Effect of 5-aza-2'-deoxycytidine in comparison to valproic acid and trichostatin A on histone deacetylase 1, DNA methyltransferase 1, and CIP/KIP family (p21, p27, and p57) genes expression, cell growth inhibition, and apoptosis induction in colon cancer SW480 cell line

Document Type : Original Article


1 .Research Center for Noncommunicable Diseases, Jahrom University of Medical Sciences, Jahrom, Fars Province, Iran

2 Research Center for Noncommunicable Diseases, Jahrom University of Medical Sciences, Jahrom, Fars Province, Iran


Background: Cancer initiation and progression depends on genetic and epigenetic alterations such as DNA methylation and histone modifications. Hypermethylation and deacetylation of the CIP/KIP family (p21, p27, and p57) lead to tumorigenesis. Our previous study indicated that DNA methyltransferase (DNMT) inhibitor and histone deacetylase (HDAC) inhibitors can inhibit cell growth and induce apoptosis. The aim of the present study was to investigate the effect of 5-Aza-2'-deoxycytidine (5-Aza-CdR) in comparison to valproic acid (VPA) and trichostatin A (TSA) on HDAC1, DNMT1, and CIP/KIP family (p21, p27, and p57) genes expression, cell growth inhibition, and apoptosis induction in colon cancer SW480 cell line. Materials and Methods: The effect of the compounds on the cell viability was measured by MTT assay. The expression of HDAC1, DNMT1, and CIP/KIP family (p21, p27, and p57) genes was evaluated by real-time quantitative reverse transcription- polymerase chain reaction (RT-PCR). For the detection of cell apoptosis, apoptotic cells were examined by the Annexin V-FITC/PI detection kit. Results: The results of MTT assay indicated that 5-Aza-CdR, VPA, and TSA significantly inhibited cell growth (P < 0.002,P < 0.001, andP < 0.001, respectively). The results of real-time RT-PCR demonstrated that all compounds significantly down-regulated DNMT1 and HDAC1, and up-regulated p21, p27, and p57 genes expression. The result of flow cytometry assay revealed that all agents induced apoptosis significantly. Conclusion: 5-Aza-CdR, VPA, and TSA can significantly downregulate DNMT1 and HDAC1 and up-regulate p21, p27, and p57 genes expression through which enhance cell apoptosis and cell growth inhibition in colon cancer.


Kanai Y, Ushijima S, Kondo Y, Nakanishi Y, Hirohashi S. DNA methyltransferase expression and DNA methylation of CPG Islands and Peri-centromeric satellite regions in human colorectal and stomach cancers. Int J Cancer 2001;91:205-12.  Back to cited text no. 1
de Ruijter AJ, van Gennip AH, Caron HN, Kemp S, van Kuilenburg AB. Histone deacetylases (HDACs): Characterization of the classical HDAC family. Biochem J 2003;370:737-49.  Back to cited text no. 2
Higashijima J, Kurita N, Miyatani T, Yoshikawa K, Morimoto S, Nishioka M, et al. Expression of histone deacetylase 1 and metastasis-associated protein 1 as prognostic factors in colon cancer. Oncol Rep 2011;26:343-8.  Back to cited text no. 3
Park MT, Lee SJ. Cell cycle and cancer. J Biochem Mol Biol 2003;36:60-5.  Back to cited text no. 4
Chim CS, Wong AS, Kwong YL. Epigenetic inactivation of the CIP/KIP cell-cycle control pathway in acute leukemias. Am J Hematol 2005;80:282-7.  Back to cited text no. 5
Shin JY, Kim HS, Park J, Park JB, Lee JY. Mechanism for inactivation of the KIP family cyclin-dependent kinase inhibitor genes in gastric cancer cells. Cancer Res 2000;60:262-5.  Back to cited text no. 6
