The Biological Roles Of PGD In Colorectal Cancer Liver Metastasis

Object: To investigate the biological roles of key enzyme 6-phosphate gluconate dehydrogenase（PGD）in the liver metastasis of colorectal cancer and its mechanism.Methods: The colorectal cancer liver metastasis experimental model was performed in nude mice with spleen injection,qPCR and western blot were used to detect the changes of PGD expression in the pentose phosphate metabolism pathway of liver metastasis tumor cells and pre-metastasis tumor cells of colorectal cancer.Database Oncomine was also used to analyze the relationship between colorectal cancer patients PGD expression level and colorectal cancer,and PGD expression level between primary colorectal cancer tissue and colorectal cancer liver metastasis.Used lentiviralbased related techniques to overexpress PGD and knock down PGD in colorectal cancer cell lines,or used PGD inhibitors for CCK8 detection experiments,Cell migration experiments and Matrigel invasion experiments to explore the effects of PGD expression on the migration and invasion ability of colorectal cancer cells.Performed the mouse spleen injection liver metastasis experimental model to verify the effects that PGD affected the colorectal cancer liver metastasis in the mice.In the mechanism part,we analyzed the effects of PGD expression level on EMT in the gene and protein levels,and used flow cytometry to detect the effects of PGD on the redox of colorectal cancer cells,also the effects of PGD expression on the antioxidant capacity of colorectal cancer cells.Results: Real-time quantitative PCR and western blot were used to detect the expression level of PGD before and after metastasis in the spleen-liver metastasis model of nude mice.The results showed that PGD was up-regulated after liver metastasis in colorectal cancer.In the Oncomine colorectal cancer database,we analyzed the PGD expression level in colorectal cancer patients,the expression level of PGD in the tumor tissue was higher than that of adjacent cancer,and also the PGD expression level of colorectal cancer in liver metastasis was higher than that of carcinoma in situ.The results of in vitro experiments showed that the migration and invasion ability of HCT116 cell line overexpressed PGD was significantly enhanced comparing with the control group.In vivo experiments,spleen injection colorectal cancer cell line HCT116,the CRC liver metastasis animal experimental model showed that increased PGD expression level promoted liver metastasis of colorectal cancer.When validated in CRC tumor cell lines LOVO and Caco2,the experimental results were consistent with HCT116 cells.Compared with the control group,the cell migration and invasion ability of HCT116 was reduced by using physcion,a specific inhibitor of PGD enzyme activity.The results of physcion related experiments in LOVO was consistent with that of HCT116 cells.Mechanism research showed that overexpression of PGD in colorectal cancer cell line HCT116 can promote EMT from gene and protein levels,and knocking down PGD inhibited EMT of colorectal cancer cells,and in LOVO,Caco2,DLD1 colorectal cancer cell lines the result was verified.The results of flow cytometry experiments showed that after knocking down PGD in HCT116 colorectal cancer cells,NADPH production reduced,the cell’s antioxidant capacity weakened,and ROS levels increased.It was further detected the resistant capacity of PGD knockdown cells to H₂O₂.The cell activity is decreased,and the tolerance capacity of H₂O₂ gradually weakened with the increase of H₂O₂ concentration.After using the inhibitor physcion on HCT116 cells,consistent results were also obtained,that is,under the condition of PGD enzyme activity inhibited,NADPH production reduced,the antioxidant capacity of HCT116 cells weakened,ROS levels increased,and after the enzyme activity was inhibited,the cell’s tolerance to H₂O₂ weakened,and this reduced tolerance was concentration-dependent for both physcion and H₂O₂.Conclusion: In colorectal cancer,the up-regulation of PGD promoted the capacity of liver metastasis and invasion of colorectal cancer cells.The mechanism exploration showed that the upregulation of PGD will promote the process of EMT in colorectal cancer cells.When the expression level of PGD is decreased or the enzyme activity is weakened,the production of NADPH reduced,and the antioxidants ability of colorectal cancer diminished,which was not beneficial to colorectal cancer liver metastasis.