Arsenic Trioxide-induced Apoptosis is Associated with Induction of SHP-1 in Cutaneous T Cell Lymphoma

The activity of As2O3 was analyzed in two cutaneous T cell lymphoma cell lines, including HuT 78 and HuT 102, and cells derived from cutaneous T cell lymphoma patients. All of the cells were treated with As2O3 of different concentration (2.5μmol/L, 5μmol/L, 7.5μmol/L) and different time (24h, 48h, 72h). The methylation status of SHP-1 promoter and the changes of methylation status after the treatment of As2O3 in the cells was detected by methylation specific polymerase chain reaction(MSP). Then the expression level of SHP-1 mRNA was analyzed by Real-time polymerase chain reaction (Real-time PCR). Meanwhile the proliferative inhibition rate was determined with MTT, and the apoptosis induced by As2O3 was detected by flow cytometry with Annexin V/PI staining. DAPI staining was also used to observe the morphological changes during the apoptosis procedure. Then we explore the possible mechanism of As2O3 in this malignancy. First, protein levels of SHP-1, STAT3, phospho-STAT3 were analyzed using Western blotting. In addition, Real-time PCR was also used to analyze the Bcl2, Bcl-xL, survivin, c-myc, Mcl-1, cyclin-D1 and VEGF mRNA expression level.

The MSP results indicated that the SHP-1 promoter in these cells was completely methylated and there was no unmethylation strap. The methylation straps of SHP-1 promoter treated with 5.0μmol/L As2O3 were weakened along with the prolonging of treatment time, meanwhile the unmethylation straps were enhanced. The expression levels of SHP-1 mRNA increased along with the increase of As2O3 concentration and treatment time(P<0.05). As2O3 induced significant inhibition on the proliferation and its inhibitory effects were enhanced along with the increase of concentration and time(P<0.05). We also certified that As2O3 could induce apoptosis of these cells and the apoptosis rates were significantly higher along with the increase of As2O3 concentration and treatment time(P<0.05). The SHP-1 protein was not detectable in untreated cells. After the treatment of As2O3, the expression of SHP-1 protein increased along with the prolonging of treatment time(P<0.05). Meanwhile, the level of STAT3 protein remained unchanged. Strikingly, phospho-STAT3 protein was constitutively expressed before treatment. With progressive demethylation and reexpression of SHP1, there was progressive down-regulation of phosphorylated STAT3. Bcl2, Bcl-xL, survivin and Mcl-1 mRNA, which were related to JAK/STAT pathway, were all downregulated. At the same time, the expression of VEGF, c-Myc and cyclin D1 mRNA were also reduced.

The SHP-1 promoter in cutaneous T cell lymphoma cells is completely methylated resulting in the transcriptional silencing of SHP-1. As2O3 can induce the re-expression of SHP-1 mRNA and protein in cutaneous T cell lymphoma cells via the demethylation effect. What's more, As2O3 can inhibit proliferation and induce apoptosis of these cells. It is reported that, SHP-1 is the negative regulator of STAT3. Our results demonstrated that the demethylation leading to re-expression of SHP1 resulted in down-regulation of phosphorylation of STAT3, so did the target genes of STAT3, including Bcl2, Bcl-xL, survivin, Mcl-1, etc. That's the probable mechanism in this study. In conclusion, As2O3 may be beneficial for advanced-stage MF/SS.

No relevant conflicts of interest to declare.