Mirza S, Sharma G, Parshad R, Gupta SD, Pandya P, Ralhan R. Expression of DNA methyltransferases in breast cancer patients and to analyze the effect of natural compounds on DNA methyltransferases and associated proteins. J Breast Cancer 2013;16:23-31.  Back to cited text no. 7
Sanaei M, Kavoosi F, Salehi H. Genistein and trichostatin A induction of estrogen receptor alpha gene expression, apoptosis and cell growth inhibition in hepatocellular carcinoma HepG 2 cells Asian Pac J Cancer Prev 2017;18:3445-50.  Back to cited text no. 8
Sanaei M, Kavoosi F, Atashpour S, Haghighat S. Effects of genistein and synergistic action in combination with tamoxifen on the HepG2 human hepatocellular carcinoma cell line Asian Pac J Cancer Prev 2017;18:2381-5.  Back to cited text no. 9
Sanaei M, Kavoosi F, Pourahmadi M, Moosavi SN. Effect of genistein and 17-β estradiol on the viability and apoptosis of human hepatocellular carcinoma HepG2 cell line. Adv Biomed Res 2017;6:163.  Back to cited text no. 10
[PUBMED]  [Full text]  
Mottamal M, Zheng S, Huang TL, Wang G. Histone deacetylase inhibitors in clinical studies as templates for new anticancer agents. Molecules 2015;20:3898-941.  Back to cited text no. 11
Habold C, Poehlmann A, Bajbouj K, Hartig R, Korkmaz KS, Roessner A, et al. Trichostatin A causes p53 to switch oxidative-damaged colorectal cancer cells from cell cycle arrest into apoptosis. J Cell Mol Med 2008;12:607-21.  Back to cited text no. 12
Sanaei M, Kavoosi F, Mansoori O. Effect of valproic acid in comparison with vorinostat on cell growth inhibition and apoptosis induction in the human colon cancer SW48 cells in vitro. Exp Oncol 2018;40:95-100.  Back to cited text no. 13
Sanaei M, Kavoosi F. Effect of curcumin and trichostatin a on the expression of DNA methyltransfrase 1 in hepatocellular carcinoma cell line hepa 1-6. Iran J Pediatr Hematol Oncol 2018;8 (4): 193-201  Back to cited text no. 14
Zhu WG, Otterson GA. The interaction of histone deacetylase inhibitors and DNA methyltransferase inhibitors in the treatment of human cancer cells. Curr Med Chem Anticancer Agents 2003;3:187-99.  Back to cited text no. 15
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.  Back to cited text no. 16
Osada H, Tatematsu Y, Saito H, Yatabe Y, Mitsudomi T, Takahashi T. Reduced expression of class II histone deacetylase genes is associated with poor prognosis in lung cancer patients. Int J Cancer 2004;112:26-32.  Back to cited text no. 17
Gao FH, Hu XH, Li W, Liu H, Zhang YJ, Guo ZY, 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;10:610.  Back to cited text no. 18
Oya M, Schulz WA. Decreased expression of p57(KIP2) mRNA in human bladder cancer. Br J Cancer 2000;83:626-31.  Back to cited text no. 19
Kavoosi F, Dastjerdi MN, Valiani A, Esfandiari E, Sanaei M, Hakemi MG. Genistein potentiates the effect of 17-beta estradiol on human hepatocellular carcinoma cell line. Adv Biomed Res 2016;5:133.  Back to cited text no. 20
[PUBMED]  [Full text]  
Kavoosi F, Sanaei M. Comparative analysis of the effects of valproic acid and tamoxifen on proliferation, and apoptosis of human hepatocellular carcinoma WCH 17 CellLin. Iran J Pediatr Hematol Oncol 2018;8:12-20.  Back to cited text no. 21
Jang B, Kim LH, Lee SY, Lee KE, Shin JA, Cho SD. Trichostatin A induces apoptosis in oral squamous cell carcinoma cell lines independent of hyperacetylation of histones. J Cancer Res Ther 2018;14:S576-82.  Back to cited text no. 22
Xu Q, Liu X, Zhu S, Hu X, Niu H, Zhang X, et al. Hyper-acetylation contributes to the sensitivity of chemo-resistant prostate cancer cells to histone deacetylase inhibitor trichostatin A. J Cell Mol Med 2018;22:1909-22.  Back to cited text no. 23
