann K, Scicchitano MS, Karathanasis SK, Butt TR, Jungbauer A. Regulation of human estrogen receptor by phytoestrogens in yeast and human cells. J Steroid Biochem Mol Biol 1998;67:421-9.  Back to cited text no. 33
Setchell KD, Cassidy A. Dietary isoflavones: Biological effects and relevance to human health. J Nutr 1999;129:758S-67.  Back to cited text no. 34
Tikkanen MJ, Adlercreutz H. Dietary soy-derived isoflavone phytoestrogens. Could they have a role in coronary heart disease prevention? Biochem Pharmacol 2000;60:1-5.  Back to cited text no. 35
Wiseman H. The therapeutic potential of phytoestrogens. Expert Opin Investig Drugs 2000;9:1829-40.  Back to cited text no. 36
Mirza S, Sharma G, Parshad R, Gupta SD, Pandya P, Ralhan R. Expression of DNA methyltransferases in breast cancer patients and to analyze the effect of natural compounds on DNA methyltransferases and associated proteins. J Breast Cancer 2013;16:23-31.  Back to cited text no. 37
Kurebayashi J, Otsuki T, Kunisue H, Tanaka K, Yamamoto S, Sonoo H. Expression levels of estrogen receptor-alpha, estrogen receptor-beta, coactivators, and corepressors in breast cancer. Clin Cancer Res 2000;6:512-8.  Back to cited text no. 38
Otsuki T, Yamada O, Kurebayashi J, Moriya T, Sakaguchi H, Kunisue H, et al. Estrogen receptors in human myeloma cells. Cancer Res 2000;60:1434-41.  Back to cited text no. 39
Li Y, Meeran SM, Patel SN, Chen H, Hardy TM, Tollefsbol TO. Epigenetic reactivation of estrogen receptor-a (ERa) by genistein enhances hormonal therapy sensitivity in ERa-negative breast cancer. Mol Cancer 2013;12:9.  Back to cited text no. 40
Gu Y, Zhu CF, Dai YL, Zhong Q, Sun B. Inhibitory effects of genistein on metastasis of human hepatocellular carcinoma. World J Gastroenterol 2009;15:4952-7.  Back to cited text no. 41
Yanagihara K, Ito A, Toge T, Numoto M. Antiproliferative effects of isoflavones on human cancer cell lines established from the gastrointestinal tract. Cancer Res 1993;53:5815-21.  Back to cited text no. 42
Entschladen F, Drell TL 4th, Lang K, Joseph J, Zaenker KS. Tumour-cell migration, invasion, and metastasis: Navigation by neurotransmitters. Lancet Oncol 2004;5:254-8.  Back to cited text no. 43
Gu Y, Zhu CF, Iwamoto H, Chen JS. Genistein inhibits invasive potential of human hepatocellular carcinoma by altering cell cycle, apoptosis, and angiogenesis. World J Gastroenterol 2005;11:6512-7.  Back to cited text no. 44
Fritz WA, Coward L, Wang J, Lamartiniere CA. Dietary genistein: Perinatal mammary cancer prevention, bioavailability and toxicity testing in the rat. Carcinogenesis 1998;19:2151-8.  Back to cited text no. 45
Lamartiniere CA, Zhao YX, Fritz WA. Genistein: Mammary cancer chemoprevention, in vivo mechanisms of action, potential for toxicity, and bioavailability in rats. J Womens Cancer 2000;2:11-9.  Back to cited text no. 46
Lamartiniere CA, Moore JB, Brown NM, Thompson R, Hardin MJ, Barnes S. Genistein suppresses mammary cancer in rats. Carcinogenesis 1995;16:2833-40.  Back to cited text no. 47
Murrill WB, Brown NM, Zhang JX, Manzolillo PA, Barnes S, Lamartiniere CA. Prepubertal genistein exposure suppresses mammary cancer and enhances gland differentiation in rats. Carcinogenesis 1996;17:1451-7.  Back to cited text no. 48
Wang J, Eltoum IE, Lamartiniere CA. Dietary genistein suppresses chemically-induced prostate cancer in Lobund-Wistar rats. Cancer Lett 2001;12:321-9.  Back to cited text no. 49
Lee JY, Kim HS, Song YS. Genistein as a potential anticancer agent against ovarian cancer. J Tradit Complement Med 2012;2:96-104.  Back to cited text no. 50
