Antiproliferative and Antitelomerase Effects of Silymarin on Human Colorectal and Hepatocellular Carcinoma Cells

Abstract

Silymarin, a widely-used hepatoprotective agent, has shown antitumor properties in both in vitro and animal studies. Currently, there is limited knowledge regarding silymarinʼs antitelomerase effects on human colorectal cancer and hepatocyte carcinoma cells. In this study, we investigated the antiproliferative and antitelomerase effects of silymarin on four human colorectal cancer and HepG2 hepatocyte carcinoma cell lines. The cell viability and telomerase activity were assessed using MTT and the telomerase repeat amplification protocol assay, respectively. We also investigated the effects of silymarin on the expression of human telomerase reverse transcriptase and its promoter methylation in HepG2 cells by real-time RT-PCR and methylation-specific PCR, respectively. Silymarin treatment inhibited cell proliferation and telomerase activity in all cancer cells. After 24 h of treatment, silymarin exhibited IC₅₀ values ranging from 19 – 56.3 µg/mL against these cancer cells. A 30-min treatment with silymarin at the IC₅₀ concentration effectively inhibited telomerase activity in cell-free extracts of both colorectal cancer and hepatocyte carcinoma cells. Treatment of HepG2 cells with 10 and 30 µg/mL of silymarin for 48 h resulted in a decrease in human telomerase reverse transcriptase expression to 75 and 35% of the level observed in the untreated control (p < 0.01), respectively. Treatment with silymarin (10, 30, and 60 µg/mL) for 48 h did not affect human telomerase reverse transcriptase promoter methylation in HepG2 cells. In conclusion, our findings suggest that silymarin inhibits cancer cell growth by directly inhibiting telomerase activity and downregulating its human telomerase reverse transcriptase catalytic subunit. However, silymarin did not affect human telomerase reverse transcriptase promoter methylation at the concentrations of 10 – 60 µg/mL used in this study.

Keywords

Silybum marianum - Asteraceae - Telomerase - hTERT - colorectal cancer cells - hepatocarcinoma cells

