Tumor Suppressor Gene XAF1 Expression And Its Regulation In Hepatocellular Carcinoma

PartⅠExpression of X-linked inhibitor of apoptosis-associated factor 1in hepatocellular carcinoma and its clinical significanceObjective Apoptosis resistance plays an important role in the initiation and progression ofhuman cancers.The inhibitors of apoptosis (IAPs) comprise a family of proteins that play acritical role in cell-acquire resistance to apoptosis.Cell life and death decisions rely on adelicate balance between pro- and anti-apoptotic factors.The upregulation of IAPexpression or function is a common feature for human hepatocellular carcinoma (HCC).X-linked inhibitor of apoptosis protein associated factor-1(XAF1) is a novel natural IAPantagonist.XAF1 mRNA is expressed in normal tissues but low or not in various cancersand thus poses a potential tumor suppressor gene.In this study,we explore the significanceof XAF1 in HCC.Methods The expression of XAF1 mRNA,the distribution of XAF1 protein in human fetalliver cell line L02,hepatoma cell linesSMMC7721,Hep3B,and HepG2,and 30 cases of hepatic carcinoma and their corresponding surrounding liver tissue specimens wereexamined by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR),streptavidin peraxidase (SP) immunohistochemical staining,and the correlation withclinical factors.Results XAF1 transcript and protein can be detected in L02 cell,whereas,XAF1 transcriptand protein were expressed at low levels in hepatoma cell lines HepG2,SMMC7721 andHep3B.The expression of XAF1 mRNA and protein in HCC tissues was lower than thosein their corresponding nontumorous liver tissue specimens (mRNA:0.587+0.064,1.013±0.159,respectivelv t=2.392,P=0.000;protein:165.17±17.85.104.74±15.7,respectively.t=17.57,P=0.000).XAF1 protein was distributed in both nuclear andcytoplasm.XAF1 expression was significantly lower in poorly differentiated than those inwell- or moderately differentiated HCCs.Conclusion The expression of XAF1 decreases in the hepatocellular carcinoma.XAF1may play a key role in the hepatocarcinogenesis PartⅡCorrelation of XAF1 methylation with drug resistanceof hepatoma cell linesObjective Aberrant promoter methylation is a fundamental mechanism of tumorsuppressor genes inactivation.It has been proposed that aberrant DNA methylation canalter cancer's response to chemotherapeutic agents.Hepatocellular carcinoma is notsensitive to chemotherapy.The purpose of the present study was to investigate therelationship between methylation of XAF1 and drug resistance and apoptosis of hepatomacell lines.Methods Methylation status of XAF1 from genomic gene of hepatocellular carcinomacell lines was determined with methylation-specific polymerase chain reaction(MSP) andwas further verified by demethylation treatment with 5-aza-2'-deoxycytidine (5-Aza-CdR),a demethylation compound.XAF1 cDNA was introduced into hepatoma cell lines;chemosensitivity to MMC and fluorouracil were analyzed using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay and flow cytometryanalysis,respectively.Apoptosis was examined by flow cytometry analysis and thealteration of caspase-3 cleavage was measured by western blot.Results MSP analysis indicated that there was aberrant methylation of XAF1 promoter inhepatoma cell lines.The expression of XAF1 mRNA from hepatoma cell lines treated with5,10μmol/L of 5-Aza-CdR for 96h was significantly up-regulated (P= 0.034,P=0.002,respectively).Compared with control vector,XAF1 transfected hepatoma cells showedenhanced sensitivity to chemotherapeutic drug.Flow cytometry analysis revealed that theapoptosis rate is increased markedly in hepatoma cell lines transfected with XAF1 compared with those with control vector response to chemotherapeutic drug.XAF1increased significantly the protein levels of active caspase-3 and cleaved PARP.Conclusion XAF1 was inactivated in hepatoma cell lines through epigenetichypermethylation.Introduction of XAF1 may increased hepatoma cell lines sensitivity tochemotherapeutic drug,and induced hepatoma cell lines apoptosis,which may provide theexperimental evidences for application of demethylation agent to HCC therapy. PartⅢHSF1 mediates regulation of XAF1 in HCCObjective Stress factors includes heat shock,heavy metals,inflammation,autoimmune reactions,and viral,et al.Some stress factors have been considered as tumorigenic agents.Heat shock factor1 (HSF1) protects from