Arsenic Trioxide-induced Apoptosis is Associated with Induction of SHP-1 in Cutaneous T Cell Lymphoma
Authors: Jin Xin , Li Fang , Guo Shanqi , Xia Bing , Zhang Ling , Zhang Yizhuo 
Published: October 2019

Abstract
Abstract 4716
Arsenic trioxide (As2O3) is an ancient drug which was used in traditional Chinese medicine. It has attracted worldwide interests because of its antitumor activity when used in acute promyelocytic leukemia (APL). What's more, the demethylation and antiapoptosis function can also be seen in many papers. The methylation status of SH2 containing phosphatase-1 (SHP-1) promoter is common in malignant hematological diseases, such as MDS. Inducing gene demethylation may be a mechanism of As2O3 in treating hematologic cancers. Whether As2O3 has the effects on demethylation of SHP-1 and on the proliferation and apoptosis of cutaneous T cell lymphoma cell lines, and what is the concrete mechanism? Our study aimed to solve these problems.

Methods:
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.

Results:
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.

Conclusion:
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.

Disclosures:
No relevant conflicts of interest to declare.