Apoptotic Induction in CCRF-CEM and HL-60 Human Leukemic Cell Lines by 5-Azacitidine and Trichostatin A

Sudin Aziee, Mohd Yassim Haiyuni, Hamid Ali Nagi Al-Jamal, Mohamed Yusoff Shafini, Ridhwan Abdul Wahab, Shaharum Shamsuddin, Muhammad Farid Johan

Abstract


The aims of the study were to investigate the anti-cancer effects of 5-Aza and TSA in two leukemic cell lines (CCRF-CEM and HL-60). Inhibition concentration of 5-Aza and TSA were measured using trypan blue exclusion assay. 5-Aza and TSA at IC50 were treated to both CCRF-CEM and HL-60 cell lines for 4-6 days. To confirm the inhibition effects of these agents, Annexin-V stained cells were analyzed using flow cytometry to evaluate the apoptotic induction. The IC50 values of CCRF-CEM were 2.01±0.1µM and 2.65±0.3µM for 5-Aza- and TSA-treated, respectively. Whereas, the IC50 values of HL-60 were 1.98±0.2µM and 2.35±0.2µM for 5-Aza- and TSA-treated, respectively. To further substantiate the findings, the time-dependent exposure of both drugs was studied. CCRF-CEM cells were reduced to 49.4%±5.0, 49.4%±2.5 and 41.5%±5.6 by 5-Aza; 56.5%±7.0, 45.3%±4.2 and 40.2%±4.2 by TSA treatment at first, third and sixth day. HL-60 cells were reduced to 72.0%±4.5, 51.0%±1.5 and 40.6%±2.6 by 5-Aza at first, third and sixth day. Meanwhile, HL-60 cells reduced to 55.6%±4.5, 45.2%±4.0 and 36.3%±2.9 by TSA at first, second and fourth day. Both cell lines were significantly inhibited (p<0.05) compared to the untreated. Furthermore, flow cytometry demonstrated that 5-Aza and TSA significantly increased the cells population positive for Annexin-V in CCRF-CEM and HL-60 cell lines. In CCRF-CEM, the total apoptotic rates were 51.7%±9.7 and 49.4%±6.0 for 5-Aza- and TSA-treated, while, in HL-60, the apoptotic rates were 51.0%±3.9 and 49.7%±9.6 for 5-Aza- and TSA-treated, in a dose- and time-dependent manner, respectively. Epigenetic modification drugs, 5-Aza and TSA have anti-leukemic effects and induce apoptosis at micro-molar concentrations in CCRF-CEM and HL-60 leukemic cell lines. These results may provide a new insight into the use of 5-Aza and TSA in inhibiting the growth of leukemic cells and useful strategy in developing an epigenetic therapy.

Keywords


Epigenetic, 5-Aza, TSA, CCRF-CEM, HL-60

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References


Qureshi AK, Hall GW. Leukaemias: a review. Paediatrics and child health. 2013;23(11):461-6.

Luczak M, Kaźmierczak M, Handschuh L, Lewandowski K, Komarnicki M, Figlerowicz M. Comparative proteome analysis of acute myeloid leukemia with and without maturation. Journal of proteomics. 2012;75(18):5734-48.

Ntziachristos P, Mullenders J, Trimarchi T, Aifantis I. Mechanisms of epigenetic regulation of leukemia onset and progression. Advances in immunology. 2013;117:1-30.

Huang YW, Kuo CT, Stoner K, Huang TH, Wang LS. An overview of epigenetics and chemoprevention. FEBS letters. 2011;585(13):2129-36.

Wouters BJ, Delwel R. Epigenetics and approaches to targeted epigenetic therapy in acute myeloid leukemia. Blood. 2016;127(1):42-52.

Matoušová M, Votruba I, Otmar M, Tloušťová E, Günterová J, Mertlíková-Kaiserová H. 2´-deoxy-5,6-dihydro-5-azacytidine - a less toxic alternative of 2´-deoxy-5-azacytidine. Epigenetics. 2014;6(6):769-76.

