Bufalin Attenuate CAFs-induced Oxaliplatin Resistance In Colorectal Cancer By STAT3/Bcl-2 Signal Pathway

ObjectiveThis study aimed at IL-6 and elucidated that IL-6 is one of the important correlations between drug resistance of CRC and CAFs cells in its TME through a series of experiments.At the same time,based on IL-6/STAT3/Bcl-2 signaling pathway,the mechanism of BU reversing CAFs-mediated multidrug resistance of CRC was explored,which provided a new scientific basis for integrated traditional Chinese and Western medicine treatment of colorectal cancer.Methods1.Collagenase digestion method was used to isolate and culture primary CAFs cells,morphological observation was carried out by microscope,and the marker protein was detected by Western blotting;2.The effect of CAFs on oxaliplatin resistance of CRC cells was studied by CCK-8 experiment of CAFs-tumor cell interaction model(co-culture,indirect effect of conditioned medium);3.The secretion of IL-6 in CAFs was detected by RT-PCR and ELISA;the activation of IL-6/STAT3/Bcl-2 signaling pathway under CAFs was detected by Western Blotting;4.The effect of BU on oxaliplatin resistance in CRC mediated by CAFs was studied by CCK-8 assay in CAFs-tumor cell interaction model(co-culture);5.Western Blotting and ELISA were used to study the effect of BU on IL-6/STAT3/Bcl-2 signaling pathway activated by CAFs and the secretion of IL-6;6.PDX model was constructed and randomly divided into normal saline(NS)group,oxaliplatin(OXA)group,bufalin(BU)group,oxaliplatin+bufalin(OXA+BU)group,5 mice in each group.After the treatment,the size of each group of tumors was measured,and the expression of IL-6 in tumor tissues were detected by immunofluorescence.The serum of mice was extracted and the expression of IL-6 in serum was detected by ELISA.Results1.The morphology of CAFs cells was observed under microscope and the protein markers of CAFs were detected by Western Blotting.The results showed that the primary CAFs accorded with the characteristics of carcinogenic fibroblasts;2.The effects of CAFs on the drug resistance of CRC cells HCT116 and HT29 to oxaliplatin were studied by CCK-8 and Western Blotting experiments.The results showed that CAFs reduced the drug resistance of CRC cells to oxaliplatin.CAFs help CRC cells to resist the killing of chemotherapeutic drugs and promote drug resistance;3.IL-6 ELISA and RT-PCR were used to detect the release of IL-6 from CAFs at protein and m RNA levels.The results showed that CAFs from CRC microenvironment overexpressed IL-6,and CAFs could activate IL-6/STAT3/Bcl-2 signal pathway that involved in oxaliplatin resistance in CRC;4.The effect of BU on oxaliplatin resistance in CRC cells mediated by CAFs was studied by CCK-8 experiment.The results showed that BU could inhibit oxaliplatin resistance in CRC cells mediated by CAFs and enhance the chemosensitivity of CRC cells to oxaliplatin;5.Western Blotting studied the molecular mechanism of BU on oxaliplatin resistance mediated by CAFs.The results showed that the apoptotic proteins of CRC cell lines HCT116 and HT29 were significantly up-regulated during oxaliplatin intervention,and the related proteins in IL-6/STAT3/Bcl-2 signaling pathway were significantly downregulated,suggesting that BU reverses the multidrug resistance of Oxaliplatin mediated by CAFs through inhibiting IL-6/STAT3/Bcl-2 signaling pathway;IL-6 ELISA assay showed that BU could inhibit the expression of IL-6 in co-culture system,but not directly inhibit the release of IL-6 in CAFs;6.After in vivo experiment,the change trend of mice tumors was observed,and the results showed that BU combined with Oxaliplatin could significantly inhibit the growth of mice tumors.At the same time,the content of IL-6 in serum of mice and immunofluorescence test showed that BU combined with oxaliplatin could significantly inhibit the expression of IL-6 in serum of mice and the secretion of IL-6 in CAFs cells,indicating that BU could enhance the drug sensitivity of oxaliplatin and inhibit drug resistance by inhibiting the secretion of IL-6 in CAFs.Conclusion1.CAFs reduce sensitivity of CRC cells to OXA by releasing IL-6 and activating STAT3/Bcl-2 signaling pathway;2.BU can reverse OXA resistance by inhibiting IL-6/STAT3/Bcl-2 signaling pathway;3.BU combined with OXA can significantly inhibit the growth of tumors in mice,the expression of IL-6 in serum and the secretion of IL-6 in CAFs cells,indicating that BU can enhance the drug sensitivity of OXA and inhibit drug resistance by inhibiting the secretion of IL-6 in CAFs.