The Effect of Decitabine Combined with Arsenic Trioxide on DAPK Gene and HL-60 Cell Proliferation and Apoptosis

Jinhai Ren, Jingjing Yao, Xiaonan Guo, Xiaoling Guo, Shengxin Cai

Department of Hematology, Secondary Hospital of Hebei Medical University, Hebei Hematology Institute, Shijiazhuang, China.

DOI: 10.4236/jct.2015.615134                

Abstract

Purpose: Our study was to detect the effect of Decitabine (DAC) combined with arsenic trioxide (AS₂O₃) on DAPK gene and HL-60 cell proliferation and apoptosis. Methods: DAC and AS₂O₃ monotherapy, combination treatment and DAC pretreatment were used in this study after incubating with HL-60 cell for 24 h, 48 h, 72 h. CCK8 was used to detect the cell proliferation of HL-60 cell. Flow cytometry was used to detect the cell apoptosis. Then, we used RT-PCR to obtain the gene expression level of DAPK. Results: HL-60 cells were treated with different concentrations of DAC (20 μmol/L, 40 μmol/L, 80 μmol/L), AS₂O₃ (1 μmol/L, 2.5 μmol/L, 5 μmol/L) monotherapy for 24 h, 48 h, 72 h; along with the extension of the drug concentration and time, proliferation inhibition rate had gradually increased. Monotherapy of DAC, AS₂O₃ could inhibit the proliferation and induce apoptosis of HL-60 cells, and was time- and dose-dependent. DAC (80 μmol/L) was firstly used for pretreatment, and then, different concentrations of AS₂O₃ (1 μmol/L, 2.5 μmol/L, 5 μmol/L) were used for 24 h, 48 h, 72 h. It was found that cell proliferation inhibition rate and apoptosis rate had increased significantly. When the two drugs were used together, the increasing proliferation inhibition rate, apoptosis rate and DAPK had become more obvious. Conclusion: DAC and AS₂O₃ had a synergetic effect for the HL-60 cell proliferation inhibition, apoptosis and expression of DAPK.

