Investigation of the effect of 5-Aza-2′-deoxycytidine on p15INK4, p16INK4, p18INK4, and p19INK4 genes expression, cell growth inhibition, and apoptosis induction in hepatocellular carcinoma PLC/PRF/5 cell line

Sanaei, Masumeh; Kavoosi, Fraidoon; Ghasemi, Ali

Investigation of the effect of 5-Aza-2′-deoxycytidine on p15INK4, p16INK4, p18INK4, and p19INK4 genes expression, cell growth inhibition, and apoptosis induction in hepatocellular carcinoma PLC/PRF/5 cell line

Authors

¹ Research Center for Non-Communicable Diseases, Jahrom University of Medical Sciences, Jahrom, Fars Province, Iran

² Student of Research Committee, Jahrom University of Medical Sciences, Jahrom, Fars Province, Iran

Abstract

Background: Cyclin-dependent kinases (CDKs) are the key regulators of cell-cycle transitions and characterized by needing a separate subunit, a cyclin, which provides domains essential for enzymatic activity. The activities of cyclin-CDK complexes are controlled by a group of molecules that inhibit CDK activity and CDK inhibitors (CKIs). Cancer often exhibits an aberrant CpG methylation of promoter regions of tumor suppressor genes such as CKIs. Treatment with the DNA demethylating agents, such as 5-aza-2′-deoxycytidine (5-Aza-CdR), can restore and upregulate CKIs. Previously, we reported the effect of 5-Aza-CdR and genistein on DNA methyltransferase (DNMTs) in hepatocellular carcinoma (HCC). The aim of the present study was to evaluate the effect of 5-Aza-CdR on p15INK4, p16INK4, p18INK4, and p19INK4 genes expression, cell growth inhibition, and apoptosis induction in HCC PLC/PRF/5 cell line. Materials and Methods: The effect of 5-Aza-CdR on the cell growth of PLC/PRF/5 cells, genes expression, and apoptosis induction were assessed by 3-[4, 5-dimethyl-2-thiazolyl]-2, 5-diphenyl-2H-tetrazolium bromide assay, real-time quantitative reverse transcription-polymerase chain reaction analysis, and flow cytometry, respectively. Results: 5-Aza-CdR (0, 1, 5, 10, 25, and 50 μM) inhibited PLC/PRF/5 cell growth at different periods significantly. This compound induced apoptosis and reactivated p15INK4, p16INK4, p18INK4, and p19INK4 genes expression at a concentration of 5 μM significantly. Conclusion: 5-Aza-CdR can inhibit cell viability and induce apoptosis by epigenetic reactivation of p15INK4, p16INK4, p18INK4, and p19INK4 genes in HCC PLC/PRF/5.

