The Effects And Mechanisms Of PRDX2 Trageting LT-TICs On Tumorigenicity,Metastasis And Drug Resistance Of Colon Cancer

Objective:to isolate and identify a subpopulation of LT-TICs and to explore the effects and molecular mechanisms of PRDX2 trageting LT-TICs on tumorigenicity,metastasis and drug resistance of colon cancer.Methods:1.based on the principle of separation and identification of cancer stem cells,and combined with the two key phenotypes of LT-TICs(maintained tumor formation in serial xenotransplants and drove metastasis in vivo),here,we isolated and identified a subpopulation of LT-TICs by a series of experiments.2.PRDX2 expression was detected in CD133(+)CD44(+)and CD133(-)CD44(-)colon cancer tissues using IHC.qRT-PCR was used to detect the mRNA levels of PRDX2,CD133 and CD44 in 40 pairs of human colon carcinomas with the matched adjacent noncancerous tissues.The functional relation between PRDX2 and CD133~+CD44~+was comfirmed by a series of experiments.3.PRDX2 expression was detected in non-metastatic tissues,matched normal colon tissues,and liver metastatic tissues using IHC,and the expression of PRDX2 in the invasion front of tumor tissues was also measured.In vitro and in vivo assays was used to investigate the effects of PRDX2 on the metastatic phenotype of CD133~+CD44~+cells.The expression of related proteins in EMT and WNT pathway was detected by West blotting.4.mock-and shPRDX2-HT29-CD133~+CD44~+cells were exposed with500ug/ml 5-FU or 100?M oxaliplatin for 24h,and the cell apoptosis was analyzed by flow cytometry with PE-labeled Annexin V containing7-amino-actinomycin(7-AAD).The endogenous production of ROS in mock-and shPRDX2-HT29-CD133~+CD44~+cells with or without following exposure to OXLP or 5-FU was measured by DCFH-DA,and DNA injury was tested using alkaline comet assay.Results:1.CD133~+CD44~+CSCs functionally comfirms the extensive tumorigenic potential of LT-TICs2.CD133~+CD44~+CSCs also functionally reproduces the LT-TICs that drives tumor metastasis formation.3.CD133~-CD44~-cells showed a high sensitivity in vitro to 5-Fu or oxaliplatin tested in a dose-dependent fashion.In contrast,CD133~+CD44~+cells were largely inert to chemotherapeutic drugs-induced apoptosis even at the highest dose concentrations used.4.There was a significant correction between PRDX2 and CD133~+CD44~+cells.5.Downregulated PRDX2 expression in CD133~+CD44~+cells inhibited the in vitro self-renewal capacity,and reduced tumor incidence and growth in vivo.6.PRDX2 was highly expressed in distant metastatic CRC tissue compared with non-metastatic CRC and matched normal colon tissues,and PRDX2 knockdown in CD133~+CD44~+cells significantly decreased the liver metastatic potential of CD133~+CD44~+cells through regulating Wnt/?-cantenin signal pathway and EMT preocess.7.PRDX2 knockdown in CD133~+CD44~+cells sensitize these cells to chemo-therapeutics,and the mechanisms associated with this effect include enhancement of ROS production,strengthened sensitivity to oxidative stress and a more DNA break in CD133~+CD44~+cells.Conclusion:CD133~+CD44~+cells is enriched in LT-TICs and can be as a typical representation of LT-TICs in colon cancer.PRDX2 targeted at regulating CD133~+CD44~+cells(LT-TICs)may have important clinical implication to eradicate colon cancer.