Silymarin inhibits Toll-like receptor 8 gene expression and apoptosis in Ramos cancer cell line

Document Type: Original Research Article

Authors

1 Student Scientific Research Center, Department of Clinical Biochemistry, School of Medicine, Zahedan University of Medical Sciences, Zahedan, Iran.

2 Department of Clinical Biochemistry, School of Medicine, Zahedan University of Medical Sciences, Zahedan, Iran.

3 Cellular and Molecular Research Center, Department of Clinical Biochemistry, School of Medicine, Zahedan University of Medical Sciences, Zahedan, Iran.

4 Department of Laboratory Sciences, Borujerd Branch, Islamic Azad University, Borujerd, Iran

Abstract

Objective: Silymarin is a herbal extract containing flavonolignans, and it has inhibitory effects against the growth of different cancer cell lines by inducing apoptosis. Toll-like receptors are suggested as a novel and attractive target to treat cancer. The current study aimed at examining the mechanism of silymarin-induced apoptosis in Ramos cells and investigating its effects on TLR8 expression.
Materials and Methods: The half maximal inhibitory concentration (IC50) of silymarin in Ramos cells was determined via MTT viability test while the type of cell death was tested by annexin V/propidium iodide (PI) double staining method. The activity of caspase-3 and expression of TLR8 were measured in a time-dependent manner (in IC50) by colorimetric assay and real-time polymerase chain reaction (RT-PCR), respectively.
Results: The results of MTT showed that IC50 of silymarin in Ramos cells was 100 μg/ml after 48 hr treatment (p<0.01). Flow cytometry by annexin V/PI, showed that silymarin induced early/late apoptosis in this cell line (p<0.05 to p<0.01). In addition, the caspase-3 colorimetric method showed that caspase-3 increased in the Ramos cell line after treatment (p<0.01). This treatment led to a reduction in TLR8 mRNA expression in a time-dependent manner (p<0.01).
Conclusion: The results indicated a new mechanism in the anticancer activity ofToll-like receptor (TLR) signaling after silymarin treatment in Ramos cancer cell line. This plant could be used to develop anticancer agents inhibiting TLRs.

Keywords

Main Subjects


Agarwal R, Agarwal C, Ichikawa H, Singh RP, Aggarwal BB. 2006. Anticancer potential of silymarin: from bench to bed side. Anticancer Res, 26: 4457-4498.

Barton GM, Kagan JC. 2009. A cell biological view of Toll-like receptor function: regulation through compartmentalization. Nat. Rev. Immunol, 9:535-542.

Bowie A, O'neill L. 2000. The interleukin-1 receptor/Toll-like receptor superfamily: signal generators for pro-inflammatory interleukins and microbial products. J Leukoc Biol, 67: 508-514.

Brosh R, Rotter V. 2009. When mutants gain new powers: news from the mutant p53 field.Nat Rev Cancer, 9: 701.

Burkitt D. 1958. A sarcoma involving the jaws in African children.Br J Surg, 46:218-23.

Burkitt DP. 1969. Etiology of Burkitt's lymphoma—an alternative hypothesis to a vectored virus. J Natl Cancer Inst, 42: 19-28.

Cherfils-vicini J, Platonova S, Gillard M, Laurans L, Validire P, Caliandro R, Magdeleinat P, Mami-chouaib F, Dieu-nosjean MC, Fridman WH. 2010a. Triggering of TLR7 and TLR8 expressed by human lung cancer cells induces cell survival and chemoresistance. J Clin Invest, 120:1285.

Cherfils-vicini J, Platonova S, Gillard M, Laurans L, Validire P, Caliandro R, Magdeleinat P, Mami-chouaib F, Dieu-nosjean MC, Fridman WH. 2010b. Triggering of TLR7 and TLR8 expressed by human lung cancer cells induces cell survival and chemoresistance. J Clin Invest, 120:1285-1297.

Chuang TH, Ulevitch RJ. 2000. Cloning and characterization of a sub-family of human toll-like receptors: hTLR7, hTLR8 and hTLR9. Eur Cytokine Netw, 11: 37.

Deep G, Oberlies NH, Kroll DJ, Agarwal R. 2008. Identifying the differential effects of silymarin constituents on cell growth and cell cycle regulatory molecules in human prostate cancer cells. Int J Cancer, 123: 41-50.

Faezizadeh Z, Mesbah-namin SAR, Allameh A. 2012. The effect of silymarin on telomerase activity in the human leukemia cell line K562. Planta Med, 78, 899-902.

Farrell PJ, Allan GJ, Shanahan F, Vousden K, Crook T. 1991. p53 is frequently mutated in Burkitt's lymphoma cell lines. EMBOJ, 10: 2879-2887.

Grimm M, Kim M, Rosenwald A, Heemann U, Germer CT, Waaga-gasser AM, Gasser M. 2010. Toll-like receptor (TLR) 7 and TLR8 expression on CD133+ cells in colorectal cancer points to a specific role for inflammation-induced TLRs in tumourigenesis and tumour progression. Eur J Cancer, 46: 2849-2857.

Grimmig T, Matthes N, Hoeland K, Tripathi S, Chandraker A, Grimm M, Moench R, Moll EM, Friess H, Tsaur I. 2015. TLR7 and TLR8 expression increases tumor cell proliferation and promotes chemoresistance in human pancreatic cancer. Int J Oncol, 47: 857-866.

