Effect of 5-aza-2ˈ-deoxycytidine on p27Kip1, p21Cip1/Waf1/Sdi1, p57Kip2, and DNA methyltransferase 1 Genes Expression, Cell Growth Inhibition and Apoptosis Induction in Colon Cancer SW 480 and SW 948 Cell Lines
DOI:
https://doi.org/10.31661/gmj.v9i.1899Keywords:
5-aza-CdR; Cip/Kip Genes; DNMT1; Colon CancerAbstract
Background: Dysregulation of the cell cycle has been reported in various cancers. Inactivation of the cyclin-dependent kinases inhibitors (CDKIs), CIP/KIP family, such as p21Cip1/Waf1/Sdi1, p27Kip1, and p57Kip2 genes because of hypermethylation has been shown in several cancers. Treatment with DNA demethylating agent 5-aza-2ˈ-deoxycytidine (5-Aza-CdR) has been indicated that affect genomic methylation and resulting in silenced genes reactivation in colon cancer. Previously, we evaluated the effect of 5-Aza-CdR on DNA methyltransferase 1 (DNMT1) gene expression in hepatocellular carcinoma (HCC) which encouraged us to design the current study. The present study aimed to evaluate the effect of 5-Aza-CdR on p21Cip1/Waf1/Sdi1, p27Kip1, p57Kip2, and DNAT1 genes expression, cell growth inhibition and apoptosis induction in colon cancer SW 480 and SW 948 cell lines. Materials and Methods: The effect of 5-aza-CdR on the SW 480 and SW 948 cells growth, apoptosis induction and genes expression were assessed by MTT assay, flow cytometry, and real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis respectively. Results: 5-aza-CdR inhibited cell growth as time- and dose-dependent manner significantly (P<0.001). The agent reactivated p15INK4, p16INK4, p18INK4, and p19INK4 genes expression and induced apoptosis at a concentration of 5 μM significantly. Besides, 5-aza-CdR had a more significant effect on the SW 480 cell line in comparison to SW 948 cell line. Conclusion: 5-Aza-CdR plays a key role in the up-regulation of p21Cip1/Waf1/Sdi1, p27Kip1, and p57Kip2 and down-regulation of DNMT1 genes resulting in cell growth inhibition and apoptosis induction. [GMJ.2020;9:e1899]
References
Park M-T, Lee S-J. Cell cycle and cancer. J. Biochem. Mol. Biol. 2003; 36(1):60-5. https://doi.org/10.5483/BMBRep.2003.36.1.060PMid:12542976 Williams GH, Stoeber K. The cell cycle and cancer. The Journal of pathology. 2012; 226(2):352-64. https://doi.org/10.1002/path.3022PMid:21990031 Collins I, Garrett MD. Targeting the cell division cycle in cancer: CDK and cell cycle checkpoint kinase inhibitors. Curr Opin Pharm. 2005; 5(4):366-73. https://doi.org/10.1016/j.coph.2005.04.009PMid:15964238 Malumbres M. Cyclin-dependent kinases. Genome Biol. 2014; 15(6):122. https://doi.org/10.1186/gb4184PMid:25180339 PMCid:PMC4097832 Lu R, Wang X, Chen Z-F, Sun D-F, Tian X-Q, Fang J-Y. Inhibition of the extracellular signal-regulated kinase/mitogen-activated protein kinase pathway decreases DNA methylation in colon cancer cells. J Biol Chem. 2007; 282(16):12249-59. https://doi.org/10.1074/jbc.M608525200PMid:17307743 Das PM, Singal R. DNA methylation and cancer. J Clin Oncol. 2004; 22(22):4632-42. https://doi.org/10.1200/JCO.2004.07.151PMid:15542813 Roulois D, Yau HL, Singhania R, Wang Y, Danesh A, Shen SY, et al. DNA-demethylating agents target colorectal cancer cells by inducing viral mimicry by endogenous transcripts. Cell 2015; 162(5):961-73. https://doi.org/10.1016/j.cell.2015.07.056PMid:26317465 PMCid:PMC4843502 Mossman D, Kim K-T, Scott RJ. Demethylation by 5-aza-2'-deoxycytidine in colorectal cancer cells targets genomic DNA whilst promoter CpG island methylation persists. BMC Cancer. 