4SC-202 Induces Apoptosis In Myelodysplastic Syndromes And The Underlying Mechanism

Objective: 4SC-202 is a new class of target drugs for histone lysine-specific demethylase 1(LSD1)and class I histone deacetylases(HDACs).In this study,the agent influenced the cell viability,apoptosis and cycle of myelodysplastic syndrome(MDS),and classical molecular biology methods were used to explore the mechanism of 4SC-202 anti-MDS.HO-1 is a protective protein against tumor apoptosis.Therefore,the study also explored the effect of HO-1 on 4SC-202 anti-MDS.Method: Clinical samples: Collected 32 newly diagnosed MDS and MDS to acute myeloid leukemia(MDS/AML)patient specimens,q RT-PCR,Western blot and cytoimmunochemistry methods to detect LSD1,HDAC3 and HO-1 m RNA and protein expression levels.In vitro experiment: MDS cell line SKM-1 cells were treated with different concentrations and different time points 4SC-202,and cells were detected cell viability by CCK-8,apoptosis rate and cycle arrest by flow cytometry and the expression of LSD1,HDAC3 by q RT-PCR.The q RT-PCR and protein immunoblotting were used to detect the expression levels of LSD1,HDAC1/2/3,H3K4me2,AC-H3,HO-1,apoptosis and cycle-related proteins and NF-κB pathway-related proteins.Up-regulated HO-1 lentivirus-transfected SKM-1 cells were treated with 4SC-202 for 24 h.Western blot was used to detect the expression levels of HO-1,apoptosis-related proteins and NF-κB pathway-related proteins and flow cytometric analysis Apoptosis rate.Cells were treated with Hemin(HO-1 activator)and Zn PP(HO-1 inhibitor)for 2 h,then add 4SC-202 to expose for 24 h,use CCK-8 to detect cell viability and flow cytometry Apoptosis rate and Western blotting were used to detect the expression level of NF-κB pathway related proteins.After LSD1 and HDAC3 were silenced with small interfering RNA,the expression levels of NF-κB pathway-related proteins and HO-1 protein were detected by Western blotting.Immunofluorescence microscopy detected the localized expression of P65.In vivo: Establish HO-1 overexpression model and no-load control model in NOD/SCID mice,the content of human CD45+ in peripheral blood of mice were analyzed by flow cytometry,and draw survival curves.Results: Compared with normal samples,LSD1,HDAC3,and HO-1 were highly expressed in MDS/AML patient samples.After 4SC-202 treated SKM-1 cells,the survival rate of SKM-1 cells was inhibited,the apoptosis rate increased,and the cell cycle was arrested in the G2/M phase.4SC-202 reduces p-p65 and p-IκB-α protein expression and inhibits the transfer of P65 into the nucleus,thereby inhibiting the activation of the NF-κB pathway.4SC-202 down-regulated the expression level of HO-1 in SKM-1 cells.Direct silencing of LSD1 and HDAC3 will down-regulate the protein levels of p-p65,p-IκB-α and HO-1.Up-regulation of HO-1 does not directly affect the expression of apoptotic protein and apoptosis rate and the expression of p-p65 and p-IκB-α,but significantly reduces the effect and effect of 4SC-202 on apoptotic protein and induced cell apoptosis rate Downregulated levels of p-p65 and p-IκB-α.On the contrary,Znpp can enhance the effect of 4SC-202 against SKM-1 cells.In the NOD/SCID mouse model of allograft SKM-1 cells,it was found that the survival rate of mice treated with oral 4SC-202 was higher than that of the non-administered group.Among the mice treated with 4SC-202,the survival rate of mice in the HO-1 high expression group was lower than that in the HO-1 unupregulated group.Conclusion: Collectively,4SC-202,an inhibitor of LSD1 and HDAC1/2/3,significantly inhibited cell growth by causing cell cycle arrest at G2/M,inducing apoptosis and decreasing HO-1 gene expression via the suppression of NF-κB pathway in MDS cells.In addition,up-regulation of HO-1 expression attenuated the inhibitory effect of 4SC-202-induced activation of NF-κB pathway,thereby attenuating the efficacy of 4SC-202,but low HO-1 expression enhanced the efficacy of 4SC-202 in MDS cells.