The Mechanism Of The Calmodulin Pathway Inhibitors Inhibit P-gp Activity By Affecting The Lipid Rafts

In recent years,Although the new anti-tumor drug was found one after another, the survival rates and quality of life of cancer patients is not significantly increased, which in addition to the anticancer efficacy and adverse reactions, tumor multidrug resistance (multidrug resistance, MDR) has become a key factors for the success to a cancer chemotherapy. P-gp is a representative member of the superfamily of ABC transporters (ATP-dependent transporter protein), is considered to be dominant in the process to cause MDR. Karin Klappe et found that P-gp is a protein with an important transport function in the membrane, is located in the area of lipid rafts, its function is related to the change of sphingolipids and cholesterol in the lipid raftes.Lipid rafts is a bilayer structure micro-region containing particular lipids and specific proteins within the cell membrane, the portion of the micro-region having a low fluidity, showing ordered liquid phase, and rich in cholesterol and sphingomyelin.So, the change of cholesterol and sphingomyelin in the lipid rafts can affect the location and transport activity of the P-gp. In an effort to over-come P-gp mediated drug resistance, much effort has been placed on the development of inhibitors to P-gp. Analysis of the existing P-gp inhibitor, including verapamil, cyclosporine A, PSC833, D-verapamil have showed an significant inhibition to the transport activity of P-gp, and the mechanism of their reversal multidrug resisitance is not clear.In our previous study on the fungal resistance, we found that the increased stability of fungal lipid rafts can promote its formation of resistance,and can be the positive regulated by the calmodulin pathway. It has been reported that the P-gp inhibitors can affect the calmodulin pathway in tumor, and cyclosporin A (CsA) is a calcium calcineurin inhibitor, and verapamil (VP) is a calcium channel blocking stagnation agent.Therefore, we choosed the cyclosporine A (CsA) and verapamil (VP) to verify whether P-gp inhibitors affect the stability of the lipid rafts, and the location of P-gp in lipid rafts, and the transport activity of P-gp, and the mechanism for the P-gp inhibitors to reverse the multidrug resistance,,in addition,giving an eye on the traditional Chinese medicine whether it works similar to the CsA or VP and providing a new idea to study the mechanism of traditional Chinese medicine to reverse multidrug resistance. The experimental methods used in this paper including:MTT assay,Real-Time-PCR,flow cytometry measuring intracellular drug accumulation,western blot and confocal microscope scanning.By using those experimental methods,we get following results:①he MCF-7/adr is a multidrug resistance cell,the CsA or VP can reverse the multidrug resistance of MCF-7/adr to Dox;②he CsA or VP can significantly increase the intracellular accumulation of Dox or rhodanmine6g in MCF-7/adr cells,while the mRNA level of P-gp increased after incubated with CsA or VP;③the stability of the lipid rafts and the location of P-gp in lipid rafts were affected by CsA or VP;④the M-(3-CD can weaken the multidrug resistance of MCF-7/adr cells to Dox and significant increase the intracellular accumulation of Rh6g in MCF-7/adr;⑤the M-β-CD can affect the location of P-gp;⑥Artesunate can reverse the multidrug resistance of MCF-7/adr to Dox,and significant increase the intracellular accumulation of Dox and Rh6g;⑦Artesunate’s effect on the lipid rafts and P-gp location is similar to CsA or VP.From those experiment results,we infer that the VP or CsA can reverse the multidrug resistance of MCF-7/adr to Dox by disturbing the lipid rafts stability,inhibiting the transport activity of P-gp,while the destruction of lipid rafts alone can inhibit P-gp transport activity, which shows that the calmodulin pathway inhibitors may affect the transport activity of P-gp through affect the stability of the lipid rafts.conclusion:The lipid rafts play an important role in the process to reversal multidrug resistance by the calmodulin pathway inhibitors, and that the calmodulin pathway inhibitors affect the position of P-gp and inhibit the transport activity of P-gp to reverse the multidrug resistance of MCF-7/adr to Dox by affect the stability of the lipid rafts; the Chinese medicine-artesunate can increase the sensitivity of MCF-7/adr to Dox, and affect the stability of lipid rafts and the location of the P-gp in the lipid rafts.