Growth Inhibitory Effects And Mechanism Of Hydralazine And5-Aza-CdR On Human Osteosarcoma Cell Line And Expression Of Gene WW Domain-containing Oxidoreductase

BackgroundOsteosarcoma is the most common type of malignant bone tumors,which tends tooccur in young adults, men more than women. Small metastases have appeared inthe distance when80%~90%osteosarcoma patients were diagnosed. These smallmetastases most occur in the lungs.The surgical treatment of pure5-year survival rateless than20%. Neoadjuvant chemotherapy which was first raised by Rosen, refers tosystemic chemotherapy given to patients before local treatment (surgery orradiotherapy). Combined with clinical, imaging and pathological evaluation, determine the sensitivity of chemotherapy and the extent of the tumor necrosis.It can control the distance in the development of metastases through the effectivepreoperative chemotherapy, increases the chances of limb-salvage surgery. The wideapplication of neoadjuvant chemotherapy, the development of surgical techniques andmaterials science, greatly reduces the disability rate and mortality in patients withosteosarcoma. The osteosarcoma5-year survival rate was up to60%~80%, somepatients can also be long-term survival with tumor, and improve the quality of life.The WWOX(ww domain containing oxidoreductase)gene is a tumor suppressorgene that is detected in chromosome16q，spanning the common fragile site FRA16D．The WWOX gene have9exons, encoding a protein containing414aminoacids.The WWOX proteins contain two Ww domains in the NH2-terminal region anda short chain dehydrogenase／reductase(SDR)domain in the central region of theprotein．Aberrations of WWOX at either genome and expression level or both werecommon in various neoplasms such as osteosarcoma, colon cancer, lung cancer,gastric cancer, breast cancer．prompt the WWOX gene belongs to the tumorsuppressor genes. It can inhibit tumor cell proliferation, transcription in genome andaffect the exception to WWOX gene may be associated with the occurrence anddevelopment of tumor. The WWOX may play an important role in osteosarcoma．The DNA methylation is an important regulation way in the genetic modification,compared with the genetic mutations, such as missing genetic change, DNAmethylation doesn’t change the genetic sequences of DNA, but the expression ofgenes is regulate.The DNA methylation modification usually occurs on the rich inCG dinucleotide sequence, through modifying individual it can regulate genetranscription. And the DNA methylation modification is reversible.5-Aza-CdR(5’-aza-2’-deoxycytidine) is a common type of methylation inhibitors,covalent binding to DNA methylation enzyme, reduce the activity of DNA methylation enzyme, reduce methylation effective reverse DNA methylation status.Atpresent it is mainly used for the treatment of malignant tumor of the blood in clinical.The application in clinical is limited for its strong cytotoxic effect.Hydralazine is common used to treat cardiovascular disease in clinically. A largenumber of studies have shown that hydralazine can be used as methylation inhibitors.It can effectively to make the T lymphocyte response related antigen function,induced the expression of P16gene mRNA and protein. In view of the treatment ofhydralazine on APC(adenomatous polyposis coli),MGMT(O6-methylguanine-DNA methyltransferase) and FHIT (fragile histidine triad), DAKgene and P16gene,the results also confirmed that the drug hydralazine can be used asmethylation inhibitors,effectively reverse the methylation of tumor suppressor genesstate. It has a good prospect for hydralazine in tumor therapy.Materials and MethodsMethodsMethyl thiazol tetrazolium(MTT) colorimetric method were used to test thegrowth inhibition of MG-63cells intervened by difernt concentrations ofHydralazine and5’-aza-2’-deoxycytidine (5-Aza-CdR).Flow cytometryAnnexinV-FITC/PI methods were used to assay apoptosis. Real-time polymerasechain reaction（Real-Time PCR）methods were used to detect amplificantion ofWWOX mRNA induced by Hydralazine combined with5-Aza-CdR oralone.Western-blotting methods were used to examine the expression of WWOX inMG-63cells.ResultsHydralazine and5-Aza-CdR could inhibit the growth of MG-63cells in aconcentration and time-dependent manner with the different being significant in compae with other groups(p<0.05).ConclusionHydralazine and5-Aza-CdR could Either Hydralazine or5-Aza-CdR alone couldinhibit the proliferation of MG-63cells and induce apoptosis. The mechanism may bethat Hydralazine/5-Aza-CdR effectively cause the demethylated of WWOX geneCpG-rich promoter regions,leading to the high expression of WWOX and inhibit thegrowth of MG-63cells.