Synergistic anti-cancer effects of silibinin-etoposide combination against human breast carcinoma MCF-7 and MDA-MB-231 cell lines

Document Type : Original Article

Authors

1 Department of Clinical Biochemistry, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran

2 Toxicology Research Center, Department of Clinical Biochemistry, Faculty of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran

3 Department of Infection, Immunity, and Inflammation, University of Leicester, LE1 7RH, UK

4 Hyperlipidemia Research Center, Department of Clinical Biochemistry, Faculty of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran

Abstract

Objective(s): Recently, there is a significant focus on combination chemotherapy for cancer using a cytotoxic drug and a phytochemical compound. We investigated the effect of silibinin on etoposide-induced apoptosis in MCF-7 and MDA-MB-231 breast carcinoma cell lines.
Materials and Methods: The cytotoxic effects of silibinin and etoposide were determined using MTT assay after 24 and 48 hr incubation with these drugs individually and combined. The mRNA expression of Bax and Bcl2, and protein levels of P53, phosphorylated p53 (P-P53), and P21 were determined using real-time PCR and western blot analysis, respectively. The caspase 9 activity was measured using an ELISA kit.
Results: Silibinin and etoposide alone and combined significantly inhibit cell growth in a dose and time-dependent manner in both cell lines. The strongest synergistic effects in terms of MCF-7 cell growth inhibition [combination index (CI) = 0.066] were evident. The silibinin-etoposide combinations cause a much powerful apoptotic death (47% and 40%) compared with each compound individually in MCF-7 and MDA-MB 231 cells, respectively. Additionally, the silibinin-etoposide combinations significantly increased the expression of P53, P-P53, and P21 in MCF-7 cells. Neither silibinin nor etoposide individually increased the level of P53 and P-P53 in MDA-MB-231 cells, but both of them individually and combined increased the level of P21.
Conclusion: Since the silibinin-etoposide combination induces apoptosis in both cell lines with and without expression of p53, thus, it is suggested that this combination may be a successful therapeutic strategy for breast cancer regardless of P53 status.