Mäkelä SI, Pylkkänen LH, Santti RS, Adlercreutz H. Dietary soybean may be antiestrogenic in male mice. J Nutr 1995;125:437-45.  Back to cited text no. 51
Messina M, Barnes S, Setchel KD. Phytoestrogens and breast cancer-commentary. Lancet 1997;350:971-72.  Back to cited text no. 52
Ingram D, Sanders K, Kolybaba M, Lopez D. Case-control study of phyto-oestrogens and breast cancer. Lancet 1997;350:990-4.  Back to cited text no. 53
Lee HP, Gourley L, Duffy SW, Estéve J, Lee J, Day NE. Dietary effects on breast-cancer risk in Singapore. Lancet 1991;337:1197-200.  Back to cited text no. 54
Adlercreutz H, Mazur W. Phyto-oestrogens and Western diseases. Ann Med 1997;29:95-120.  Back to cited text no. 55
Nikov GN, Hopkins NE, Boue S, Alworth WL. Interactions of dietary estrogens with human estrogen receptors and the effect on estrogen receptor-estrogen response element complex formation. Environ Health Perspect 2000;108:867-72.  Back to cited text no. 56
Hsieh CY, Santell RC, Haslam SZ, Helferich WG. Estrogenic effects of genistein on the growth of estrogen receptor-positive human breast cancer (MCF-7) cells in vitro and in vivo. Cancer Res 1998;58:3833-8.  Back to cited text no. 57
Maggiolini M, Bonofiglio D, Marsico S, Panno ML, Cenni B, Picard D. Estrogen receptor α mediates the proliferative but not the cytotoxic dose-dependent effects of two major phytoestrogens on human breast cancer cells. Mol Pharmacol 2001;60:595-602.  Back to cited text no. 58
Wang TT, Sathyamoorthy N, Phang JM. Molecular effects of genistein on estrogen receptor mediated pathways. Carcinogenesis 1996;17:271-5.  Back to cited text no. 59
Santell RC, Chang YC, Nair MG, Helferich WG. Dietary genistein exerts estrogenic effects upon the uterus, mammary gland and the hypothalamic/pituitary axis in rats. J Nutr 1997;127:263-9.  Back to cited text no. 60
Powis G, Hill SR, Frew TJ, Sherrill KW. Inhibitors of phospholipid intracellular signaling as antiproliferative agents. Med Res Rev 1995;15:121-38.  Back to cited text no. 61
Toi M, Mukaida H, Wada T, Hirabayashi N, Toge T, Hori T, et al. Antineoplastic effect of erbstatin on human mammary and esophageal tumors in athymic nude mice. Eur J Cancer 1990;26:722-4.  Back to cited text no. 62
Constantinou A, Kiguchi K, Huberman E. Induction of differentiation and DNA strand breakage in human HL-60 and K-562 leukemia cells by genistein. Cancer Res 1990;50:2618-24.  Back to cited text no. 63
Yamashita Y, Kawada S, Nakano H. Induction of mammalian topoisomerase II dependent DNA cleavage by nonintercalative flavonoids, genistein and orobol. Biochem Pharmacol 1990;39:737-44.  Back to cited text no. 64
Alhasan SA, Pietrasczkiwicz H, Alonso MD, Ensley J, Sarkar FH. Genistein-induced cell cycle arrest and apoptosis in a head and neck squamous cell carcinoma cell line. Nutr Cancer 1999;34:12-9.  Back to cited text no. 65
Li Y, Upadhyay S, Bhuiyan M, Sarkar FH. Induction of apoptosis in breast cancer cells MDA-MB-231 by genistein. Oncogene 1999;18:3166-72.  Back to cited text no. 66
Pagliacci MC, Smacchia M, Migliorati G, Grignani F, Riccardi C, Nicoletti I. Growth-inhibitory effects of the natural phyto-oestrogen genistein in MCF-7 human breast cancer cells. Eur J Cancer 1994;30A: 1675-82.  Back to cited text no. 67
Yousefi S, Blaser K, Simon HU. Activation of signaling pathways and prevention of apoptosis by cytokines in eosinophils. Int Arch Allergy Immunol 1997;112:9-12.  Back to cited text no. 68