Zhang XF, Huang FH, Zhang GL, Bai DP, Massimo DF, Huang YF, et al. Novel biomolecule lycopene-reduced graphene oxide-silver nanoparticle enhances apoptotic potential of trichostatin A in human ovarian cancer cells (SKOV3). Int J Nanomedicine 2017;12:7551-75.  Back to cited text no. 24
Yamashita Y, Shimada M, Harimoto N, Rikimaru T, Shirabe K, Tanaka S, et al. Histone deacetylase inhibitor trichostatin A induces cell-cycle arrest/apoptosis and hepatocyte differentiation in human hepatoma cells. Int J Cancer 2003;103:572-6.  Back to cited text no. 25
Chen X, Wong P, Radany E, Wong JY. HDAC inhibitor, valproic acid, induces p53-dependent radiosensitization of colon cancer cells. Cancer Biother Radiopharm 2009;24:689-99.  Back to cited text no. 26
Ma XJ, Wang YS, Gu WP, Zhao X. The role and possible molecular mechanism of valproic acid in the growth of MCF-7 breast cancer cells. Croat Med J 2017;58:349-57.  Back to cited text no. 27
Terranova-Barberio M, Roca MS, Zotti AI, Leone A, Bruzzese F, Vitagliano C, et al. Valproic acid potentiates the anticancer activity of capecitabinein vitro andin vivo in breast cancer models via induction of thymidine phosphorylase expression. Oncotarget 2016;7:7715-31.  Back to cited text no. 28
Shan Z, Feng-Nian R, Jie G, Ting Z. Effects of valproic acid on proliferation, apoptosis, angiogenesis and metastasis of ovarian cancerin vitro and in vivo. Asian Pac J Cancer Prev 2012;13:3977-82.  Back to cited text no. 29
Li X, Qin B, Liu BO. Delineating the effect of demethylating agent 5-aza-2'-deoxycytidine on human Caco-2 colonic carcinoma cells. Oncol Lett 2016;12:139-43.  Back to cited text no. 30
Liu WJ, Ren JG, Li T, Yu GZ, Zhang J, Li CS, et al. 5-aza-2 and lt;-deoxycytidine induces hepatoma cell apoptosis via enhancing methionine adenosyltransferase 1A expression and inducing S-adenosylmethionine production. Asian Pac J Cancer Prev 2013;14:6433-8.  Back to cited text no. 31
Tao SF, Zhang CS, Guo XL, Xu Y, Zhang SS, Song JR, et al. Anti-tumor effect of 5-aza-2'-deoxycytidine by inhibiting telomerase activity in hepatocellular carcinoma cells. World J Gastroenterol 2012;18:2334-43.  Back to cited text no. 32
Yao TT, Mo SM, Liu LY, Lu HW, Huang ML, Lin ZQ. 5-aza-2'-deoxycytidine may influence the proliferation and apoptosis of cervical cancer cells via demethylation in a dose- and time-dependent manner. Genet Mol Res 2013;12:312-8.  Back to cited text no. 33
Liu Y, He G, Wang Y, Guan X, Pang X, Zhang B. MCM-2 is a therapeutic target of trichostatin A in colon cancer cells. Toxicol Lett 2013;221:23-30.  Back to cited text no. 34
Wang X, Xu J, Wang H, Wu L, Yuan W, Du J, et al. Trichostatin A, a histone deacetylase inhibitor, reverses epithelial-mesenchymal transition in colorectal cancer SW480 and prostate cancer PC3 cells. Biochem Biophys Res Commun 2015;456:320-6.  Back to cited text no. 35
Xiong H, Du W, Zhang YJ, Hong J, Su WY, Tang JT, et al. Trichostatin A, a histone deacetylase inhibitor, suppresses JAK2/STAT3 signaling via inducing the promoter-associated histone acetylation of SOCS1 and SOCS3 in human colorectal cancer cells. Mol Carcinog 2012;51:174-84.  Back to cited text no. 36
Bi G, Jiang G. The molecular mechanism of HDAC inhibitors in anticancer effects. Cell Mol Immunol 2006;3:285-90.  Back to cited text no. 37
Kostrouchová M, Kostrouch Z, Kostrouchová M. Valproic acid, a molecular lead to multiple regulatory pathways. Folia Biol (Praha) 2007;53:37-49.  Back to cited text no. 38