Keywords

Decitabine, Arsenic Trioxide, HL-60, Proliferation, Apoptosis, DAPK

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	Roman-Gomez, J., Jimenez-Velasco, A., Castillejo, J.A., Agirre, X., et al. (2004) Promoter Hypermethylation of Cancer-Related Genes: A Strong Independent Prognostic Factor in Acute Lymphoblastic Leukemia. Blood, 104, 2492-2498. http://dx.doi.org/10.1182/blood-2004-03-0954
[2]	Rossi, D., Capello, D., Gloghini, A., Franceschetti, S., et al. (2004) Aberrant Promoter Methylation of Multiple Genes throughout the Clinico-Pathologic Spectrum of B-Cell Neoplasia. Haematologica, 89, 154-64.
[3]	Jabbour, E., Issa, J.P., Garcia-Manero, G. and Kantarjian, H. (2008) Evolution of Decitabine Development: Accomplishments, Ongoing Investigations, and Future Strategies. Cancer, 112, 2341-2351. http://dx.doi.org/10.1002/cncr.23463
[4]	Schmelz, K., Wagner, M., Dorken, B. and Tamm, I. (2005) 5-Aza-2'-deoxycytidine Induces p21WAF Expression by Demethylation of p73 Leading to p53-Independent Apoptosis in Myeloid Leukemia. International Journal of Cancer, 114, 683-695. http://dx.doi.org/10.1002/ijc.20797
[5]	Wijermans, P., Lubbert, M., Verhoef, G., Bosly, A., et al. (2000) Low-dose 5-Aza-2'-deoxycytidine, a DNA Hypomethylating Agent, for the Treatment of High-Risk Myelodysplastic Syndrome: A Multicenter Phase II Study in Elderly Patients. Journal of Clinical Oncology, 18, 956-962.
[6]	Kantarjian, H.M., O’Brien, S., Cortes, J., Giles, F.J., et al. (2003) Results of Decitabine (5-Aza-2'deoxycytidine) Therapy in 130 Patients with Chronic Myelogenous Leukemia. Cancer, 98, 522-528. http://dx.doi.org/10.1002/cncr.11543
[7]	Khan, H., Vale, C., Bhagat, T. and Verma, A. (2013) Role of DNA Methylation in the Pathogenesis and Treatment of Myelodysplastic Syndromes. Seminars in Hematology, 50, 16-37. http://dx.doi.org/10.1053/j.seminhematol.2013.01.001
[8]	Blum, W., Klisovic, R.B., Hackanson, B., Liu, Z., et al. (2007) Phase I Study of Decitabine Alone or in Combination with Valproic Acid in Acute Myeloid Leukemia. Journal of Clinical Oncology, 25, 3884-3891. http://dx.doi.org/10.1200/JCO.2006.09.4169
[9]	Cashen, A.F., Schiller, G.J., O’Donnell, M.R. and DiPersio, J.F. (2010) Multicenter, Phase II Study of Decitabine for the First-Line Treatment of Older Patients with Acute Myeloid Leukemia. Journal of Clinical Oncology, 28, 556-561. http://dx.doi.org/10.1200/JCO.2009.23.9178
[10]	Plimack, E.R., Kantarjian, H.M. and Issa, J.P. (2007) Decitabine and Its Role in the Treatment of Hematopoietic Malignancies. Leukemia & Lymphoma, 48, 1472-1481. http://dx.doi.org/10.1080/10428190701471981
[11]	Malik, P. and Cashen, A.F. (2014) Decitabine in the Treatment of Acute Myeloid Leukemia in Elderly Patients. Cancer Management and Research, 6, 53-61.
[12]	Ho, S.Y., Wu Jr., W., Chiu, H.W., et al. (2011) Arsenic Trioxide and Radiation Enhance Apoptotic Effects in HL-60 Cells through Increased ROS Generation and Regulation of JNK and p38 MAPK Signaling Pathways. Chemico-Biological Interactions, 193, 162-171. http://dx.doi.org/10.1016/j.cbi.2011.06.007
[13]	Zhao, D., Jiang, Y., Dong, X., et al. (2011) Arsenic Trioxide Reduces Drug Resistance to Adriamycin in Leukemic K562/A02 Cells via Multiple Mechanisms. Biomedicine & Pharmacotherapy, 65, 354-358. http://dx.doi.org/10.1016/j.biopha.2011.04.016
[14]	Yang, L., Luo, J.M., Li, Y., et al. (2009) As2O3 Induces Demethylation and Upregulates Transeription of SHP-1 Gene in Human Lymphoma Cell Line T2 Cells. Chinese Journal of Oncology, 31, 423-427.
[15]	Gandesiri, M., Chakilam, S., Ivanovska, J., Benderska, N., et al. (2012) DAPK Plays an Important Role in Panobinostat-Induced Autophagy and Commits Cells to Apoptosis under Autophagy Deficient Conditions. Apoptosis, 17, 1300-1315. http://dx.doi.org/10.1007/s10495-012-0757-7
[16]	Gade, P., Ramachandran, G., Maachani, U.B., Rizzo, M.A., et al. (2012) An IFN-Gamma-Stimulated ATF6-C/EBP-Beta-Signaling Pathway Critical for the Expression of Death Associated Protein Kinase 1 and Induction of Autophagy. Proceedings of the National Academy of Sciences of the United States of America, 109, 10316-10321. http://dx.doi.org/10.1073/pnas.1119273109
