The Effects And Mechanisms Of Ndrg1 On The Response Of Advanced Rectal Cancer To Neoadjuvant Therapy

Objectives N-myc downstream-regulated gene 1(NDRG1)is a member of N-Myc down-stream regulated gene superfamily.Previous studies reported that NDRG1 is necessary for P53-induced apoptosis and plays potent role in promoting apoptosis in colorectal,lung and pancreatic cancer cells.However,the molecular mechanisms have been far from fully elucidation.The aim of this study is to investigate the effects and mechanisms of NDRG1 on apoptosis and potential clinical value of this effect.Methods 1.97 patients with locally advanced rectal cancer were enrolled in our study.Oxaliplatin-based chemotherapy was then delivered to all the patients for two cycles before surgery.The patients were divided into two groups as treatment effective(complete regression and partial regression)or non-effective(steady and progression).The expression level of NDRG1 of each patient was determined by two independent pathologists.Correlation of NDRG1 levels and treatment effects was then analyzed SPSS statistic software.2.Two CRC cell lines,HCT116 and SW620 cells were cultured.After over-expressed or knockdown of NDRG1,The hallmarks of apoptosis,cleaved-PARP and-caspase-3 were detected using western blot assay.3.Ectopic expression os NDRG1 was performed in sh TP53 cells and the effects of NDRG1 on apoptosis were assessed.4.Several main apoptosis regulating factors were analyzed with enzyme-linked immune-sorbent assay(ELISA)in NDRG1 over-expressed cells.5.Immunoprecipitation assay were performed to analyze the effect of NDRG1 on Bcl-2 protein.6.In vivo study was conducted by establishing nude mice xenograft model.Results 1.In all 97 enrolled patients receiving neoadjuvant therapy,58 patients were classified as effective(complete regression and partial regression).39 other patients were non-effective(steady and progression).67.2% patients in effective group showed positive signal of NDRG1 while only 46.1% patients showed positive expression in non-effective group.There was a positive correlation between NDRG1 expression and downstaging rate.2.The protein levels of cleaved-PARP and –caspase-3 was elevated after over-expression of NDRG1,which suggested NDRG1 promoted apoptosis in CRC cells.3.Silencing of TP53 gene led to resistance of HCT116 cells to oxaliplatin-induced apoptosis which could be rescued by over-expression of NDRG1.4.Upon NDRG1 expression,Bcl-2 protein showed significant decrease.These results suggested NDRG1 might promote apoptosis by targeting Bcl-2.5.Immunoprecipitation assay revealed the ubiquitination of Bcl-2 remarkably increased in NDRG1 cells,whereas the knockdown of NDRG1 resulted in the opposite.6.HCT116NDRG1 or-sh NDRG1 cells were injected into nude mice subcutaneously.Oxaliplatin was infused from the tail vein.After the infusion,the growth of NDRG1 xenografts was significantly suppressed.Moreover,xenograft tumors formed by control and sh NDRG1 cells still underwent rapid growth even after drug treatment.Conclusion NDRG1 enhances the degradation of Bcl-2 and promotes apoptosis in CRC.In the presence of NDRG1,oxaliplatin-based chemotherapy can yield great killing activity,resulting in higher downstaging rates and better prognosis of rectal cancer patients.As far as we are concerned,our study is the first demonstration that rectal cancer can be stratified by NDRG1 status in terms of the response to chemotherapy.