The Mechanisms Of Apoptosis Induction By Deubiquitylatinase Inhibitor P5091 In Esophageal Squamous Cell Carcinoma Cells

Protein deubiquitination mediated by deubiquitinatinases(DUBs)is the inverse process of the ubiquitination process.The dynamic balance of ubiquitination and deubiquitination,affects or regulates cell growth,development,signal transduction and many other cell physiological and pathological processes.The ubiquitination enzyme regulates the stability of the protein by hydrolyzing the ubiquitin chain on the protein of the substrate.Its activity directly affects the turnover rate,activity,regeneration and localization of various proteins in the cell.P5091 is one kind of USP7 inhibitors,which can strongly inhibit the activity of USP7 enzyme.In the treatment of multiple myeloma,P5091 showed good anti-tumor effect,and it can also inhibit the growth of multiple myeloma cells which are resistant to Velcade,doxorubicin.Besides,P5091 inhibited the proliferation and induced apoptosis of colorectal cancers cells.However,the role and mechanism of P5091 in esophageal cancer remain to be further investigated.In this paper,the inhibitory effect of P5091 on the proliferation of esophageal cancer cells was determined in vitro.Esophageal squamous cell carcinoma(ESCC)cell lines KYSE450,KYSE510 and KYSE30 were selected as experimental subjects to evaluate the effect of P5091 on the proliferation,apoptosis and clonal formation of esophageal carcinoma.Rescue assay was used to investigate the mechanism of P5091 on esophageal cancer cells.Our study will provide a theoretical basis for the development of new drugs for the clinical treatment of esophageal cancer.Methods1.Detecting the expression of USP7 in esophageal cancer cells and tissues: A number of esophageal cancer cell lines were selected and tissue array from ESCC were used to detect the level of USP7 protein,and then The representative cell lines were selected out as the cell line used in this experiment.2.Drug concentration screening: The effect of P5091 on different cell lines were detected by ATP-dependent luciferase cell activity assays.IC50 values was calculated the experimental concentration of the drug in different cell lines.3.Plate colony formation assay and cell morphology image analysis were used to evaluate the inhibitory effect of different concentrations of P5091 on proliferation of esophageal squamous cell carcinoma KYSE450,KYSE510 and KYSE30.4.Detection of apoptosis: The apoptosis effects of different concentrations of P5091 on esophageal squamous cell carcinoma KYSE450,KYSE510 and KYSE30 were detected by Annexin V-FITC and PI staining.5.Activation of Caspase-3: The activation of caspase-3 were detected by FITC-DEVD-FMK staining.The expression levels of c-PARP and c-caspase-3 in esophageal squamous cell carcinoma KYSE450,KYSE510 and KYSE30 were detected by Western Blot.6.Effect of P5091 on the expression of ant-apoptotic and pro-apoptotic proteins:,After treatment with P5091,Western Blot was used to detect the effect of P5091 on the expression of ant-apoptotic and pro-apoptotic proteins in esophageal squamous cell carcinoma during the apoptosis of KYSE450,KYSE510 and KYSE30.The pro-apoptotic protein Noxa changed significantly in this experiment.7.The effect of P5091 on the m RNA level of Noxa: The m RNA levels of pro-apoptotic protein Noxa were measured by Q-PCR in different concentrations of P5091 on esophageal cancer cells.8.The role of Noxa in P5091-induced apoptosis: The changes of apoptosis rate of esophageal cancer cells induced by different concentrations of P5091 were detected by flow cytometry after the knockdown of Noxa gene.9.Noxa was regulated by ATF4: Noxa and ATF4,the upstream regulatory molecule regulating the expression of Noxa,were simultaneously silenced by si RNA,and then the changes of apoptotic rate induced by different concentrations of P5091 were detected by flow cytometry.Western blotting was used to detect the expression changes of Noxa protein and apoptotic protein c-PARP after the knockdown of Noxa and ATF4.Results1.P5091 inhibits the proliferation of KYSE450,KYSE510 and KYSE30 cells in esophageal squamous cell carcinoma.Results of ATPlite,plate colony formation assay and morphological observation showed that P5091 could significantly inhibit the proliferation of esophageal cancer cells in a concentration-dependent manner.2.P5091 induces apoptosis of esophageal carcinoma cell line.Annexin V/PI results showed that P5091 induced apoptosis of the cells,and the apoptosis rate increased with the increase of drug concentration,which was conformed by the decting of caspase-3 activation.Besides,Cleaved PARP and caspases were increased in a dose-dependent manner.3.P5091 promotes apoptosis of esophageal cancer cells by up-regulating the expression of Pro-apoptotic protein Noxa.Results showed that the proten level of Noxa was significantly increased after P5091 treatment in a dose-dependent manner.To determine the role of Noxa in the P5091-induced apoptosis,the expression of Noxa was downregulated by si RNA.Rescue showed that apoptosis and the expression level of cleaved PARP protein induced by P5091 was significantly reduced after silencing Noxa.These results suggest that Noxa may be a key molecule in P5091-induced apoptosis process.4.P5091 induced endoplasmic reticulum(ERS)in esophageal cancer cell linesThe results of Western blot showed that after treatment with P5091,the protein expression levels of p-EIF2?,ATF4,CHOP were significantly increased.These results suggested that P5091 could induce ERS in esophageal carcinoma cells.5.P5091 upregulated the expression of ATF4,which caused the accumulation of Noxa,and ultimately induced apoptosis.The results of Western blot showed that ATF4 and Noxa protein levels were significantly increased after P5091 treatment.At the same time,the m RNA level of Noxa was also increased with P5091 treatment.To further investigated weather Noxa was regulated by ATF4,ATF4 and Noxa were stimultaneously silenced and treated with P5091.Results showed that the cell apoptosis rate was significantly decreased in ATF4 knockdown cell lines,and the expression levels of c-PARP and Noxa proteins were significantly reduced.These results suggested that ATF4 was likely to be a major regulator of Noxa accumulation in esophageal cancer cell apoptosis induced by P5091.Conclusion1.P5091 significantly inhibit cell proliferation of ESCC in vitro.2.P5091 induced the upregulation of ATF4 expression by ERS,which promoted apoptosis by increasing the expression of pro-apoptotic protein Noxa.