Effects of Hydralazine and 5-Aza-CdR on cell proliferation of human osteosarcoma cell line MG-63 and expression of gene WW domain-containing oxidoreductase
Li Jiazhen, Xu Zongchao, Zhang Yan, Lu Xinchang, Shi Hailong, Liu Yongkui
Published 2014-05-01
Cite as Chin J Orthop, 2014,34(05): 593-597. DOI: 10.3760/cma.j.issn.0253-2352.2014.05.013
Abstract
Objective To investigate the growth inhibition of human osteosareoma cell line(MG-63) intervened by Hydralazine and 5'-aza-2'-deoxycytidine (5-Aza-CdR),and the effect on the mRNA expression of gene WW domain-containing oxidoreductase (WWOX).Methods Certain volume of 5 × 104/ml of human osteosarcoma cell line MG-63 in logarithmic growth phase were added into 96-well plate.There were Hydralazine group (drug concentration,0.1,1.0,10 μmol/L),5-Aza-CdR group (drug concentration,5,10,20 μmol/L),Hydralazine combined with 5-Aza-CdR group (drug concentration,0.1 μmo/L + 5 μmol/L,1.0 μmol/L + 10 μmol/L,10 μmol/L + 20 μmol/L) and control group (culture medium).Methyl thiazol tetrazolium(MTT) colorimetric methods were used to test the growth inhibition of MG-63 cells intervened by different concentrations of Hydralazine and 5'-aza -2'-deoxycytidine (5-Aza-CdR).Flow cytometry AnnexinV-FITC/PI methods were used to assay the effects of Hydralazine and 5-Aza-CdR inducing apoptosis in osteosarcoma cells in vitro.Real-time polymerase chain reaction (Real-Time PCR)methods were used to detect amplification of WWOX mRNA induced by Hydralazine combined with 5-Aza-CdR or alone.Western-blotting methods were used to examine the expression of WWOX in MG-63 cells.Results Hydralazine and 5-Aza-CdR effectively inhibited the growth of MG-63 cells in a concentration and time-dependent manner.Combined effect was more obvious.Further more the expression levels of WWOX mRNA and protein were increased significantly in combined groups as compared with other groups.Conclusion Hydralazine and 5-Aza-CdR could effectively inhibit the proliferation of MG-63 cells and induce apoptosis which is concurrent with the promotion of the expression of WWOX.The mechanism may be that Hydralazine/5-Aza-CdR effectively cause the demethylated of WWOX gene CpG-rich promoter regions,leading to the high expression of WWOX and inhibit the growth of MG-63 cells.The use of hydralazine in the treatment of osteosarcoma is worthy of further investigation.
Key words:
Osteosarcoma; Hydralazine; Deoxycytidine Monophosphate; Genes, tumor suppressor
Contributor Information
Li Jiazhen
Department of Orthopeadics, The First Affliated Hospital of Zhengzhou University, Zhengzhou 450052, China
Xu Zongchao
Department of Orthopeadics, The First Affliated Hospital of Zhengzhou University, Zhengzhou 450052, China
Zhang Yan
Department of Orthopeadics, The First Affliated Hospital of Zhengzhou University, Zhengzhou 450052, China
Lu Xinchang
Department of Orthopeadics, The First Affliated Hospital of Zhengzhou University, Zhengzhou 450052, China
Shi Hailong
Department of Orthopeadics, The First Affliated Hospital of Zhengzhou University, Zhengzhou 450052, China
Liu Yongkui
Department of Orthopeadics, The First Affliated Hospital of Zhengzhou University, Zhengzhou 450052, China