The Role of Milk Thistle Extract in Breast Carcinoma Cell Line (MCF-7) Apoptosis with Doxorubicin

  • Hussein Rastegar Food and Drug Research Center, Ministry of Health and Medical Education, Tehran, Iran.
  • Hamidreza Ahmadi Ashtiani Department of Biochemistry and Nutrition, School of Medicine, Zanjan University of Medical Sciences, Zanjan, Iran. AND Department of Clinical Biochemistry, School of Medicine, Tarbiat Modares University and Institue of Medicinal Plants, Tehran, Iran. AND Department of Nutritional Hygiene, Faculty of Veterinary, Tehran University of Medical Sciences, Tehran, Iran.
  • Soghra Anjarani Reference Research Center Lab, Ministry of Health and Medical Education, Tehran, Iran.
  • Saeed Bokaee Faculty of Veterinary Medicine, Tehran University of Medical Sciences, Tehran, Iran.
  • Arash Khaki Women's Reproductive Health Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.
  • Leila Javadi Mail Department of Nutrition Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.
Keywords:
Apoptosis, Doxorubicin, Human carcinoma, MCF-7 cells, .Silymarin, Trypan Blue

Abstract

Breast cancer is the most commonly diagnosed invasive malignancy and first leading cause of cancer-related deaths in Iranian women. Based on silymarin's unique characteristics, its application in chemotherapy combined with doxorubicin can be effective to enhance the efficacy together with a reduced toxicity on normal tissues. The present study focus on evaluate the efficacy of silymarin in combination with doxorubicin, on viability and apoptosis of estrogen-dependent breast carcinoma cell line (MCF-7). After being cultured, MCF-7 cells were divided into 8 groups and treated as follows: 1st group received 75 μg silymarin, groups 2, 3, and 4 were treated with 10, 25, and 50 nM doxorubicin, respectively, and groups 5, 6, and 7 respectively received 10, 25, and 50 nM doxorubicin as well as 75 μg silymarin. Viability percentage and apoptosis of the cells were assessed with Trypan Blue staining after 16, 24, and 48 hours. Silymarin has a synergistic effect on the therapeutic potential of doxorubicin. Use of silymarin in combination with doxorubicin can be more effective on the therapeutic potential of doxorubicin and decreases its dose-limiting side effects.

References

Hortobagyi GN, de la Garza Salazar J, Pritchard K, Amadori D, Haidinger R, Hudis CA, Khaled H, Liu MC, Martin M, Namer M, O'Shaughnessy JA, Shen ZZ, Albain KS. The global breast cancer burden: variations in epidemiology and survival. Clinical Breast Cancer 2005;6(5): 391-401.

Eliassen AH, Missmer SA, Tworoger SS, Spiegelman D, Barbieri RL, Dowsett M, Hankinson SE. Endogenous steroid hormone concentration and risk of breast cancer among premenopousal women. Janti Cancer Inst 2006;98(19):1406-15

Pourhoseingoli MA, Mehrabi Y, Alavi-majd H, Yavari P, Safaee A. Association between risk of breast cancer and fertility factors-a latent variable approach. Asian Pac J Center Prev 2008;9(2):309-12.

Tyagi A.K, Agarwal C, Chan D.C.F, Agarwal R. Synergistic anti-cancer effects of silibinin with conventional cytotoxic agents doxorubicin, cisplatin and carboplatin against human breast carcinoma MCF-7 and MDA-MB468 cells. Oncology Reports 2004;11:493-9.

Fournier DB, Erdman JW, Gordon GB. Soy, its components, and cancer prevention: a review of the in vitro, animal, and human data. Cancer Epidemiol Biomark Prev 1998;7:1055-65.

Hortobagyi GN. Chemotherapy of breast cancer, a historical perspective. Semin Oncol 1997;1(7):S1-4.

Martin M. Platinum compounds in the treatment of advanced breast cancer. Clin Breast Cancer 2001;2(3):190-208.

Tyagi AK, Singh RP, Agarwal C, Chan DCF, Agarwal R. Silibinin strongly synergizes human prostate carcinoma DU145 cells to Dox-induced growth inhibition, G2-M arrest and apoptosis. Clin Cancer Res 2002;8:3512-19.