Qiu X, Hother C, Ralfkiaer UM, Sogaard A, Lu Q, Workman CT, et al. Equitoxic doses of 5-azacytidine and 5-aza-2'deoxycytidine induce diverse immediate and overlapping heritable changes in the transcriptome. Plos one. 2010;5(9):1-10.

Sripayap P, Nagai T, Uesawa M, Kobayashi H, Tsukahara T, Ohmine K, et al. Mechanisms of resistance to azacitidine in human leukemia cell lines. Exp hematol. 2014;42(4):294-306 e2.

Jasek E, Lis GJ, Jasińska M, Jurkowska H, Litwin JA. Effect of histone deacetylase inhibitors trichostatin a and valproic acid on etoposide-induced apoptosis in leukemia cells. Anticancer research 2012;32:2791-800.

Yu WP, Scott SA, Dong WF. Induction of ID1 expression and apoptosis by the histone deacetylase inhibitor (trichostatin A) in human acute myeloid leukaemic cells. Cell Proliferation. 2008;41(1):86-97.

Hollenbach PW, Nguyen AN, Brady H, Williams M, Ning Y, Richard N, et al. A comparison of azacitidine and decitabine activities in acute myeloid leukemia cell lines. Plos one. 2010;5(2):e9001.

Horing E, Podlech O, Silkenstedt B, Rota I, Alesxandros, Adamopoulou E, Naumann U, Naumann. The histone deacetylase inhibitor trichostatin a promotes apoptosis and antitumor immunity in glioblastoma cells. Anticancer research. 2013;33:1351-60.

Fard SS, Tehrani MJ, Ardekani MA. Prostaglandin E2 induces growth inhibition, apoptosis and differentiation in T and B cell-derived acute lymphoblastic leukemia cell lines (CCRF-CEM and Nalm-6). Prostaglandins, leukotrienes and essential fatty acids. 2012;87(1):17-24.

Yang H, Hoshino K, Sanchez-Gonzalez B, Kantarjian H, Garcia-Manero G. Antileukemia activity of the combination of 5-aza-2'-deoxycytidine with valproic acid. Leukemia research. 2005;29(7):739-48.

Gocek E, Marcinkowska E. Differentiation therapy of acute myeloid leukemia. Cancers 2011;3(2):2402-20.

Hotfilder M, Rottgers S, Rosemann A, Schrauder A, Schrappe M, Pieters R, et al. Leukemic stem cells in childhood high-risk all/t(9;22) and t(4;11) are present in primitive lymphoid-restricted cd34+cd19– cells. Cancer research. 2005;65(4):1442-9.

Christman JK, Mendelsohn N, Herzog D, Schneiderman N. Effect of 5-azacytidine on differentiation and DNA methylation in human promyelocytic leukemia cells (hl-60). Cancer research. 1983;43:763-9.

Stankov MV, El Khatib M, Kumar Thakur B, Heitmann K, Panayotova-Dimitrova D, Schoening J, et al. Histone deacetylase inhibitors induce apoptosis in myeloid leukemia by suppressing autophagy. Leukemia. 2014;28(3):577-88.

Martina MS, Impiombatoa AA, Qin Y, Herranza D, Bansal M, Girardic T, et al. Synergistic antileukemic therapies in NOTCH1-induced T-ALL. PNAS. 2017;114(8):2006–11.

Chambers AE, Banerjee S, Chaplin T, Dunne J, Debernardi S, Joel SP, et al. Histone acetylation-mediated regulation of genes in leukaemic cells. European journal of cancer. 2003;39(8):1165-75.

Chen J, Bai H, Wang C, Kang J. Trichostatin A improves the anticancer activity of low concentrations of curcumin in human leukemia cells. Pharmazie. 2006;61(8):710-6.

Lu MC, Du YC, Chuu JJ, Hwang SL, Hsieh PC, Hung CS, et al. Active extracts of wild fruiting bodies of Antrodia camphorata (EEAC) induce leukemia HL 60 cells apoptosis partially through histone hypoacetylation and synergistically promote anticancer effect of trichostatin A. Arch toxicol. 2009;83:121–9.

Jie Chen, Kang J-h. Quercetin and trichostatin A cooperatively kill human leukemia cells. Pharmazie. 2005;60:856-60.




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