Mechanism Of Prodigiosin Enhancing Colorectal Tumors Sensitivity To 5-Fu Via Inhibiting Autophagy

Background and purpose:Colorectal cancer(CRC)are canceres that occur in the lower gastrointestinal tract and with a gradually increased mobidity year by year,and most of which are transformed from intestinal polyps.CRC-induced deaths account for about 10% of cancer-related mortality each year.The chemotherapeutic drug 5-fluorouracil(5-fluorouracil,5-Fu)has an extremely long history and is one of the earliest discovered anti-cancer drugs.It is mainly used in the treatment of gastrointestinal tumors.However,at present,almost all cancer treatments except surgical operations have the problem of drug resistance.The failure of chemotherapy due to drug resistance is one of the main causes of poor prognosis and death of CRC patients.Autophagy is a cell-mediated degradation process in eukaryotes.At present,a large number of studies have shown that autophagy is one of the mechanisms by which tumor cells resist external survival pressure.Autophagy in cancer cells can lead to drug resistance and inhibiting autophagy can enhance the cytotoxicity of chemotherapy drugs.It is an effective cancer treatment strategy.Prodigiosin(PG)is a bacterial metabolite with antitumor biological activity and may be a potential autophagy inhibitor,but the specific functional mechanism is not yet clear.This study used PG and combined 5-Fu to treat CRC cells or transplanted tumor animal models,aiming to explore the effect and mechanism of PG in the treatment of CRC and combined therapy,and to provide a new theoretical basis for the treatment of CRC.Methods1.Observe whether PG affects the autophagy level of human CRC cells.PG were used to set concentration gradients(0.0,0.1 μM,0.2 μM,0.4 μM)and time gradients(0h,3h,6h,12h)to treat HCT116、SW480、HT-29、Caco-2 and Lo Vo cells respectively,and WB detected changes in proteins LC3B-I to LC3B-II.In addition,the SW480 with EGFP-LC3 stable transfected was also used for verification.2.To detect the inhibitory effect of PG on the autophagic flux of human CRC cells.HCT116 cell and SW480 cell were treated with PG,and the protein levels of SQSTM1 and LC3B-I to LC3B-II were detected by WB,and the autophagy inhibitor chloroquine(CQ)was used as a control.Then use GFP-LC3B-RFP-LC3BΔG fusion protein fluorescent autophagy probe SW480 cells,PG / CQ / autophagy inducer Rapamycin(Rapa)treatment and WB detection GFP-LC3B-1,GFP-LC3-BII And RFP-LC3BΔG protein level changes,and fluorescent staining to detect the relationship between GFP-LC3 and SQSTM1.3.To explore the potential mechanism of PG inhibiting the autophagic flux of human CRC cells.After treating SW480 cells with PG / CQ / Rapa,Lyso Tracker Red staining was used to localize EGFP-LC3 and lysosomes.WB was used to detect the expression levels of cathepsins CTSB and CTSD and their precursor zymogens in PG-treated cells.Finally,use pyridine orange(AO)staining to observe the lysosomal p H of SW480 cells after PG treatment.4.The effect of PG combined with 5-Fu treatment on CRC cell apoptosis was examined.HCT116 cell and SW480 cell were treated with 5-Fu,and the autophagy-related genes ATG5,ATG7 and BECN1 were knocked down and then treated with 5-Fu to verify the effect of 5-Fu on autophagy and apoptosis of CRC cells.Subsequently,5-Fu combined with PG treatment was used to detect the changes of SQSTM1 and LC3,as well as apoptosis-related proteins caspase-3,caspase-9 and PARP by WB,and using CCK8 to detect the cell proliferation ability,and flow cytometry to detect cell apoptosis.5.BALB / C nude mice were used to xenograft model.The mice were divided into control group,PG group,5-Fu group,PG + 5-Fu group,and after xenograft 72 h,using PG1 mg / kg,5-Fu 30 mg / kg twice a week for a total of 6 times,the experiment lasted 3weeks.At the same time,record the changes in the body weight and tumor size of mice.After the experiment,weighed the tumors,and the blood and other important organs and tissues were preserved.After sampling,to detect the main changes of SQSTM1,LC3 and cleared caspase-3 protein levels.Results1.PG treatment can lead to an increase in the conversion of LC3B-I to LC3B-II in CRC cells,this change is PG concentration-dependent and time-dependent,showing that PG can nonspecifically induce autophagy.2.The use of the autophagy inhibitor CQ did not increase the accumulation of LC3B-II and SQSTM1 already caused by PG.In the fusion probe cells treated with CQ /PG,GFP-LC3B-1 was transformed into GFP-LC3-BII,while RFP-LC3BΔG could only detect its LC3B-1 form.In addition,CQ / PG could prevent GFP-LC3 B Lysosomal degradation to significantly increased the GFP / RFP ratio and caused SQSTM1 to co-localize with GFP,indicating that autophagy inhibition occurs at the autophagosome level.3.Lyso Tracker Red staining showed that PG / CQ treatment resulted in accumulation of EGFP-LC3 in the cytoplasm.PG also significantly reduced the levels of mature CTSB and CTSD in a time-and dose-dependent manner,and increased the levels of pro-CTSB and pro-CTSD.In addition,PG also down-regulated the levels of CSTB and CSTD m RNA.AO staining showed that PG treatment did not significantly change the fluorescence signal.Taken together,PG inhibits lysosomal proteolytic activity without changing the p H of the vesicles.4.5-Fu will lead to the accumulation of LC3B-II in SW480 cells and the reduction of SQSTM1 levels.The use of PG combined with 5-Fu will further increase the5-Fu-induced LC3B-II increase and restore SQSTM1 levels,while the combination will reduce CRC cells.Proliferation,increase the level of cleaved caspase-3,caspase-9 and PARP,promote cancer cell apoptosis.5.In nude mice with transplanted tumors,the use of PG and 5-Fu alone had the least effect on tumor growth.The combined treatment of PG and 5-Fu can significantly reduce the size and weight of tumors.Consistent with the in vitro results,the synergistic treatment of PG and 5-Fu increased the level of LC3B-II.Compared with the control group,the in situ level of SQSTM1 in tumors treated with PG alone and combination treatment also increased significantly,while in 5-Fu treatment The in situ level of SQSTM1 in the tumor was significantly reduced.In the group,any drug treatment did not cause abnormal increase and decrease of the body weight of the mice.ConclusionProdigiosin can be used as a new type of autophagy inhibitor,weakening cathepsin activity without affecting the p H of lysosomes,blocking autophagosome-lysosome fusion to inhibit autophagy flux.PG combined with 5-Fu can increase the sensitivity to 5-Fu chemotherapy by inhibiting CRC cell autophagy and promote cancer cell apoptosis.Through this research,we provide a new basis for Prodigiosin as an autophagy inhibitor in the treatment of CRC and clinical application.