Keywords

References

1.	Satyanarayana A, Kaldis P. Mammalian cell-cycle regulation: Several Cdks, numerous cyclins and diverse compensatory mechanisms. Oncogene 2009;28:2925-39.
2.	Malumbres M. Cyclin-dependent kinases. Genome Biol 2014;15:122.
3.	Li G, Ji Y, Liu C, Li J, Zhou Y. Reduced levels of p15INK4b, p16INK4a, p21cip1 and p27kip1 in pancreatic carcinoma. Mol Med Rep 2012;5:1106-10.
4.	Aravalli RN, Steer CJ, Cressman EN. Molecular mechanisms of hepatocellular carcinoma. Hepatology 2008;48:2047-63.
5.	Shim YH, Yoon GS, Choi HJ, Chung YH, Yu E. p16 Hypermethylation in the early stage of hepatitis B virus-associated hepatocarcinogenesis. Cancer Lett 2003;190:213-9.
6.	Morishita A, Masaki T, Yoshiji H, Nakai S, Ogi T, Miyauchi Y, et al. Reduced expression of cell cycle regulator p18(INK4C) in human hepatocellular carcinoma. Hepatology 2004;40:677-86.
7.	Muscarella P, Knobloch TJ, Ulrich AB, Casto BC, Moniaux N, Wittel UA, Melvin WS, et al. Identification and sequencing of the Syrian Golden hamster (Mesocricetus auratus) p16INK4a and p15INK4b cDNAs and their homozygous gene deletion in cheek pouch and pancreatic tumor cells. Gene 2001;278:235-43.
8.	Ishiguro A, Takahata T, Saito M, Yoshiya G, Tamura Y, Sasaki M, et al. Influence of methylated p15 and p16 genes on clinicopathological features in colorectal cancer. J Gastroenterol Hepatol 2006;21:1334-9.
9.	Zheng Q, Fan H, Meng Z, Yuan L, Liu C, Peng Y, et al. Histone demethylase KDM2B promotes triple negative breast cancer proliferation by suppressing p15INK4B, p16INK4A, and p57KIP2 transcription. Acta Biochim Biophys Sin (Shanghai) 2018;50:897-904.
10.	Tischoff I, Tannapfel A. DNA methylation in hepatocellular carcinoma. World J Gastroenterol 2008;14:1741-8.
11.	Suh SI, Pyun HY, Cho JW, Baek WK, Park JB, Kyu Kwon T, et al. 5-Aza-2′-deoxycytidine leads to down-regulation of aberrant p16INK4A RNA transcripts and restores the functional retinoblastoma protein pathway in hepatocellular carcinoma cell lines. Cancer Lett 2000;160:81-8.
12.	Sanaei M, Kavoosi F. Effects of 5-aza-2′-deoxycytidine and Valproic acid on Epigenetic-modifying DNMT1 Gene Expression, Apoptosis Induction and Cell Viability in Hepatocellular Carcinoma WCH-17 cell line. IJPHO 2019;9:83-90.
13.	Sanaei M, Kavoosi F, Roustazadeh A, Golestan F. Effect of genistein in comparison with trichostatin a on reactivation of DNMTs genes in hepatocellular carcinoma. J Clin Transl Hepatol 2018;6:141-6.
14.	Azad M, Kaviani S, Noruzinia M, Mortazavi Y, Mobarra N, Alizadeh S, et al. Gene Expression Status and Methylation Pattern in Promoter of P15INK4b and P16INK4a in Cord Blood CD34 (+) Stem Cells. Iran J Basic Med Sci 2013;16:822-8.
15.	Saegusa M, Machida BD, Okayasu I. Possible associations among expression of p14ARF, p16INK4a, p21WAF1/CIP1, p27KIP1, and p53 accumulation and the balance of apoptosis and cell proliferation in ovarian carcinomas. Am Cancer Soc 2001;92:1177-89.
16.	Pesce F, Mezzomo L, Gonzales Filho P, NK FN, Leães C. Decreased of CDK Inhibitor p18INK4C mRNA expression in sporadic clinically non-functioning pituitary adenomas. J Oncol Res Forecast 2018;1:1011-7.
17.	Lin S, Wang MJ, Tseng KY. Polypyrimidine tract-binding protein induces p19Ink4d expression and inhibits the proliferation of H1299 cells. PLoS One 2013;8:58227-38.
18.	Vermeulen K, Van Bockstaele DR, Berneman ZN. The cell cycle: A review of regulation, deregulation and therapeutic targets in cancer. Cell Prolif 2003;36:131-49.
19.	Fang JY, Lu YY. Effects of histone acetylation and DNA methylation on p21WAF1 regulation. World J Gastroenterol 2002;8:400-5.
20.	Tan HH, Porter AG. p21WAF1 negatively regulates DNMT1 expression in mammalian cells. BBRC 2009;382:17117-6.
21.	Otterson GA, Khleif SN, Chen W, Coxon AB, Kaye FJ. CDKN2 gene silencing in lung cancer by DNA hypermethylation and kinetics of p16INK4 protein induction by 5-aza 2′deoxycytidine. Oncogene 1995;11:1211-6.
22.	Zhu WG, Hileman T, Ke Y, Wang P, Lu S, Duan W, et al. 5-aza-2′-deoxycytidine activates the p53/p21Waf1/Cip1 pathway to inhibit cell proliferation. J Biol Chem 2004;279:15161-6.
23.	Khamas A, Ishikawa T, Shimokawa K, Mogushi K, Iida S, Ishiguro M, et al. Screening for epigenetically masked genes in colorectal cancer Using 5-Aza-2′-deoxycytidine, microarray and gene expression profile. Cancer Genomics Proteomics 2012;9:67-75.
24.	Viallard JF, Lacombe F, Belloc F, Pellegrin JL, Reiffers J. Molecular mechanisms controlling the cell cycle: Fundamental aspects and implications for oncology. Cancer Radiother 2001;5:109-29.
25.	Cánepa ET, Scassa ME, Ceruti JM, Marazita MC, Carcagno AL, Sirkin PF, et al. INK4 proteins, a family of mammalian CDK inhibitors with novel biological functions. IUBMB Life 2007;59:419-26.
26.	Katayama K, Nakamura A, Sugimoto Y, Tsuruo T, Fujita N. FOXO transcription factor-dependent p15(INK4b) and p19(INK4d) expression. Oncogene 2008;27:1677-86.
27.	Ohtani N, Yamakoshi K, Takahashi A, Hara E. The p16INK4a-RB pathway: Molecular link between cellular senescence and tumor suppression. J Med Invest 2004;51:146-53.
28.	Pestell RG, Albanese C, Reutens AT, Segall JE, Lee RJ, Arnold A. The cyclins and cyclin-dependent kinase inhibitors in hormonal regulation of proliferation and differentiation. Endocr Rev 1999;20:501-34.

Advanced Biomedical Research