Knockdown Of PRDX2 Senstitizes Colon Cancer Cells To 5-FU By Suppressing The PI3K/AKT Signaling Pathway

Colon cancer is one of the most common tumors in the world.In our country of new cancer cases and tumor are ranked fifth in the death cause,and the incidence and mortality of colon cancer is still on the rise.At present,the treatment of colon is surgery,supplemented by systemic chemotherapy.The purpose of systemic adjuvant chemotherapy is to further eliminate the micrometastatic lesions in patients after surgery,in order to extend the survival time of patients,and adjuvant chemotherapy is considered an important part of comprehensive treatment of patients with advanced colon cancer.5-fluorouracil(5-FU)is the first choice for colon cancer chemotherapy,and it has been widely used in adjuvant chemotherapy for colon cancer.The patient's sensitivity to chemotherapeutic agents determines the efficacy of chemotherapy,and many patients often fail because of their resistance to 5-FU.So the chemotherapy drug resistance molecular biological mechanism,further screening better targets to improve the sensitivity of the effective in the treatment of colon cancer is particularly important.Recent studies showthat the active oxygen(ROS)scavenging system can enhance the body's resistance to 5-FU.Peroxiredoxin(PRDXs)in the process of tumor resistance to ROS plays a very important role.PRDX2 is an important member of them.Studies have shown that PRDX2 over-expression in colon cancer,and the down-regulation of PRDX2 can promote colon cancer cell apoptosis.These studies suggest that PRDX2 may have an important effect on the sensitivity of 5-FU in colon cancer chemotherapy,but there is no further study on the relationship between PRDX2 and 5-FU in colon cancer chemotherapy.Aims:To explore the sensitivity effect of 5-FU in colon cancer chemotherapy by PRDX2 knockdown and its further molecular mechanism.Method:1.We established PRDX2 knockdown cell line with RNAi technique in HCT116 and HT29 colon cancer cell lines,examined transfection efficiency with fluorescence microscope,Western blot and QRT-PCR.2.Cells were seeded in 96-well plates,treated by 5-FU with different concentrations for 48 h.The viability of cells was evaluated using the Cell Counting Kit-8(CCK-8)assay.3.After PRDX2 knockdown,wo detected apoptosis rate in colon cancercells treated with 5-FU by flow cytometry and analys protein levels of cleaved PARP and caspase-3 by Western blot.4.We divided nude mice into four groups(shCont+PBS,shPRDX2+PBS,shCont+5-FU and shPRDX2+5-FU)and examined the in vivo efficacy of5-FU in mice bearing abdominal tumors that originated from HCT116-shPRDX2 or HCT116-shCont cells.5.We used immunohostochemistry to detect PRDX2 and p-AKT protein levels in tumor tissues and their corresponding normal colorectal mucosal tissues from 50 colon cancer patients.6.We analyzed the protein expressions of AKT,p-AKT and p-PI3 K by Western blotting in HT-29-shCont or HT-29-shPRDX2 cells.Furthermore,5-FU treated HT29 and HCT116 cells were infected with overexpressed PRDX2,LV-PRDX2,and protein expressions of p-AKT and Bcl-2/Bax protein were observed by Western blot.We treated HT-29 cells with the AKT inhibitor,MK-2206 2HCl.Then we detected protein expressions of AKT/PI3 K,Bcl-2/Bax,p-AKT,p-PI3 K,C-PARP,caspase-3 by Western blot and analyzed apoptosis by using flow cytometry after cells were treated5-FU in the presence or absence of MK-2206 HCl.Results:1.The mRNA and protein expressions of PRDX2 were significantly decreased in the shPRDX2 group compared with the shCont group after lentivirus transfection,p < 0.05.2.We used the CCK-8 assay to detect the survival rate of colon cancer cells that were stably transfected with NC-shRNA-LV and PRDX2-shRNA-LV and treated with 5-FU at different concentrations for48 h.We observed a lower survival rate in the shPRDX2 group,compared with the shCont group,p < 0.05.3.We analyzed the apoptosis rate by flow cytometry and found that knocking down PRDX2 expression in FU-treated colon cancer cells markedly increased apoptosis and protein levels of cleaved PARP and caspase-3,p < 0.05.4.We found that the mice in the shCont group treated with 5-FU and the untreated shPRDX2 group had longer survival times than the mice in the untreated shCont group.However,the mice in the shPRDX2 group treated with 5-FU exhibited the longest survival time of the four groups,p < 0.05.5.We found that PRDX2 and p-AKT were strongly expressed in the cytoplasm of colon cancer cells,and were weakly expressed in normal colon mucosal tissues.The proportion of Prdx2-positive cells and p-AKT-positive cells were 86%(43/50)and 88%(44/50),respectively.On further analysis,we found that PRDX2 and p-AKT showed a significant positive correlation(r = 0.738,p < 0.05)through Pearson correlation.6.PRDX2 knockdown resulted in reduced p-AKT and p-PI3 K expressions.Furthermore,5-FU treated HT29 and HCT116 cells were infected with overexpressed PRDX2,LV-PRDX2,and protein expressionsof p-AKT and Bcl-2/Bax protein were observed.We treated HT-29 cells with the AKT inhibitor,MK-2206 2HCl and then observed the resistance of HT-29 cells to 5-FU.MK-2206 inhibited AKT/PI3 K protein expression.However,elevated expressions of caspase 3 and Bax were detected,along with suppressed Bcl-2 expression post MK-2206 treatment.Furthermore we observed higher C-PARP and Bax protein expression and a higher rate of apoptosis in colon cancer cells treated with MK-2206,p <0.05.Conclusion:Inhibiting PRDX2 expression promotes 5-FU-induced apoptosis in colon cancer cells via the PI3K/AKT signal pathway.