Compound Kushen Injection Sensitizes P53-mutant Colorectal Cancer Cells To Cisplatin

Rationales:Colorectal cancer（CRC）is the most frequent malignant tumor.The surgery with chemotherapy is the main treatment strategy for CRC,but the emergence of drug resistance makes the failure of chemotherapy.P53 mutation-mediated chemotherapy resistance is one of the main factors that attribute to the chemotherapy resistance of colorectal cancer.Therefore,it is meanful to mine clinical drugs to overcome the p53mutation-mediated chemotherapy resistance,which promotes the development of drugs against p53-mutant CRC and increases the success of CRC treatment.Compound Kushen Injection（CKI）is a Chinese patent medicine with the highest usage rate among adjuvant drugs for the clinical treatment of colorectal cancer.The frequency of combined with cisplatin and5-fluorouracil is as high as 45.34%and 40.90%.However,there is no reports that the relationship between the adjuvant effect of CKI and p53 mutation.This article focusses on the effect of CKI on the antitumor activity of cisplatin and 5-fluorouracil against colorectal cancer,and explores its mechanism.Therefore,the article develops a therapeutic strategy against p53-mutant colon cancer.Methods and Results:1.Based on two CRC cell lines:HCT116 and SW480 cells,the sulforhodamine B staining method was used to study the effect of CKI on the anti-CRC activity of cisplatin（Cis）and 5-fluorouracil（5FU）in vitro,and evaluated by combenefit software synergistic/antagonistic effects of drug combination.The results showed that CKI enhances the cell growth inhibition rate of Cis against HCT116 and SW480 cells,weakens the cell growth inhibition rate of 5FU against HCT116 cells,and enhances the cell growth inhibition rate of 5FU against SW480 cells.Among them,CKI showed the greatest effect on the cell growth inhibition of Cis against SW480cells,indicating that CKI selectively enhances the cell growth inhibition of Cis against SW480 cells.2.Based on the fact that HCT116 is a p53 wild-type cell and SW480 cells carry p53 R273H/P309S,lentiviral transfection technology is used to construct HCT116(p53^-/-)^{p53 WT}and HCT116(p53^-/-)^{p53 R273H/P309S}cell lines.The in vitro anti-tumor activity of drug combination were evaluated by using these cell lines.The results showed that CKI had no effect on the growth inhibition of Cis against HCT116(p53^-/-)^{p53 WT}cells,enhanced the growth inhibition of Cis against HCT116(p53^-/-)^{p53 R273H/P309S},and weakened the growth inhibition of 5FU against HCT116(p53^-/-)^p53WTand HCT116(p53^-/-)^{p53R273H/P309S}.These results showed that CKI selectively enhances the antitumor activity of Cis against p53-mutant CRC.3.In order to explore the mechanism that CKI selectively enhances the antitumor activity of Cis against p53-mutant CRC,transcriptomics technology was used to evaluate the effect of CKI,Cis,5FU and their combination on the transcriptomics of SW480 cells.The results showed that CKI reprogrammed the transcriptome of SW480 cells treated by Cis,while had a weaker effect on the transcriptome of SW480 cells treated by 5FU.4.Western blot was used to verify the key proteins of cytokine-cytokine receptor interaction.The results showed that Cis induced the expression of RIPK1,indicating that Cis induced necroptosis in SW480 cells.CKI enhanced the expression of DR5 and caspase-3 in SW480 cells treated by Cis,and induced PARP cleavage,indicating that CKI boosted Cis-induced apoptosis in SW480 cell.The inhibitors of apoptosis and necroptosis can significantly attenuate cell death induced by CKI combined with Cis,indicating that CKI’s potentiation of Cis against SW480 cell growth depends on the induction of cell apoptosis.In summary,CKI selectively increase the growth inhibition of cisplatin on p53-mutant CRC cells,and the underlying mechanism may be that:CKI increases the expression of death receptor DR5 and apoptotic protein caspase-3 in Cis-treated SW480 cells,resulting in the activation of apoptosis,which is a pathway to enhance the anti-tumor activity of Cis.This study enrich therapeutic strategies for p53-mutant CRC,and promote the precision clinical application of CKI.