Jiang C, Agarwal R, Lu J. Anti-Angiogenic Potential of a Cancer Chemopreventive Flavonoid Antioxidant, Silymarin: Inhibition of Key Attributes of VascularEndothelial Cells and Angiogenic Cytokine Secretion by Cancer Epithelial Cells. Biochemical and Biophys Res Commun 2000;16;276(1): 371–8.

Katiyar SK. Silymarin and skin cancer prevention: Antiinflammatory, antioxidant and immunomodulatory effects (Review). Int J Oncol 2005;26(1):169-76.

Jamshidi A, Ahmadi-Ashtiani HR, Naderi MM, Bokaei S,m Gholamhouseyni B. Study on topical use of Silybum marianum extract (Silymarin) on U.V. irradiated guinea pig skin. Medical Plants 2007;6(24):92-100

Zhang S, Morris M, E. Effects of the Flavonoids Biochanin A, Morin, Phloretin, and Silymarin on P-Glycoprotein- Mediated Transport. Pharm Res 2003;304(3):1258-67.

Zi X, Feyes DK, Agarwal R. Anticarcinogenic effect of a flavonoid antioxidant, silymarin, in Human Breast Cancer Cells MDA-MB 468: Induction of G1 Arrest through an increase in Cip/p21 Concomitant with a Decrease in Kinase Activity of Cyclin-dependent Kinases and Associated Cyclins. Clinical Cancer Research 1998;4:1055-64.

Crown JP. The platinum agents. a role in breast cancer treatment. Semin Oncol 2001;2(8):28-37.

Kelloff GJ. Perspectives on cancer chemoprevention research and drug development. Adv Cancer Res 2000;7(8):199-334.

Dhanalakshmi S, Singh RP، Agarwal C, Agarwal R. Silibinin inhibits constitutive and TNF·-induced activationof NF-κB and sensitizes human prostate carcinoma DU145 cells to TNF·-induced apoptosis. Oncogene 2002;2(1):1759-67.

Singh RP, Tyagi AK, Zhao J, Agarwal R. Silymarin inhibits growth and causes regression of established skin tumors in SENCAR mice via modulation of mitogenactivated protein kinases and induction of apoptosis. Carcinogenesis 2002;2(3):499-510.

Singh RP, Dhanalakshmi S, Tyagi AK, Chan DCF, Agarwal C, Agarwal R. Dietary feeding of silibinin inhibits advance human prostate carcinoma growth in athymic nude mice، and increases plasma insulin-like growth factor-binding protein-3 levels. Cancer Res. 2002;6(2):3063-9.

Wellington K, Jarwis B. Silymarin: a review of its clinical properties in the management of hepatic disorders. Bio Drugs 2001;1(5):465-89.

Azari S, Ahmadi N, Tehrani MJ, Shokri F. Profiling and authentication of human cell lines using shattandem repeat(STR)loci: Report from the national cell bank of Iran. Biologicals 2007;35(3):196-202.

Zhang S, Yang X, E. Morris M. Flavonoids Are Inhibitors of Breast Cancer Resistance Protein (ABCG2)-Mediated Transport. Mol Pharmacol 2004;65(5):1208-16.

Pontes H, Carvalho M, De Pinho P.G, Carmo H, Remião F, Carvalho F, Bastos ML. Ethanol, the forgotten artifact in cell culture. Arch Toxicol 2008;82(3):197-8.

Jain PT, Pento JT. A vehicle for the evaluation of hydrophobic compounds in cell culture. Res Chem Pathol Pharmacol 1991;74(1):105-16.

Koyuturk M, Ersoz M, Altiok N. Simvastatin induces apoptosis in human breast cancer cells: p53 and estrogen receptor independent pathway requiring signalling through JNK. Cancer Letters 2007;250:220–8.

Bentrari1 F, Arnould L, Jackson A.P, Jeannin J, Pance A. Progesterone enhances cytokine-stimulated nitric oxide synthase II expression and cell death in human breast cancer cells. Laboratory Investigation (LI) 2005;85: 624–32.

Dhillon NK, Mudryj M. Cyclin E overexpression enhances cytokine-mediated apoptosis in MCF7 breast cancer cells. Genes and Immunity 2003;4:336-42.