cell death against various kinds of stresses and these effects aremediated by regulation of heat shock proteins expression.Recent studies have shown thatHSF1 could also act as a negative regulator of certain genes through binding to heat-shock element(HSE).Our previous works revealed that XAF1 expression was lower in HCC tissues.Theaim of this study was to investigate the relationship between XAF 1 and HSF1 in HCC.Methods 1395bp-promoter fragment of XAF1 gene was amplified by PCR and cloned intopGL3-basic to form pGL3-XAFlp.The promoter activity was determined bydual-luciferase reporter assay.Two RNAs were designed according to the coding sequenceofHSF1 gene,and cloned into the downstream of H1 promoter ofpsiRNA-hH1neo to formpsiRNA 1,psiRNA 2,psiRNA3.The best effective silencing plasmid was screened.Afterhepatoma cell lines were treated with heat stress,the HSF1 and XAF1 expression weremeasured with RT-PCR,western blot,respectively.Meanwhile,Hepatoma cell lines weretransfected with psiRNA2 which targeted HSF1;24h after transfection,followed by heatstress treatment,the alteration of HSF1 and XAF1 expression were also detected.Hepatoma cell lines were transfected with pGL3-XAF1 p,followed by heat stress treatment.The XAF1 promoter activity was determined by dual-luciferase report assay.Theexpression of HSF1 and XAF1 in HCC tissues and matched non-tumor liver tissues wereanalyzed by immunohistochemical staining.Results Recombinant plasmids pGL3-XAFlp and psiRNA1-3 were confirmed byrestriction enzyme digestions and sequencing analysis.The psiRNA2 had the best silencingeffect.The HSF1 expression was significantly upregulated and XAF1 expression wassignificantly downregulated in the HepG2 cells treated with heat stress.The XAF1expression was significantly upregulated in the stress cells when transfecting with the psiRNA2 which effectively targeted HSF1.The activities of pGL3-XAF1 p in hepatoma celllines after treatment with heat stress were significantly lower than those in the unstressedcells.HSF1 expression level was found to be upregulated in the HCC tissues compared withmatched non-tumor liver tissues.The expression of XAF1 and HSF1 was negativelycorrelated in the HCC tissues.Conclusion HSF1 downregulates XAF1 through binding to heat-shock element (HSE) of XAF1promoter,which is the mechanism of XAF1 transcriptional silencing in HCC. PartⅣTriptolide induces hepatoma cells apoptosis via up-regulation ofXAF1 transcriptional activityObjective Triptolide (TPL) is a diterpenoid triepoxide derived from the herb Tripterygiumwilfordii that has been used as a natural medicine in China for hundreds of years,particularly in the treatment of autoimmune and inflammatory diseases,includingrheumatoid arthritis.Several reports have indicated that triptolide could inhibit theproliferation of cancer cells in vitro and reduce the growth and metastases of tumor in vivo.Recent study has shown that TPL is a potent inhibitor of HSF1 activation.Our previousstudy have implicated that HSF1 could down-regulated XAF1 in hepatoma cell lines.Therebefore,Our aim was to examine the effect of TPL on the stressed HepG2 cellsapoptosis and XAF1 expression in this study.Methods Human hepatoma cell line HepG2 was treated with different concentrations ofTPL followed by a heat shock exposure.Cell viability was analyzed by MTT assay.Cellapoptosis were detected by flow cytometry using Annexin V/PI.The expression of HSF1and XAF1 protein were determined by RT-PCR,Western blot,respectively.HepG2 weretransfected with pGL3-XAF1p,24h after transfection,cells were treated sequentially withTPL followed by a heat shock exposure.The XAF1 promoter activity was determined bydual-luciferase report assay.XAF1 cDNA was introduced into hepatoma cell lines followedby a heat shock exposure.Cell viability and cell apoptosis were determined by flowcytometry analysis.Results TPL significantly reduced viability of HepG2 cells in dose dependent manner.TPLinduced apoptosis and increased XAF 1 expression and its promoter activity in the stressedHepG2 cells.Flow cytometry analysis revealed that the apoptosis rate is increasedmarkedly in hepatoma cell lines transfected with XAF1 compared with those with controlvector response to a heat shock exposure.Conclusion TPL causes HepG2 cells death by induction of apoptosis and its mechanism ofaction is mediated via up-regulation of XAF1 transcriptional activity.