Huang Q, Wul J, Tashiro SI, Onodera S, Li LH, Ikejima T. 2005. Silymarin augments human cervical cancer HeLa cell apoptosis via P38/JNK MAPK pathways in serum-free medium. J Asian Nat Prod RES, 7: 701-709.

Hussein MR. 2005. Apoptosis in the ovary: molecular mechanisms. Hum Reprod Update, 11: 162-178.

Kawai T, Akira S. 2006. TLR signaling. Cell Death Differ, 13: 816-825.

Kawai T, Akira S. 2011. Toll-like receptors and their crosstalk with other innate receptors in infection and immunity. Immunity, 34: 637-650.

Kellym G, Alvero AB, Chen R, Silasi DA, Abrahams VM, Chan S, Visintin I, Rutherford T, Mor G. 2006. TLR-4 signaling promotes tumor growth and paclitaxel chemoresistance in ovarian cancer. Cancer Res, 66: 3859-3868.

Kren V, Walterova D. 2005. Silybin and silymarin-new effects and applications. Biomedical Pap, 149:29-41.

Li ZJ, Yao C, Liu SF, Chen L, Xi YM, Zhang W, Zhang GS. 2014. Cytotoxic effect of icaritin and its mechanisms in inducing apoptosis in human burkitt lymphoma cell line. Biomed Res Int, 2014.

Marx J. 2004. Inflammation and cancer: the link grows stronger. Science, 306: 966.

Molavi O, Samadi N, Wu C, Lavasanifar A, Lai R. 2016. Silibinin suppresses NPM-ALK, potently induces apoptosis and enhances chemosensitivity in ALK-positive anaplastic large cell lymphoma. Leuk Lymphoma, 57: 1154-1162.

Pradere JP, Dapito DH, Schwabe RF. 2014. The Yin and Yang of Toll-like receptors in cancer. Oncogene, 33: 3485.

Pradhan S, Girish C. 2006. Hepatoprotective herbal drug, silymarin from experimental pharmacology to clinical medicine. Indian J Med Res, 124: 491.

Ramakrishnan G, Lo Muzio L, Elinos-baez CM, Jagan S, Augustine TA, Kamaraj S, Anandakumar P, Devaki T. 2009. Silymarin inhibited proliferation and induced apoptosis in hepatic cancer cells. Cell Proliferation, 42: 229-240.

Ramasamy K, Agarwal R. 2008. Multitargeted therapy of cancer by silymarin. Cancer Lett, 269, 352-362.

Rivlin N, Brosh R, Oren M, Rotter V. 2011. Mutations in the p53 tumor suppressor gene: important milestones at the various steps of tumorigenesis. Genes Cancer, 2: 466-474.

Romano B, Pagano E, Montanaro V, Fortunato AL, Milic N, Borrelli F. 2013. Novel insights into the pharmacology of flavonoids. Phytother Res, 27: 1588-1596.

Sato Y, Goto Y, Narita N, Hoon DS. 2009. Cancer cells expressing toll-like receptors and the tumor microenvironment. Cancer Microenviron, 2: 205-214.

Shanmugam M K, Kannaiyan R, Sethi G. 2011. Targeting cell signaling and apoptotic pathways by dietary agents: role in the prevention and treatment of cancer. Nutr Cancer, 63: 161-173.

Su CH, Chen LJ, Liao J F, Cheng JT. 2013. Increase of phosphatase and tensin homolog by silymarin to inhibit human pharynx squamous cancer. J Med Food,16:778-784.

Tyagi A, Raina K, Singh RP, Gu M, Agarwal C, Harrison G, Glode LM, Agarwal R. 2007. Chemopreventive effects of silymarin and silibinin on N-butyl-N-(4-hydroxybutyl) nitrosamine–induced urinary bladder carcinogenesis in male ICR mice. Mol Cancer Ther, 6: 3248-3255.

Unitt J, Hornigold D. 2011. Plant lectins are novel Toll-like receptor agonists. Biochem Pharmacol, 81: 1324-1328.

Vousden KH, Crook T, Farrell PJ. 1993. Biological activities of p53 mutants in Burkitt's lymphoma cells. J Gen Virol, 74: 803-810.

Wang CY, Guttridge DC, Mayo MW, Baldwin S. 1999. NF-κB induces expression of the Bcl-2 homologue A1/Bfl-1 to preferentially suppress chemotherapy-induced apoptosis. Mol Cell Biol, 19: 5923-5929.

Wang R, Miyahara Y, Wang H. 2008. Toll-like receptors and immune regulation: implications for cancer therapy. Oncogene, 27: 181.

Wen Z, Dumas TE, Schrieber SJ, Hawke RL, Fried MW, Smith PC. 2008. Pharmacokinetics and metabolic profile of free, conjugated, and total silymarin flavonolignans in human plasma after oral administration of milk thistle extract. Drug Metab Dispos, 36: 65-72.

Yu HC, Chen LJ, Cheng KC, Li YX, Yeh CH, Cheng JT. 2012. Silymarin inhibits cervical cancer cell through an increase of phosphatase and tensin homolog. Phytother Res, 26: 709-715.

Zhang Y, Yang H, Barnie PA, Yang P, Su Z, Chen J, Jiao Z, Lu L, Wang S, Xu H. 2014. The expression of Toll-like receptor 8 and its relationship with VEGF and Bcl-2 in cervical cancer. Int J Med Sci, 11: 608.