Keywords


1. Momenimovahed Z, Salehiniya H. Epidemiological characteristics of and risk factors for breast cancer in the world. Breast Cancer (Dove Med Press) 2019; 11: 151–164.
2. Ji X, Lu Y, Tian H, Meng X, Wei M, Chod WC. Chemoresistance mechanisms of breast cancer and their countermeasures. Biomed Pharmacother 2019; 114:108800.
3. Muley H, Fadó R, Rodríguez-Rodríguez R, Casals N. Drug uptake-based chemoresistance in breast cancer treatment. Biochem Pharmacol 2020; 177:113959.
4. Wang X, Haiyun Zhang H, Chen X. Drug resistance and combating drug resistance in Cancer. Cancer Drug Resist 2019; 2:141-160.
5. Senapati S, Mahanta AK, Kumar S, Maiti P. Controlled drug delivery vehicles for cancer treatment and their performance. Signal Transduct Target Ther 2018; 3:7.
6. Pucci C, Martinelli C, Ciofani G. Innovative approaches for cancer treatment: current perspectives and new challenges. Ecancermedicalscience 2019; 13:961.
7. Bayat Mokhtari R, Homayouni TS, Baluch N, Morgatskaya E, Kumar S, Das B, et al. Combination therapy in combating cancer. Oncotarget 2017; 8: 38022-38043.
8. Al-Mahayri ZN, Patrinos GP, Ali BR. Toxicity and pharmacogenomic biomarkers in breast cancer chemotherapy. Front Pharmacol 2020; 11: 445.
9. Abotaleb M, Samuel SM, Varghese E, Varghese S. Kubatka P, Liskova A, et al. Flavonoids in Cancer and Apoptosis. Cancers (Basel) 2019; 11: 28.
10. Taleb A, Ahmad KA, Ihsan AU, Qu J, Lin N, Hezam K, et al. Anti-oxidant effects and mechanism of silymarin in oxidative stress induced cardiovascular diseases. Biomed Pharmacother 2018; 102: 689-698.
11. Yang N, Jia X, Wang D, Wei C, He Y, Chen L, et al. Silibinin as a natural anti-oxidant for modifying polysulfone membranes to suppress hemodialysis-induced oxidative stress. J Memb Sci 2019;574: 86-99.
12. Binienda A, Ziolkowska Z, Pluciennik E. The Anticancer properties of silibinin: Its molecular mechanism and therapeutic effect in breast cancer. Anticancer Agents Med Chem 2020; 20:1787-1796.
13. Binienda A, Ziolkowska S, Pluciennik E. The anticancer properties of silibinin: its molecular mechanism and therapeutic effect in breast cancer. Anticancer Agents Med Chem 2020; 20:1787-1796.
14. Bayram D, Çetin ES, Kara M, Özgöçmen M, Candan IA. The apoptotic effects of silibinin on MDA-MB-231 and MCF-7 human breast carcinoma cells. Hum Exp Toxicol 2017; 36:573- 586.
15. Singh RP, Agarwal R. Prostate cancer chemoprevention by silibinin: Bench to bedside. Mol Carcinog 2006; 45:436-42.
16. Flaig TW, Gustafson AL, Su LJ, Zirrolli JA, Crighton F, Harrison GS, et al. A phase I and pharmacokinetic study of silybin-phytosome in prostate cancer patients. Invest New Drugs 2007; 25:139-146.
17. Molavi O, Narimani F, Asiaee F, Sharifi S, Tarhriz V, Shayanfar A. Silibinin sensitizes chemo-resistant breast cancer cells to chemotherapy. Pharm Biol 2017; 55:729-739.
18. Delmas D, Xiao J, Vejux A, Aires V. Silymarin and cancer: A dual strategy in both in chemoprevention and chemosensitivity. Molecules 2020; 25:2009.
19. Khedri A, Khaghani S, Kheirollah A, Babaahmadi-Rezaei H, Shadboorestan A, Zangooei M, et al. Signaling crosstalk of FHIT, p53, and p38 in etoposide-induced apoptosis in MCF-7 cells. J Cell Biochem 2019; 120:9125-9137.
20. Montecucco A, Zanetta F, Biamonti G. Molecular mechanisms of etoposide. EXCLI Journal 2015; 14: 95-108.
21. Feroz W, Sheikh MA. Exploring the multiple roles of guardian of the genome: P53: Egypt  J Med Hum Genet 2020; 21:49.
22. Chou TC. Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies. Pharmacol Rev 2006;58: 621–681.
23. Zhao CY, Cheng R, Yang Z, Tian ZM. Nanotechnology for cancer therapy based on chemotherapy. Molecules 2018; 23: 826.
24. Bukowski K, Kciuk M, Kontek R. Mechanisms of multidrug resistance in cancer chemotherapy. Int J Mol Sci 2020; 21: 3233.
25. Mohammad RM, Muqbil I, Lowe L, Yedjou C, Hsu HY, Lin LT, et al. Broad targeting of resistance to apoptosis in cancer. Seminars in Cancer Biology 2015;35: S78–S103.
26. Hu Q, Sun W, Wang C, Gu Z. Recent advances of cocktail chemotherapy by combination drug delivery systems. Adv Drug Deliv Rev 2016; 98: 19–34.
27. Wang X, Simpson ER, Brown KA. p53: Protection against tumor growth beyond effects on cell cycle and apoptosis. Cancer Res 2015; 75: 5001-5007.
28. Hientz K, Mohr A, Bhakta-Guha D, Efferth T. The role of p53 in cancer drug resistance and targeted chemotherapy. Oncotarget 2017;8: 8921-8946.
29. Lieb MC, Hofmann TG. The role of p53 signaling in colorectal cancer. Cancers 2021; 13: 2125.
30. Michel M, Kaps L, Maderer A, Galle PR, Moehler M. The role of p53 dysfunction in colorectal cancer and its implication for therapy. Cancers 2021; 13: 2296.  
31. Agarwal C, Singh RP, Dhanalakshmi S, Tyagi AK, Tecklenburg M, Sclafani RA, et al. Silibinin upregulates the expression of cyclin-dependent kinase inhibitors and causes cell cycle arrest and apoptosis in human colon carcinoma HT-29 cells. Oncogene 2003;22: 8271–8282.
32. Sameri S, Saidijam M, Bahreini F, Najafi R. Cancer chemopreventive activities of silibinin on colorectal cancer through regulation of ecadherin/β-Catenin 33. pathway. Nutr Cancer 2020; 4:1-11.
33. Lu S, Zhang Z, Chen M, Li C, Liu L, Li Y. Silibinin inhibits the migration and invasion of human gastric cancer SGC7901 cells by downregulating MMP-2 and MMP-9 expression via the p38MAPK signaling pathway. Oncol Lett 2017; 14: 7577-7582.
34. Pfeffer CM, T. K. Singh A. Apoptosis: A target for anticancer therapy. Int J Mol Sci 2018; 19: 448.
35. Bruni E, Reichle A, Scimeca M, Bonanno E, Ghibelli L. Lowering etoposide doses shifts cell demise from caspase- dependent to differentiation and caspase-3-independent apoptosis via DNA damage response, inducing AML culture extinction. Front Pharmacol 2018; 9:1307.
36. Maleki Zadeh M, Motamed N, Ranji N, Majidi M, Falahi F. Silibinin-induced apoptosis and downregulation of microRNA-21 and microRNA-155 in MCF-7 human breast cancer cells. J Breast Cancer 2016; 19:45-52.
37. Nam C, Doi K, Nakayama H. Etoposide induces G2/M arrest and apoptosis in neural progenitor cells via DNA damage and an ATM/p53-related pathway. Histol Histopathol 2010;25: 485-493.
38. Kluska M, Katarzyna Wo┼║niak K. Natural polyphenols as modulators of etoposide anti-cancer activity. Int J Mol Sci 2021; 22: 6602.  
39. Aubrey BJ, Kelly GL, Janic A, Herold MJ, Strasser A. How does p53 induce apoptosis and how does this relate to p53- mediated tumor suppression? Cell Death Differ 2018; 25: 104–113.
40. Huang Y, Liu N, Liu J, Liu Y, Zhang C, Long S, et al. Mutant p53 drives cancer chemotherapy resistance due to loss of function on activating transcription of PUMA. Cell Cycle 2019; 18:3442-3455.
41. MB Barros T, PB Lima A, C Almeida T, N da Silva G. Inhibition of urinary bladder cancer cell proliferation by silibinin. Environ Mol Mutagen 2020; 61:445-455.