Moldeus T, Hogberg J, Orrhenius S, Fleischer S, Packer L. Trypan blue dye exclusion method. Methods in Enzymology 1978;52:60-71.

Syntichaki P, Tavernarakis N. Death by necrosis, Uncontrollable catastroph or is there order behind the chaos? EMBO 2002;3 (7):604-9.

Katiyar SK, Roy AM, Baliga MS. Silymarin induces apoptosis primarily through a p53-dependent pathway involving Bcl-2/Bax, cytochrome c release, and caspase activation. Molecular Cancer Therappy 2005;4(2):207-16.

Hortobagyi G.N. Progress in Systemic Chemotherapy of Primary Breast Cancer: an Overview. J Natl Cancer Inst Monogr 2001;30:72-9.

Figg W. D, Arlen P, Gulley J, Fernandez P, Noone M, Fedenko K, Hamilton M, Parker C, Kruger E. A, Pluda J, Dahut WL. A randomized Phase II trial of docetaxel (taxotere) plus thalidomide in androgen-independent prostate cancer. Semin Urol Oncol 2001;2(8):62-6.

Haas N. B.Can chemotherapy alters the course of prostate cancer. Semin Urol Oncol 2001;1(9):212-21.

Kren V, Walterova D. Silibin and silimarin-new effects and applications. Biomedical Papers 2005;149(1):29-41.

Liu D S, Krebs C E, Liu SJ. Prolifration of human breast cancer cells and anti-cancer action of doxorubicin and vinblastine are independent of PKC-alpha. J Cell Biochem 2007;101(2):517-28.

Hortobagyi GN. Anthracyclines in the treatment of cancer. Drugs 1997;54(4):1-7.

Seidman A, Hudis C, Pierri MK, Shak S, Paton V, Ashby M. Cardiac dysfunction in the trastuzumab clinical trials experience. J Clin Oncol 2002;20(5):1215-21.

Pai VB, Nahata MC. Cardiotoxicity of chemotherapeutic agents: incidence, treatment and prevention. Drug Saf 2000;22:263-302.

Swain S, Whaley FS, Ewer MS. Congestive heart failure in patients treated with doxorubicin: a retrospective analysis of three trials. Cancer 2003; 97:2869-79.

O'Brien, Mary ER. Single-agent treatment with pegylated liposomal doxorubicin for metastatic breast cancer. Anti- Cancer Drugs 2008; 19(1):1-7.

Jamshidi A, Ahmadi-Ashtiani H, Oliazadeh N, Jafarzadeh M, Taheri-Brojerdy M, Naderi M. A key for the thousand lock (Complete review of silymarin and introduction of Silybum marianum). No Avar Publisher 1386:187-323.

Chlopčíková A, Psotová J, Miketová P, Šimánek V. Chemoprotective effect of plant phenolics against anthracycline-induced toxicity on rat cardiomyocytes. Part I. Silymarin and its flavonolignans. Phytoterapy Res 2004;1(8):107-10.

Psotová J, Chlopčíková S, Grambal F, Šimánek V, Ulrichová J. Influence of silymarin and its flavonolignans on doxorubicin-iron induced lipid peroxidation in rat heart microsomes and mitochondria in comparison with quercetin. Phytother Res 2002;1(6):S63-7.

Thigpen JT. Innovations in anthracycline therapy: overview. Community Oncol 2005;2(1):3-7.

Orditura M, Quaglia F, Morgillo F, Martinelli E, Lieto E, de Rosa G. Pegylated liposomal doxorubicin: pharmacologic and clinical evidence of potent antitumor activity with reduced anthracycline-induced cardiotoxicity (review). Oncol Rep 2004;12:549. 45. Bosisio E, Benelli C, Pirola O. Effect of the flavanolignans of Silybum marianum L. on lipid peroxidation in rat livermicrosomes and freshly isolated hepatocytes. Pharmacol Res 1992;25: 147-54.

Basaga H, Poli G, Tekkaya C. Free radical scavenging and antioxidative properties of ‘silibin’ complexes on microsomal,lipid peroxidation. Cell Biochem 1997;15:27-33.

Baer-Dubowska W, Szaefer H, Krajka-Kuzniak V. Inhibition of murine hepatic cytochrome P450 activities by natural and synthetic phenolic compounds. Xenobiotica1998;28:735-43.

