Study On The Mechanism Of Verapamil Enhancing Chemosensitivity Of SW620/Ad300 Cells To Paclitaxel

Background:Chemotherapy is the most important treatment for advanced colon cancer.However,multidrug resistance could cause unsatisfactory curative effect.Therefore,improving chemosensitivity of colon cancer is very important.P-glycoprotein is a main mechanism of MDR.Verapamil,as a classic P-gp inhibitor,can enhance the chemosensitivity on tumors.But the mechanism of verapamil inhibiting P-gp is not very clear in colon cancer.Objective:In this research,the objective is to investigate the mechanism of verapamil on enhancing chemosensitivity of paclitaxel in SW620/Ad300 cells.Methods:1.The effects of proliferation and MDR on drug-resistant colon cancer cells induced by verapamil were detected by MTT assay and flow cytometry.2.Western blot was applied to detect the expression of P-gp and BCRP affected by verapamil in SW620/Ad300 cells.3.In order to investigate the effect of verapamil on activity of P-gp,UPLC was established to detect the accumulation of paclitaxel in SW620/Ad300 cells.4.The mechanism of verapamil enhancing chemosensitivity of drug-resistant colon cancer cells was researched by metabolomics based on UHPLC-Q Exactive.Results:1.MTT showed that different concentrations of paclitaxel were applied to SW620 and SW620/Ad300 for 48 h.The IC₅₀ of SW620 cells was 4.74±0.11 nM,and the IC₅₀ of SW620/Ad300 cells was 2915.67±116.45 nM.The multiple drug resistance of SW620/Ad300 was 604.91.After different concentrations of verapamil affected on SW620/Ad300 cells,the IC₅₀ of 24 h was 153.37±1.74μM,and the IC₅₀of 48 h was 104.77±0.78μM.The IC₅₀ of paclitaxel was 59.06±0.70 nM in united group（paclitaxel plus verapamil）,while the IC₅₀ of paclitaxel was 2915.67±116.45nM in control group.The reversing fold of verapamil against SW620/Ad300 cells was 47.97.The apoptosis of SW620/Ad300 cells detected by flow cytometry showed that the united group induced more apoptosis than other groups.2.Western blot showed that the expression of P-gp in SW620/Ad300 cells was significantly higher than SW620 cells.There was no significant difference in the expression of BCRP between SW620/Ad300 cells and SW620 cells.It indicated that MDR of SW620/Ad300 cells was associated with excessive expression of P-gp.Verapamil did not significantly change the expression of P-gp in SW620/Ad300 cells.3.The results of UPLC detection showed that verapamil could significantly increase accumulation of paclitaxel in SW620/Ad300 cells via inhibiting the function of P-gp.Nevertheless,verapamil had no obvious effect on accumulation of paclitaxel in SW620 cells.4.Metabolomics based on UHPLC-Q Exactive revealed the mechanism of verapamil inhibiting P-gp in SW620/Ad300 cells.It shows the mechanism of verapamil inhibiting P-gp transport activity may be mainly through the influence of glycerophospholipid metabolism and sphingolipid metabolism.Conclusion:1.Verapamil can enhance the sensitivity of SW620/Ad300 cells to paclitaxel by inhibiting the transport activity of P-gp.2.Metabolomics reveals that verapamil may affect sphingolipid metabolism and glycerophospholipid metabolism to inhibit the transport activity of P-gp,thereby enhancing the sensitivity of SW620/Ad300 cells to chemotherapy.