2010; 10(1):366 https://doi.org/10.1186/1471-2407-10-366PMid:20618997 PMCid:PMC2912869 Zhu W-G, 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(15):15161-6. https://doi.org/10.1074/jbc.M311703200PMid:14722112 Bott S, Arya M, Kirby R, Williamson M. p21 WAF1/CIP1 gene is inactivated in metastatic prostatic cancer cell lines by promoter methylation. Prostate Cancer Prostatic Dis. 2005; 8(4):321. https://doi.org/10.1038/sj.pcan.4500822PMid:16276353 Ling Y, Zhang C, Xu Y, Zhu J, Zhu C, Lu M, et al. Promoter methylation‑associated silencing of p27kip1 gene with metastasis in esophageal squamous cell carcinoma. Mol Med Report. 2014; 9(3):1075-9 https://doi.org/10.3892/mmr.2014.1887PMid:24402707 Kobatake T, Yano M, Toyooka S, Tsukuda K, Dote H, Kikuchi T, et al. Aberrant methylation of p57KIP2 gene in lung and breast cancers and malignant mesotheliomas. Oncol Rep. 2004; 12(5):1087-92. https://doi.org/10.3892/or.12.5.1087PMid:15492797 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. Iranian Journal of Pediatric Hematology & Oncology. 2019; 9(2). https://doi.org/10.18502/ijpho.v9i2.607 Sanaei M, Kavoosi F, Roustazadeh A, Golestan F. Effect of Genistein on DNMT1Gene Expression and Cell Proliferation of Hepatocellular Carcinoma HepG2 Cell Line. Global Journal of Medicine Researches and Studies. 2017; 4(1). Sanaei M, Kavoosi F, Roustazadeh A, Golestan F. Effect of genistein in comparison with trichostatin a on reactivation of DNMTs genes in hepatocellular carcinoma. Journal of clinical and translational hepatology. 2018; 6(2):141. https://doi.org/10.14218/JCTH.2018.00002PMid:29951358 PMCid:PMC6018304 Gao F-H, Hu X-H, Li W, Liu H, Zhang Y-J, Guo Z-Y, et al. Oridonin induces apoptosis and senescence in colorectal cancer cells by increasing histone hyperacetylation and regulation of p16, p21, p27 and c-myc. BMC Cancer. 2010; 10(1):610. https://doi.org/10.1186/1471-2407-10-610PMid:21054888 PMCid:PMC2992521 Oya M, Schulz W. Decreased expression of p57 KIP2 mRNA in human bladder cancer. Br. J. Cancer. 2000; 83(5):626. https://doi.org/10.1054/bjoc.2000.1298PMid:10944603 PMCid:PMC2363522 Chim C, Wong A, Kwong Y. Epigenetic inactivation of the CIP/KIP cell-cycle control pathway in acute leukemias. Am J Hematol. 2005; 80(4):282-7. https://doi.org/10.1002/ajh.20503PMid:16315255 Brakensiek K, Länger F, Kreipe H, Lehmann U. Absence of p21CIP1, p27KIP1 and p57KIP2 methylation in MDS and AML. Leukemia Res. 2005; 29(11):1357-60. https://doi.org/10.1016/j.leukres.2005.04.012PMid:15936816 Xu X-L, Yu J, Zhang H-Y, Sun M-H, Gu J, Du X, et al. Methylation profile of the promoter CpG islands of 31 genes that may contribute to colorectal carcinogenesis. World journal of gastroenterology: WJG. 2004; 10(23):3441. https://doi.org/10.3748/wjg.v10.i23.3441PMid:15526363 PMCid:PMC4576225 Peng Y-T, Wu W-R, Chen L-R, Kuo K-K, Tsai C-H, Huang Y-T, et al. Upregulation of cyclin-dependent kinase inhibitors CDKN1B and CDKN1C in hepatocellular carcinoma-derived cells via goniothalamin-mediated protein stabilization and epigenetic modifications. Toxicology reports. 2015; 2:322-32. https://doi.org/10.1016/j.toxrep.2015.01.010PMid:28962365 PMCid:PMC5598353 Zhao Y, Li Q, Wu X, Chen P. Upregulation of p27Kip1 by demethylation sensitizes cisplatin-resistant human ovarian cancer SKOV3 cells. Mol Med Report. 