Publication History

Received: 23 September 2023

Accepted after revision: 14 January 2024

Accepted Manuscript online:
14 January 2024

Article published online:
08 February 2024

• References

• 1 Gazák R, Walterová D, Kren V. Silybin and silymarin–new and emerging applications in medicine. Curr Med Chem 2007; 14: 315-338
• 2 Féher J, Lengyel G. Silymarin in the prevention and treatment of liver diseases and primary liver cancer. Curr Pharm Biotechnol 2012; 13: 210-217
• 3 Wang Y, Yuan AJ, Wu YJ, Wu LM, Zhang L. Silymarin in cancer therapy: Mechanisms of action, protective roles in chemotherapy-induced toxicity, and nanoformulations. J Funct Foods 2023; 100: 105384
• 4 Velmurugan B, Gangar SC, Kaur M, Tyagi A, Deep G, Agarwal R. Silibinin exerts sustained growth suppressive effect against human colon carcinoma SW480 xenograft by targeting multiple signaling molecules. Pharm Res 2010; 27: 2085-2097
• 5 Hogan FS, Krishnegowda NK, Mikhailova M, Kahlenberg MS. Flavonoid, silibinin, inhibits proliferation and promotes cell-cycle arrest of human colon cancer. J Surg Res 2007; 143: 58-65
• 6 Kauntz H, Bousserouel S, Gossé F, Raul F. Epigenetic effects of the natural flavonolignan silibinin on colon adenocarcinoma cells and their derived metastatic cells. Oncol Lett 2013; 5: 1273-1277
• 7 Mateen S, Raina K, Agarwal C, Chan D, Agarwal R. Silibinin synergizes with histone deacetylase and DNA methyltransferase inhibitors in upregulating E-cadherin expression together with inhibition of migration and invasion of human non-small cell lung cancer cells. J Pharmacol Exp Ther 2013; 345: 206-214
• 8 Kumar S, Raina K, Agarwal C, Agarwal R. Silibinin strongly inhibits the growth kinetics of colon cancer stem cell-enriched spheroids by modulating interleukin 4/6-mediated survival signals. Oncotarget 2014; 5: 4972-4989
• 9 Kauntz H, Bousserouel S, Gossé F, Raul F. Silibinin triggers apoptotic signaling pathways and autophagic survival response in human colon adenocarcinoma cells and their derived metastatic cells. Apoptosis 2011; 16: 1042-1053
• 10 Zvereva MI, Shcherbakova DM, Dontsova OA. Telomerase: structure, functions, and activity regulation. Biochemistry (Mosc) 2010; 75: 1563-1583
• 11 Harrington L. Biochemical aspects of telomerase function. Cancer Lett 2003; 194: 139-154
• 12 Corey DR. Telomeres and telomerase: From discovery to clinical trials. Chem Biol 2009; 16: 1219-1223
• 13 Iliopoulos D, Satra M, Drakaki A, Poultsides GA, Tsezou A. Epigenetic regulation of hTERT promoter in hepatocellular carcinomas. Int J Oncol 2009; 34: 391-399
• 14 Jäger K, Walter M. Therapeutic targeting of telomerase. Genes (Basel) 2016; 7: 39
• 15 Ganesan K, Xu B. Telomerase Inhibitors from natural products and their anticancer potential. Int J Mol Sci 2017; 19: 13
• 16 Nasiri M, Zarghami N, Koshki KN, Mollazadeh M, Moghaddam MP, Yamchi MR, Esfahlan RJ, Barkhordari A, Alibakhshi A. Curcumin and silibinin inhibit telomerase expression in T47D human breast cancer cells. Asian Pac J Cancer Prev 2013; 14: 3449-3453
• 17 Thelen P, Wuttke W, Jarry H, Grzmil M, Ringert RH. Inhibition of telomerase activity and secretion of prostate specific antigen by silibinin in prostate cancer cells. J Urol 2004; 171: 1934-1938
• 18 Faezizadeh Z, Mesbah-Namin SA, Allameh A. The effect of silymarin on telomerase activity in the human leukemia cell line K562. Planta Med 2012; 78: 899-902
• 19 Lee DD, Komosa M, Nunes NM, Tabori U. DNA methylation of the TERT promoter and its impact on human cancer. Curr Opin Genet Dev 2020; 60: 17-24
• 20 Lee DD, Leão R, Komosa M, Gallo M, Zhang CH, Lipman T, Remke M, Heidari A, Nunes NM, Apolónio JD, Price AJ, De Mello RA, Dias JS, Huntsman D, Hermanns T, Wild PJ, Vanner R, Zadeh G, Karamchandani J, Das S, Taylor MD, Hawkins CE, Wasserman JD, Figueiredo A, Hamilton RJ, Minden MD, Wani K, Diplas B, Yan H, Aldape K, Akbari MR, Danesh A, Pugh TJ, Dirks PB, Castelo-Branco P, Tabori U. DNA hypermethylation within TERT promoter upregulates TERT expression in cancer. J Clin Invest 2019; 129: 223-229
• 21 Raina K, Rajamanickam S, Singh RP, Deep G, Chittezhath M, Agarwal R. Stage-specific inhibitory effects and associated mechanisms of silibinin on tumor progression and metastasis in transgenic adenocarcinoma of the mouse prostate model. Cancer Res 2008; 68: 6822-6830
• 22 Kaur M, Velmurugan B, Tyagi A, Deep G, Katiyar S, Agarwal C, Agarwal R. Silibinin suppresses growth and induces apoptotic death of human colorectal carcinoma LoVo cells in culture and tumor xenograft. Mol Cancer Ther 2009; 8: 2366-2374
• 23 Ioset JR, Brun R, Wenzler T, Kaiser M, Yardley V. Drug Screening for Kinetoplastids Diseases: A Training Manual for Screening in Neglected Diseases. London, UK; Department for International Development; 2009
