The Effect And Mechanism Of Chidamide In Acute Myeloid Leukemia

Part ⅠDownregulation of SP1/NFκB/DNMT1 and FLT3/STAT5 pathway and DNA methylation with the treatment of Chidamide in AMLObjective:To explore the anti-leukemic effect of the novel HDAC inhibitor Chidamide on AML and the mechanism of DNA methylation.Methods:The cellular function of inhibition of proliferation, cell apoptosis and cell cycle arrest were measured after the treatment of 0, 1μM,3μM Chidamide in MV4-11 and Kasumi-1 by CCK-8 analysis and flow cytometry. After exposure of 0, 1uM,3μM Chidamide for 48h in MV4-11 and 72h in Kasumi-1, Western Blot were performed to test the protein expression of SP1, NFκB, FLT3, STAT5, p-STAT5 and DNMT1; DNMT1 and P15 gene mRNA expressions were examined by Real-time PCR; and Global DNA methylation was determined by DNA Dotblot. The patients’ primary blast cells were treated with Chidamide at the above concentration,DNMT1 and P15 expression were examined.Results:After the treatment of 0, 1μM,3μM Chidamide, the inhibition of proliferation, cell apoptosis were showed in MV4-11 and Kasumi-1 with time and dose-dependence. At 48h IC50 of 1.8μM in MV4-11 and 1.48μM in Kasumi-1 were observed. The cell cycle was arrested at the G0/G1 phase after incubation of Chidamide for 48h. Chidamide induced acetylation of histon H3 and H4. The protein expressions of SP1, FLT3, STAT5, p-STAT5 and DNMT1 were reduced. The mRNA expression DNMT1 was down-regulated. DNA hypomethylation was induced and P15 gene was up-regulated after the treatment of Chidamide. The expression of DNMT1 mRNA and protein of patients’ primary blast cells were decreased. On the contrary, P15 mRNA was up-regulated following the treatment of Chidamde.Conlusion:Chidamide is an efficient anti-leukemic HDAC inhibitor in AML not only through histon acetylation but DNA demethylation by down-regulation of SP1/NFκB/DNMT1, and eventually leads to reactivation of antitumor gene. Moreover Chidamide reduces the protein expression of FLT3/STAT5/p-STAT5 pathway. Taken together, Chidamide will be a potent HDAC inhibitor agent for AML, especially for FLT3/ITD mutation AML.Part Ⅱepigenetic effect of Chidamide in AML1-ETO positive AMLObjective:To explore the specific anti-leukemic effect and the mechanism of the novel HDAC inhibitor Chidamide on AML1-ETO positive AMLMethods:The cellular function of inhibition of proliferation, cell apoptosis and cell cycle arrest were measured after the treatment of 0, 1μM,3μM Chidamide in AML1-ETO positive cell lines Kasumi-1, Skno-1 and AML1-ETO negative cell lines U937, Skno-1-si-AE-by CCK-8 analysis and flow cytometry. Western Blot and Real time PCR were performed for the protein expression of AML1-ETO, c-KIT,DNMT3A and DNMT1 and mRNA expression of DNMT3A and DNMT1; Global DNA methylation was determined by DNA Dotblot; P15 gene mRNA expression was examined by Real time PCR. After 48h transfection of AMLl-ETO plasmid into 293T, Scramble,siRNA into Kasumi-1 and Skno-1, protein expression of AML1-ETO, DNMT3A was examined by Western Blot. Lusciferase analysis on the promoter activity of DNMT3A was meaured after the 48h cotransfection of PGL3-3A, PCMV-AML1-ETO, PGL3-Basic plasmid into 293T and Kasumi-1 with treatment of 3μM Chidamide after 6h cotransfection.Results:After the treatment of 0, 1μM,3μM Chidamide, the inhibition of proliferation and cell apoptosis in AML1-ETO positive cell lines Kasumi-1, Skno-1 were higher significantly than AML1-ETO negative cell lines U937, Skno-1-si-AE. IC50 of 1.13μM, 2.58μM,3.9μM,6.71μM for Kasumi-1, Skno-1, U937, Skno-1-si-AE were discovered, respectively. The cell cycle was arrested at the G1/G0 phase in Kasumi-1 and Skno-1 following the incubation of Chidamide for 48h. The protein expressions of AML1-ETO, c-KIT,DNMT3A and DNMT1 were repressed and the mRNA expressions of DNMT3A and DNMT1 were down-regulated. Chidamide didn’t decrease the expression level of DNMT3A protein and mRNA in U937. DNA hypomethylation was observed and P15 gene increased after the treatment of Chidamide in Kasumi-1 and Skno-1. Gain and loss of function were concomitantbetween AML1-ETO and DNMT3A. Luscifrease analysis showed that AML1-ETO downregulated the promoter activity of DNMT3A after the exposure of 3μM Chidamide.Conlusion:HDAC inhibitor Chidamide can induce proliferation inhibition and cell apoptosis more sensitively in AML1-ETO positive cell lines Kasumi-1, Skno-1 than AML1-ETO negative cell lines U937, Skno-1-si-AE. Cell cycle is arrested at the G1/G0 phase in Kasumi-1 and Skno-1. Chidamide has more specific and sensitive anti-leukemic effect on AML1-ETO positive than AML1-ETO negative AML cells by repressing AML1-ETO which downregulates DNMT3A. Chidamide can reactivate anit-tumor gene P15 expression by inducing DNA demethylation and histon acetylation.Chidamide inhibits DNMT3A promoter activity by down-regulation AML1-ETO expression. Chidamide will be a potent HDAC inhibitor and DNA demethylation agent for AML1-ETO positive AML.