TPL Enhances TRAIL Sensitivity Of Pancreatic Cancer Cells Via Downregulation Of PUM1 And The Role Of PUM1 In Pancreatic Cancer Cells

Background:Pancreatic cancer is extremely malignant,and ranks as the seventh leading cause of cancer-related deaths and the second leading cause of digestive tract malignancy-related deaths.According to global cancer statistics for 2012,the age-standardized rate per 100,000 in terms of the incidence and mortality is 5.9 and 5.5,respectively.The curative effect of surgery is not satisfactory in case of pancreatic cancer,and the 5-year postoperative survival rate is less than 5%.As a member of the tumor necrosis factor(TNF)superfamily,TNF related apoptosis-inducing ligand(TRAIL)can trigger apoptosis in cancer cells,but not in normal cells.Thus,it is considered a promising anticancer drug.However,recent studies revealed that many malignant tumors are resistant to TRAIL,creating a bottleneck in clinical applications.Triptolide(TPL)is a unique structural form of diterpene isolated from the traditional Chinese medicine herb Tripterygium wilfordii.TPL has also been demonstrated to have good antitumor activity both in vivo and in vitro.Our previous study indicates that treatment of PC cells with a combination of TRAIL and TPL at low concentrations results in cell death and increases the apoptosis rate of PC cells.However,current knowledge is far from adequate to fully understand the mechanisms by which TPL increases TRAIL sensitivity of pancreatic cancer.Purposes:We aimed to find TPL-regulated upstream components of the signaling pathways of TRAIL to further understand the regulatory mechanism by which TPL increases the sensitivity to TRAIL.And to understand the important molecular mechanism underlying the occurrence and development of PDAC.Methods:The proliferation activity of pancreatic cancer cells was detected by CCK-8,Celigo.The expression of diverse proteins was examined by using western blot analysis.Immunohistochemistry analysis was employed to detect the expression of proteins in tissue microarray.Immunoprecipitation analysis was employed to detect the interaction between related proteins.Transwell was employed to detect levels of cell migration and invasion.The apoptosis or ROS were detected by using flow cytometry.Subcutaneous tumorigenesis in nude mice was used to detect the growth ability of tumor cells in vivo.And the ability of tumor invasion and metastasis in vivo was detected by lung metastasis test in nude miceResults:This study includes three parts:Part One: Triptolide enhances TRAIL sensitivity of pancreatic cancer cells via downregulation of PUM11.PUM1 is a key factor in TPL enhances TRAIL sensitivity of pancreatic cancer cellsAfter treatment with low-concentration TRAIL and/or TPL,cells were harvested for microarray analysis to identify differentially expressed mRNAs.23 candidate target genes were identified by the formula gene set I(TPL vs DMSO)+ gene set II(TPL + TRAIL vs DMSO)– gene set III(TRAIL vs DMSO).Celigo results show that,after treatment with low-concentration TRAIL,PUM1 gene plays the most obvious role in suppressing cell growth compared with the control group.Therefore,PUM1 may be the key factor for TPL enhancing TRAIL sensitivity.2.Effects of PUM1 expression level on the proliferation and apoptosis of pancreatic cancer cells with the treatment of low TRAIL concentration.After treatment with low-concentration TRAIL,knock down PUM1 can suppress pancreatic cancer cells proliferation,promote apoptosis,while enhancing the expression of apoptosis related proteins cleaved caspase 3 and cleaved caspase 9.On the contrary,after treatment with low-concentration TRAIL pancreatic cancer cells,the overexpression of PUM1 can promote cell proliferation,inhibit apoptosis,and inhibiting the expression of apoptosis related proteins cleaved caspase 3 and cleaved caspase 9.The tumorigenesis experiment in nude mice also proved that in the pancreatic cancer cells treated with low concentration TRAIL,the tumor volume of shPUM1 group was significantly reduced compared with that of the control group.3.Knockdown PUM1 can enhance