[17]	Schneider-Stock, R., Kuester, D., Ullrich, O., Mittag, F., et al. (2006) Close Localization of DAP-Kinase Positive Tumour-Associated Macrophages and Apoptotic Colorectal Cancer Cells. Journal of Pathology, 209, 95-105. http://dx.doi.org/10.1002/path.1951
[18]	Luo, X.J., Li, L.L., Deng, Q.P., Yu, X.F., et al. (2011) Grifolin, a Potent Antitumour Natural Product Upregulates Death-Associated Protein Kinase 1 DAPK1 via p53 in Nasopharyngeal Carcinoma Cells. European Journal of Cancer, 47, 316-325. http://dx.doi.org/10.1016/j.ejca.2010.09.021
[19]	Inbal, B., Bialik, S., Sabanay, I., Shani, G., et al. (2002) DAP Kinase and DRP-1 Mediate Membrane Blebbing and the Formation of Autophagic Vesicles during Programmed Cell Death. Journal of Cell Biology, 157, 455-468. http://dx.doi.org/10.1083/jcb.200109094
[20]	Eisenberg-Lerner, A. and Kimchi, A. (2007) DAP Kinase Regulates JNK Signaling by Binding and Activating Protein Kinase D under Oxidative Stress. Cell Death & Differentiation, 14, 1908-1915. http://dx.doi.org/10.1038/sj.cdd.4402212
[21]	Tahara, T., Shibata, T., Nakamura, M., Okubo, M., et al. (2011) Polymorphisms of DNA Repair and Xenobiotic Genes Predispose to CpG Island Methylation in Non-Neoplastic Gastric Mucosa. Helicobacter, 16, 99-106. http://dx.doi.org/10.1111/j.1523-5378.2011.00821.x
[22]	Su, Y., Xu, H., Xu, Y., Yu, J., et al. (2012) Azacytidine Inhibits the Proliferation of Human Promyelocytic Leukemia Cells (HL60) by Demethylation of MGMT, DAPK and p16 Genes. Hematology, 17, 41-46. http://dx.doi.org/10.1179/102453312X13221316477624
[23]	Niu, Y.M., Wang, P.P., Wang, Y., Wang, Y.Z., et al. (2014) Expression of Death-Associated Protein Kinase Gene and Methylation Status of Promoter Region in Acute Leukemia. Journal of Experimental Hematology, 22, 30-34.
[24]	Liu, X.F., Tang, K., Yu, S.P., Wang, Z.Q., et al. (2012) Correlation between Promoter Methylation of p14(ARF), TMS1/ASC, and DAPK, and p53 Mutation with Prognosis in Cholangiocarcinoma. World Journal of Surgical Oncology, 10, 5. http://dx.doi.org/10.1186/1477-7819-10-5
[25]	Liu, Y., Gao, W., Siegfried, J.M., Weissfeld, J.L., et al. (2007) Promoter Methylation of RASSF1A and DAPK and Mutations of K-ras, p53, and EGFR in Lung Tumors from Smokers and Never-Smokers. BMC Cancer, 7, 74. http://dx.doi.org/10.1186/1471-2407-7-74
[26]	Wu, J., Hu, C.P., Gu, Q.H., Li, Y.P., et al. (2010) Trichostatin A Sensitizes Cisplatin-Resistant A549 Cells to Apoptosis by Up-Regulating Death-Associated Protein Kinase. Acta Pharmacologica Sinica, 31, 93-101. http://dx.doi.org/10.1038/aps.2009.183
[27]	Nair, S., Hagberg, H., Krishnamurthy, R., Thornton, C., et al. (2013) Death Associated Protein Kinases: Molecular Structure and Brain Injury. International Journal of Molecular Sciences, 14, 13858-13872. http://dx.doi.org/10.3390/ijms140713858
[28]	Widau, R.C., Jin, Y., Dixon, S.A., Wadzinski, B.E., et al. (2010) Protein Phosphatase 2A (PP2A) Holoenzymes Regulate Death-Associated Protein Kinase (DAPK) in Ceramide-Induced Anoikis. Journal of Biological Chemistry, 285, 13827-13838. http://dx.doi.org/10.1074/jbc.M109.085076
[29]	Soncini, M., Santoro, F., Gutierrez, A., Frige, G., et al. (2013) The DNA Demethylating Agent Decitabine Activates the TRAIL Pathway and Induces Apoptosis in Acute Myeloid Leukemia. Biochimica et Biophysica Acta, 1832, 114-120. http://dx.doi.org/10.1016/j.bbadis.2012.10.001
[30]	Ge, F., Lu, X.P., Zeng, H.L., He, Q.Y., et al. (2009) Proteomic and Functional Analyses Reveal a Dual Molecular Mechanism Underlying Arsenic-Induced Apoptosis in Human Multiple Myeloma Cells. Journal of Proteome Research, 8, 3006-3019. http://dx.doi.org/10.1021/pr9001004
[31]	Morales, A.A., Gutman, D., Cejas, P.J., Lee, K.P., et al. (2009) Reactive Oxygen Species Are Not Required for an Arsenic Trioxide-Induced Antioxidant Response or Apoptosis. Journal of Biological Chemistry, 284, 12886-12895. http://dx.doi.org/10.1074/jbc.M806546200