Blaheta RA, Cinatl J Jr. Anti-tumor mechanisms of valproate: A novel role for an old drug. Med Res Rev 2002;22:492-511.  Back to cited text no. 39
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;312:525-36.  Back to cited text no. 40
Xiong H, Chen ZF, Liang QC, Du W, Chen HM, Su WY, et al. Inhibition of DNA methyltransferase induces G2 cell cycle arrest and apoptosis in human colorectal cancer cells via inhibition of JAK2/STAT3/STAT5 signalling. J Cell Mol Med 2009;13:3668-79.  Back to cited text no. 41
Groh IA, Chen C, Lüske C, Cartus AT, Esselen M. Plant polyphenols and oxidative metabolites of the herbal alkenylbenzene methyleugenol suppress histone deacetylase activity in human colon carcinoma cells. J Nutr Metab 2013;2013:821082.  Back to cited text no. 42
Dagdemir A, Durif J, Ngollo M, Bignon YJ, Bernard-Gallon D. Histone lysine trimethylation or acetylation can be modulated by phytoestrogen, estrogen or anti-HDAC in breast cancer cell lines. Epigenomics 2013;5:51-63.  Back to cited text no. 43
Cheishvili D, Boureau L, Szyf M. DNA demethylation and invasive cancer: Implications for therapeutics. Br J Pharmacol 2015;172:2705-15.  Back to cited text no. 44
Sun G, Mackey LV, Coy DH, Yu CY, Sun L. The histone deacetylase inhibitor vaproic acid induces cell growth arrest in hepatocellular carcinoma cells via suppressing notch signaling. J Cancer 2015;6:996-1004.  Back to cited text no. 45
Xiong Y, Dowdy SC, Podratz KC, Jin F, Attewell JR, Eberhardt NL, et al. Histone deacetylase inhibitors decrease DNA methyltransferase-3B messenger RNA stability and down-regulate de novo DNA methyltransferase activity in human endometrial cells. Cancer Res 2005;65:2684-9.  Back to cited text no. 46
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.  Back to cited text no. 47
Zheng QH, Ma LW, Zhu WG, Zhang ZY, Tong TJ. P21Waf1/Cip1 plays a critical role in modulating senescence through changes of DNA methylation. J Cell Biochem 2006;98:1230-48.  Back to cited text no. 48
Phan NL, Trinh NV, Pham PV. Low concentrations of 5-aza-2'-deoxycytidine induce breast cancer stem cell differentiation by triggering tumor suppressor gene expression. Onco Targets Ther 2016;9:49-59.  Back to cited text no. 49
Tatebe H, Shimizu M, Shirakami Y, Sakai H, Yasuda Y, Tsurumi H, et al. Acyclic retinoid synergises with valproic acid to inhibit growth in human hepatocellular carcinoma cells. Cancer Lett 2009;285:210-7.  Back to cited text no. 50
Kaiser M, Zavrski I, Sterz J, Jakob C, Fleissner C, Kloetzel PM, et al. The effects of the histone deacetylase inhibitor valproic acid on cell cycle, growth suppression and apoptosis in multiple myeloma. Haematologica 2006;91:248-51.  Back to cited text no. 51
Fortson WS, Kayarthodi S, Fujimura Y, Xu H, Matthews R, Grizzle WE, et al. Histone deacetylase inhibitors, valproic acid and trichostatin-A induce apoptosis and affect acetylation status of p53 in ERG-positive prostate cancer cells. Int J Oncol 2011;39:111-9.  Back to cited text no. 52
Cucciolla V, Borriello A, Criscuolo M, Sinisi AA, Bencivenga D, Tramontano A, et al. Histone deacetylase inhibitors upregulate p57Kip2 level by enhancing its expression through Sp1 transcription factor. Carcinogenesis 2008;29:560-7.  Back to cited text no. 53
Park WH, Jung CW, Park JO, Kim K, Kim WS, Im YH, et al. Trichostatin inhibits the growth of ACHN renal cell carcinoma cells via cell cycle arrest in association with p27, or apoptosis. Int J Oncol 2003;22:1129-34.  Back to cited text no. 54