Chang KL, Kung ML, Chow NH, Su SJ. Genistein arrests hepatoma cells at G2/M phase: Involvement of ATM activation and upregulation of p21waf1/cip1 and Wee1. Biochem Pharmacol 2004;67:717-26.  Back to cited text no. 69
Ravindranath MH, Muthugounder S, Presser N, Viswanathan S. Anticancer therapeutic potential of soy isoflavone, genistein. Adv Exp Med Biol 2004;546:121-65.  Back to cited text no. 70
Chodon D, Banu SM, Padmavathi R, Sakthisekaran D. Inhibition of cell proliferation and induction of apoptosis by genistein in experimental hepatocellular carcinoma. Mol Cell Biochem 2007;297:73-80.  Back to cited text no. 71
Huang EJ, Wu CC, Huang HP, Liu JY, Lin CS, Chang YZ, et al. Apoptotic and anti-proliferative effects of 17beta-estradiol and 17beta-estradiol-like compounds in the Hep3B cell line. Mol Cell Biochem 2006;290:1-7.  Back to cited text no. 72
Rajah TT, Peine KJ, Du N, Serret CA, Drews NR. Physiological concentrations of genistein and 17ß-estradiol inhibit MDA-MB-231 breast cancer cell growth by increasing BAX/BCL-2 and reducing pERK1/2. Anticancer Res 2012;32:1181-91.  Back to cited text no. 73
Moutsatsou P. The spectrum of phytoestrogens in nature: Our knowledge is expanding. Hormones (Athens) 2007;6:173-93.  Back to cited text no. 74
Adlercreutz H. Phyto-oestrogens and cancer. Lancet Oncol 2002;3:364-73.  Back to cited text no. 75
Barone M, Di Leo A. Estrogen receptor beta in colorectal cancer prevention: Do we have conclusive proof? J Genet Syndr Gene Ther 2013;4:11-9.  Back to cited text no. 76
Cotterchio M, Boucher BA, Manno M, Gallinger S, Okey A, Harper P. Dietary phytoestrogen intake is associated with reduced colorectal cancer risk. J Nutr 2006;136:3046-53.  Back to cited text no. 77
Saxena S, Jyoti, Sharma A. Soybean seeds – An approach to treatment of breast cancer. World J Pharm Pharma Sci 2009;3:1972-82.  Back to cited text no. 78
Huang YT, Cha TL, Sun GH. Estrogen inhibits renal cell carcinoma cell progression through estrogen receptor-β activation. PLoS One 2013;10:137-46.  Back to cited text no. 79
Luo Y, Waladali W, Li S, Zheng X, Hu L, Zheng H, et al. 17beta-estradiol affects proliferation and apoptosis of rat prostatic smooth muscle cells by modulating cell cycle transition and related proteins. Cell Biol Int 2008;32:899-905.  Back to cited text no. 80
Kim MJ, Cho SI, Lee KO, Han HJ, Song TJ, Park SH. Effects of 17ß-estradiol and estrogen receptor antagonists on the proliferation of gastric cancer cell lines. J Gastric Cancer 2013;13:172-8.  Back to cited text no. 81
Messa C, Russo F, Pricci M, Di Leo A. Epidermal growth factor and 17beta-estradiol effects on proliferation of a human gastric cancer cell line (AGS). Scand J Gastroenterol 2000;35:753-8.  Back to cited text no. 82
Messa C, Pricci M, Linsalata M, Russo F, Di Leo A. Inhibitory effect of 17beta-estradiol on growth and the polyamine metabolism of a human gastric carcinoma cell line (HGC-27). Scand J Gastroenterol 1999;34:79-84.  Back to cited text no. 83
Hall JM, Couse JF, Korach KS. The multifaceted mechanisms of estradiol and estrogen receptor signaling. J Biol Chem 2001;276:36869-72.  Back to cited text no. 84
Cross HS, Kallay E, Lechner D, Gerdenitsch W, Adlercreutz H, Armbrecht HJ. Phytoestrogens and vitamin D metabolism: A new concept for the prevention and therapy of colorectal, prostate, and mammary carcinomas. Am Soc Nutr Sci 2004;13:54-61.  Back to cited text no. 85
Miodini P, Fioravanti L, Di Fronzo G, Cappelletti V. The two phyto-oestrogens genistein and quercetin exert different effects on oestrogen receptor function. Br J Cancer 1999;80:1150-5.  Back to cited text no. 86