the sensitivity of TRAIL by inhibiting the G1-S transition of pancreatic cancer cells.After treatment with low-concentration TRAIL,the cell cycle experiment showed that shPUM1 could inhibit the G1-S transition of pancreatic cancer cells.Western blot showed that the expression levels of p27 and p27-cdk2 complexes were significantly increased,and the expression levels of cyclin A and cyclin E were significantly reduced.4.TPL enhances the sensitivity of TRAIL by enhancing autophagy of pancreatic cancer cells via the expression of PUM1.Transmission electron microscopy(TEM)found that compared with the TRAIL group,the number of autophagosomes in TPL+TRAIL group increased.Western Blot showed that the expression of ATG5 ULK1 VPS34 was increased,while the expression of mTOR decreased in pancreatic cancer cells,suggesting that TPL+TRAIL can promote autophagy in pancreatic cancer cells.Meanwhile,we found that after treatment with TRAIL,shPUM1 group can also enhance autophagy of pancreatic cancer cells,and the overexpression of PUM1 can partially reverse the effect of TPL enhance on TRAIL autophagy.Inhibition of autophagy by 3-MA can partially reverse the effect of PUM1 knock down after treatment with low-concentration TRAIL that suppression of pancreatic cancer cell proliferation and promote of apoptosis.Part Two: The mechanism of PUM1 in pancreatic cancer.1.The high expression of PUM1 protein indicates poor prognosis of patients.GEPIA bioinformatics analysis indicated that the expression of PUM1 mRNA in tumor tissues was higher than that in adjacent tissues,and tissue chip analysised by immunohistochemical indicated that the expression of PUM1 protein in PDAC patients was higher in tumor tissues than that in adjacent tissues.2.Inhibiting the expression of PUM1 can suppress proliferation,migration,invasive and epithelial–mesenchymal transition(EMT)of pancreatic cancer cells while promote the apoptosis.In vitro,MTS show that knockdown PUM1 can suppress cell proliferation,transwell assay demonstrated that knockdown PUM1 can suppress cell migration and invasion,flow cytometry analysis show that knockdown PUM1 can promote apoptosis,Western Blot showed that knockdown PUM1 can suppress the expression of MMP9,VEGF,vimentin and enhance the expression of E-cadherin.On the contrary,over expression PUM1 can promote cell proliferation,migration,invasive and EMT,while suppress cell apoptosis.In vivo,subcutaneous tumor formation in nude mice show that knock down PUM1 can suppress the growth of subcutaneous tumor.The lung metastasis model proves that knocking down PUM1 can reduce the number of ki67 positive cells in lung tissue.3.PUM1 promotes the proliferation and migration of pancreatic cancer cells to invade EMT and suppresss apoptosis by inhibiting the PERK/eIF2/ATF4 signaling pathway.IPA analysis found that knocking PUM1 can activate eIF2 signaling pathway.Western Blot showed that PUM1 knockdown promoted the expression of p-PERK,p-EIF2 A,ATF4.Tissue chip correlation analysis showed that the expression level of PUM1 was negatively correlated with the expression of p-PERK.After the overexpression of PERK in pancreatic cancer cells,MTS showed that the cell proliferation was suppressed,transwell assay showed that cell migration and invasion was suppressed,western blot analysis showed that the expression of MMP9,VEGF,vimentin was decreased,while the expression of E-cadherin was increased.The use of PERK inhibitors in PUM1 knockdown pancreatic cancer cells can partially reverse the effects of PUM1 on the proliferation,migration,invasive,EMT and apoptosis of pancreatic cancer cells.Conclusion:1.Knocking down PUM1 can enhance the sensitivity of pancreatic cancer cells to TRAIL both in vivo and in vitro,suggesting that PUM1 may become a new target to enhance the sensitivity of pancreatic cancer cells to TRAIL.Our results indicate that TPL enhances TRAIL sensitivity of pancreatic cancer cells by activating autophagy via downregulation of PUM1.2.PUM1 knockdown suppressed cell growth,invasion,and metastasis,and promoted apoptosis by activating the PERK/eIF2/ATF4 signaling pathway in PDAC cells.