• 24 Heshmati M, Hasani-Reza Abad N, Pashapour S. Evaluating the effects of silymarin on expressing SBDSP1 and CASC11 genes in HCT116 colon cancer cells. J Kermanshah Univ Med Sci 2022; 26: e122802
• 25 Yassin NYS, AbouZid SF, El-Kalaawy AM, Ali TM, Almehmadi MM, Ahmed OM. Silybum marianum total extract, silymarin and silibinin abate hepatocarcinogenesis and hepatocellular carcinoma growth via modulation of the HGF/c-Met, Wnt/β-catenin, and PI3K/Akt/mTOR signaling pathways. Biomed Pharmacother 2022; 145: 112409
• 26 Widodo H, Rohman A. Ethyl acetate crude fraction of Macaranga subpeltata and silymarin increase Vero cell survival and HepG2 cell death due to oxidative stress. J Appl Pharm Sci 2023; 13: 108-115
• 27 Ramakrishnan G, Lo Muzio L, Elinos-Báez CM, Jagan S, Augustine TA, Kamaraj S, Anandakumar P, Devaki T. Silymarin inhibited proliferation and induced apoptosis in hepatic cancer cells. Cell Prolif 2009; 42: 229-240
• 28 Yurtcu E, İşeri Ö, Sahin F. Genotoxic and cytotoxic effects of doxorubicin and silymarin on human hepatocellular carcinoma cells. Hum Exp Toxicol 2014; 33: 1269-1276
• 29 Tatsumoto N, Hiyama E, Murakami Y, Imamura Y, Shay JW, Matsuura Y, Yokoyama T. High telomerase activity is an independent prognostic indicator of poor outcome in colorectal cancer. Clin Cancer Res 2000; 6: 2696-2701
• 30 Amirsaadat S, Pilehvar-Soltanahmadi Y, Zarghami F, Alipour S, Ebrahimnezhad Z, Zarghami N. Silibinin-loaded magnetic nanoparticles inhibit hTERT gene expression and proliferation of lung cancer cells. Artif Cells Nanomed Biotechnol 2017; 45: 1649-1656
• 31 Chatran M, Pilehvar-Soltanahmadi Y, Dadashpour M, Faramarzi L, Rasouli S, Jafari-Gharabaghlou D, Asbaghi N, Zarghami N. Synergistic anti-proliferative effects of metformin and silibinin combination on T47D breast cancer cells via hTERT and cyclin D1 inhibition. Drug Res (Stuttg) 2018; 68: 710-716
• 32 Bagherpour B, Gharagozloo M, Moayedi B. The influence of iron loading and iron chelation on the proliferation and telomerase activity of human peripheral blood mononuclear cells. Iran J Immunol 2009; 6: 33-39
• 33 Parzonko A, Naruszewicz M. Silymarin inhibits endothelial progenitor cellsʼ senescence and protects against the antiproliferative activity of rapamycin: preliminary study. J Cardiovasc Pharmacol 2010; 56: 610-618
• 34 Horikawa I, Michishita E, Barrett JC. Regulation of hTERT transcription: A target of cellular and viral mechanisms for immortalization and carcinogenesis. Cytotechnology 2004; 45: 23-32
• 35 Gaylor DW. Are tumor incidence rates from chronic bioassays telling us what we need to know about carcinogens?. Regul Toxicol Pharmacol 2005; 41: 128-133
• 36 Salmani-Javan E, Jafari-Gharabaghlou D, Bonabi E, Zarghami N. Fabricating niosomal-PEG nanoparticles co-loaded with metformin and silibinin for effective treatment of human lung cancer cells. Front Oncol 2023; 13: 1193708
• 37 Ito H, Kyo S, Kanaya T, Takakura M, Inoue M, Namiki M. Expression of human telomerase subunits and correlation with telomerase activity in urothelial cancer. Clin Cancer Res 1998; 4: 1603-1608
• 38 Dogan F, Forsyth NR. Telomerase regulation: A role for epigenetics. Cancers (Basel) 2021; 13: 1213
• 39 Ramlee MK, Wang J, Toh WX, Li S. Transcription Regulation of the Human Telomerase Reverse Transcriptase (hTERT) Gene. Genes (Basel) 2016; 7: 50
• 40 Kim W, Ludlow AT, Min J, Robin JD, Stadler G, Mender I, Lai TP, Zhang N, Wright WE, Shay JW. Regulation of the human telomerase gene TERT by telomere position effect-over long distances (TPE-OLD): Implications for aging and cancer. PLoS Biol 2016; 14: e2000016
• 41 Leão R, Apolónio JD, Lee D, Figueiredo A, Tabori U, Castelo-Branco P. Mechanisms of human telomerase reverse transcriptase (hTERT) regulation: clinical impacts in cancer. J Biomed Sci 2018; 25: 22
• 42 Nemos C, Rémy-Martin JP, Adami P, Arbez-Gindre F, Schaal JP, Jouvenot M, Delage-Mourroux R. Improved TRAP-silver staining versus conventional radioactive TRAP assays: Quantification of telomerase activity during immortalization and in pathological human endometrium. Clin Biochem 2003; 36: 621-628
• 43 Tao SF, Zhang CS, Guo XL, Xu Y, Zhang SS, Song JR, Li R, Wu MC, Wei LX. Anti-tumor effect of 5-aza-2′-deoxycytidine by inhibiting telomerase activity in hepatocellular carcinoma cells. World J Gastroenterol 2012; 18: 2334-2343
• 44 Pakdel A, Naghibalhossaini F, Mokarram P, Jaberipour M, Hosseini A. Regulation of carcinoembryonic antigen release from colorectal cancer cells. Mol Biol Rep 2012; 39: 3695-3704
• 45 Sarabi MM, Naghibalhossaini F. The impact of polyunsaturated fatty acids on DNA methylation and expression of DNMTs in human colorectal cancer cells. Biomed Pharmacother 2018; 101: 94-99