Kim DH, Jin YH, Park JB, Kobashi K. Silymarin and its components are inhibitors of beta-glucuronidase. Biol Pharm Bull 1994;17:443-5.

Sonnenbichler J, Goldberg M, Hane L. Stimulatory effect of silibinin on the DNA synthesis in partially hepatectomized rat livers, non-response in hepatoma and other malignant cell lines. Biochem Pharmacol1986;35:538-41.

Zi X, Mukhtar H, Agarwal R. Novel cancer chemopreventive effects of a flavonoid antioxidant silymarin, inhibition of mRNA expression of an endogenous tumor promoter TNF-alpha. Biochem Biophys Res Commun 1997;239:334-9.

Lang I, Nekam K, Gonzalez-Cabello R. Hepatoprotective and immunological effects of antioxidant drugs. Tokai J Exp Clin Med 1990;15:123-7.

Dehmlow C, Murawski N, de Groot H . Scavenging of reactive oxygen species and inhibition of arachidonic acidmetabolism by silibinin in human cells. Life Sci 1996;58:1591-600.

Dehmlow C, Erhard J, de Groot H. Inhibition of Kupffer cell functions as an explanation for the hepatoprotectiveproperties of silibinin. Hepatology 1996;23:749-54.

Boigk G, Stroedter L, Herbst H. Silymarin retardscollagen accumulation in early and advanced biliary fibrosis secondary to complete bile duct obliteration inrats. Hepatology 1997;26:643-9.

Lee S.O, Jeong YJ, Gwon Im H, Kim C.H, Chang YC, Lee IS. Silibinin suppresses PMA-induced MMP-9 expression by blocking the AP-1 activation via MAPK signaling pathways in MCF-7 human breast carcinoma cells. BBRC 2007;354:165-71.

Raina K, Agarwal R. Combinatorial strategies for cancer eradication by silibinin and cytotoxic agents: efficacy and mechanisms. Acta Pharmacol Sin 2007;28(9):1466-75.

Malewicz B, Wang Z, Jiang C, Guo J, P.Cleary M, P. Grande J, Lu J. Enhancement of mammary carcinogenesis in two rodent models by silymarin dietary supplements. Carcinogenesis 2006;27(9):1739-47.

Darzynkiewicz Z , Pozarowski P, Lee BV, Johnson GL. Florochrome-labeied inhibitors of caspases;convenient in vitro and in vivo markers of apoptotic cells for cytometric analysis. Metods Mol Biol 2011;682:103-14.

Gain P, Thuret G, Chiquet G, Dumollard J.M, Mosnier J.F,Burillon C, Delbosc B, Hervé P. Value of two mortality assessment techniques for organ cultured corneal endothelium: trypan blue versus TUNEL technique. L Campos Ophthalmol 2002;86:306–310.

Perry SW, Epstein LG, Gelbard HA. Simultaneous in situdetection of apoptosis and necrosis in monolayer cultures by TUNEL and trypan blue staining. Biotechniques 1997;22(6):1102-6.

Yanga HL, Chenb CS, Changc WH, Luc FJ, Laia YC, Chend CC, Hseud TH, Kuoe CT, Hseue YC. Growthinhibition and induction of apoptosis in MCF-7 breast cancer cells by Antrodia camphorate. Cancer Letters 2006;231:215-22.

Singh RP, Mallikarjuna GU, Sharma G, Dhanalakshmi S, Tyagi AK, Chan DC, Agarwal C, Agarwal R. Oral silibinin inhibits lung tumor growth in athymic nude mice and forms a novel chemocombination with doxorubicin targeting nuclear factor B–mediated inducible chemoresistance. CCR 2004;10(24):8641-7.

Mereish KA, Bunner DL, Ragland DR, Creasia DA. Protection against microcystin-LR-induced hepatotoxicity by silymarin: biochemistry, histopathology, and lethality. Pharm Res 1991;8(2):273-7.

How to Cite
1.
Rastegar H, Ahmadi Ashtiani H, Anjarani S, Bokaee S, Khaki A, Javadi L. The Role of Milk Thistle Extract in Breast Carcinoma Cell Line (MCF-7) Apoptosis with Doxorubicin. Acta Med Iran. 51(9):591-598.
QRcode
Section
Articles