2016; 14(2):1659-66. https://doi.org/10.3892/mmr.2016.5399PMid:27314502 Lee K-H, Lotterman C, Karikari C, Omura N, Feldmann G, Habbe N, et al. Epigenetic silencing of MicroRNA miR-107 regulates cyclin-dependent kinase 6 expression in pancreatic cancer. Pancreatology. 2009; 9(3):293-301. https://doi.org/10.1159/000186051PMid:19407485 PMCid:PMC2835374 Schneider-Stock R, Diab-Assef M, Rohrbeck A, Foltzer-Jourdainne C, Boltze C, Hartig R, et al. RETRACTION: 5-aza-Cytidine Is a Potent Inhibitor of DNA Methyltransferase 3a and Induces Apoptosis in HCT-116 Colon Cancer Cells via Gadd45-and p53-Dependent Mechanisms. J Pharmacol Exp Ther. 2005; 312(2):525-36. https://doi.org/10.1124/jpet.104.074195PMid:15547111 Tao S-F, Zhang C-S, Guo X-L, Xu Y, Zhang S-S, Song J-R, et al. Anti-tumor effect of 5-aza-2'-deoxycytidine by inhibiting telomerase activity in hepatocellular carcinoma cells. World journal of gastroenterology: WJG. 2012; 18(19):2334. https://doi.org/10.3748/wjg.v18.i19.2334PMid:22654424 PMCid:PMC3353367 Bott S, Arya M, Kirby R, Williamson M. p21 WAF1/CIP1 gene is inactivated in metastatic prostatic cancer cell lines by promoter methylation. Prostate Cancer Prostatic Dis. 2005;8(4):321. https://doi.org/10.1038/sj.pcan.4500822PMid:16276353 WANG H-l, ZHANG J, LI Y, WANG X-q. Experimental studies of 5-Aza-CdR on p27kip1 gene's abnormal methylation in gastric cancer cell line [J]. Practical Pharmacy and Clinical Remedies. 2011; 2. Kobatake T, Yano M, Toyooka S, Tsukuda K, Dote H, Kikuchi T, et al. Aberrant methylation of p57KIP2 gene in lung and breast cancers and malignant mesotheliomas. Oncol Rep. 2004;12(5):1087-92. https://doi.org/10.3892/or.12.5.1087PMid:15492797 Sandal T. Molecular aspects of the mammalian cell cycle and cancer. The oncologist. 2002;7(1):73-81. https://doi.org/10.1634/theoncologist.7-1-73PMid:11854549 Cheetham S, Tang M, Mesak F, Kennecke H, Owen D, Tai I. SPARC promoter hypermethylation in colorectal cancers can be reversed by 5-Aza-2′ deoxycytidine to increase SPARC expression and improve therapy response. Br J Cancer. 2008;98(11):1810. https://doi.org/10.1038/sj.bjc.6604377PMid:18458674 PMCid:PMC2410109 Wang L-S, Kuo C-T, Cho S-J, Seguin C, Siddiqui J, Stoner K, et al. Black raspberry-derived anthocyanins demethylate tumor suppressor genes through the inhibition of DNMT1 and DNMT3B in colon cancer cells. Nutr Cancer. 2013;65(1):118-25. https://doi.org/10.1080/01635581.2013.741759PMid:23368921 PMCid:PMC3570951 Hsi LC, Xi X, Wu Y, Lippman SM. The methyltransferase inhibitor 5-aza-2-deoxycytidine induces apoptosis via induction of 15-lipoxygenase-1 in colorectal cancer cells. Mol Cancer Ther. 2005;4(11):1740-6. https://doi.org/10.1158/1535-7163.MCT-05-0218PMid:16275995 Kaminski R, Kozar K, Niderla J, Grzela T, Wilczynski G, Skierski JS, et al. Demethylating agent 5-aza-2'-deoxycytidine enhances expression of TNFRI and promotes TNF-mediated apoptosis in vitro and in vivo. Oncol Rep. 2004;12(3):509-16. https://doi.org/10.3892/or.12.3.509PMid:15289829 Yu J, Ma X, Cheung K, Li X, Tian L, Wang S, et al. Epigenetic inactivation of T-box transcription factor 5, a novel tumor suppressor gene, is associated with colon cancer. Oncogene. 2010; 29(49):6464. https://doi.org/10.1038/onc.2010.370PMid:20802524 Satoh A, Toyota M, Itoh F, Kikuchi T, Obata T, Sasaki Y, et al. DNA methylation and histone deacetylation associated with silencing DAP kinase gene expression in colorectal and gastric cancers. Br J Cancer. 2002; 86(11):1817. https://doi.org/10.1038/sj.bjc.6600319PMid:12087472 PMCid:PMC2375414
