Effect Of Arsenic Trioxide On Demethylation Of SHP-1Gene In Hut78Cells

Objective:1. To investigate the methylation status of SHP-1promoter in Hut78cells.2. If the SHP-1promoter is hypermethylated, we treated Hut78cells with AS2O3and analysed the changes of the methylation status of SHP-1promoter, SHP-1mRNA and protein levels and the proliferation and apoptosis of Hut78cells before and after the treatment. We investigated the effects of AS2O3on demethylation of SHP-1and on the proliferation and apoptosis of Hut78cells and these results may provide helpful information for AS2O3used to the treatment of advanced-stage MF/SS.Methods:1. The methylation status of SHP-1promoter in Hut78cells was detected by methylation specific polymerase chain reaction(MSP).2. The changes of methylation status of SHP-1promoter in Hut78cells before and after AS2O3treatment were detected by MSP.3. The expression levels of SHP-1mRNA in Hut78cells before and after AS2O3treatment were analysed by Real-time polymerase chain reaction (Real-time PCR).4. The expression levels of SHP-1protein in Hut78cells before and after AS2O3treatment were analysed by Western Blot.5. The changes of Hut78cells proliferation induced by AS2O3were detected by MTT method.6. The changes of Hut78cells apoptosis induced by AS2O3were detected by flow cytometry with Annexin V/PI staining.7. All data were processed with SPSS for windows software version19.0. The means of different samples were compared by using one-way ANOVA. If the difference was statistically significant, further LSD-t tests were used to analyzed the means. The level of significance was set at0.05.Results:1. The MSP results indicated that the SHP-1promoter in Hut78cells was completely methylated and there was no unmethylation strap.2. The methylation straps of SHP-1promoter in Hut78cells treated with5.0μmol/L AS2O3were weakened along with the prolonging of treatment time, meanwhile the unmethylation straps were enhanced.3. The expression levels of SHP-1mRNA in Hut78cells increased along with the increase of AS2O3concentration and treatment time(P<0.05).4. The SHP-1protein was not detectable in untreated Hut78cells. After treating Hut78cells with5.0μmol/L AS2O3, the expression of SHP-1protein increased along with the prolonging of treatment time(P<0.05).5. AS2O3elicited significant inhibition of Hut78cells proliferation and its inhibitory effects were enhanced along with the increase of concentration and time(P<0.05).6. AS2O3could induce apoptosis of Hut78cells and the apoptosis rates were significantly higher along with the increase of AS2O3concentration and treatment time(P<0.05).Conclusion:1. The SHP-1promoter in Hut78cells is completely methylated resulting in the transcriptional silencing of SHP-1.2. AS2O3can cause re-expression of SHP-1mRNA and protein in Hut78cells via the demethylation effect, and inhibit proliferation and induce apoptosis of Hut78cells. AS2O3maybe active in treatment of advanced-stage MF/SS.