Small Molecular Protein Kinase Inhibitor Midostaurin, Sorafenib And Vatalanib Reverse Multidrug Resistance And Its Mechanism

Background: Protein kinase inhibitors (PKIs) are a relatively new class of antineoplastic drugs. Thesecompounds could affect the crucial signaling pathways involved in cancer proliferation, invasion,metastasis and angiogenesis by inhibiting the function of several protein kinases, which play essential rolesin cellular signaling transduction. However, the therapeutic potential of PKIs could be significantlyhindered by the emerging resistance against these compound. ATP binding cassette (ABC) transporters,transmembrane proteins, facilitate the unidirectional translocation of structurally unrelated compoundsacross membrane barriers via the energy supplied by ATP. These efflux pumps were considered to be themain contributing elements lead to the occurrence of MDR phenotype during cancer chemotherapy.Moreover, it has been recently reported that some ABC transporters also confers to the resistance againstsome PKIs. And it is quite interesting that, some PKIs were even demonstrated to be ideal candidates aschemo-sensitizing agent targeting to MDR mediated by these ABC-transporters, due to their interactionwith ABC transporters. Therefore, it is a meaningful work to make it clear the relationship between PKIsand MDR-ABC transporter, it may facilitate the discovery of new MDR-ABC transporter inhibitor andprovide more effective and safer combination chemotherapeutic strategies in the clinic.Objective： This study was designed to determine the potency of Midostaurin, Vatalanib andSorafenib on sensitize multidrug resistance mediated by MDR-ABC transporters. Method: MTS and SRB assay was used to evaluate the cytotoxicity and the reversal activity inresistant and sensitive cells. The intracellular accumulation of fluorescence substrates (Rh-123, MX andADR for P-gp, BCRP, MRP1, respectively) were analysed by flow cytometry. Western blotting was used todetermine the protein expression level. qPCR assay was used to assess the expression of MDR-ABCtransporters on mRNA level. The effect of compounds on ATPase activity was tested with P-gp-GloTMAssay Sysetem.Result:(1) P-gp-overexpressing cell line K562/A02and its parental sensitive cell K562were used toassess the reversal effect of Midostaurin in vitro. Midostaurin in non-toxic concentration (0.5μM)significantly potentiated the cytotoxicity of doxorubicin, paclitaxel and vincristine to K562/A02，but not toP-gp negative parental cell K562； Midostaurin remarkably inhibited the efflux of Rh-123in K562/A02cells，but had no effect on sensitive cells. However, the ATPase activity assay show that the P-gp-mediatedATP hydrolyzing could be inhibited by Midostaurin.0.5μM Midostaurin did not influence the expressionof P-gp on protein and mRNA level, and had little impact on the total and phosphorylated forms of AKTand ERK1/2in both cells.(2) Sorafenib produced a chemo-sensitivity increase of BCRP substrates, such asMX、ADR and TOPT, in a dose-dependent manner in the HEK293/BCRP while having little influence onthe parental cell HEK293/Vec. Sorafenib at the same concentration did not have reversal affect on P-gp andMRP1overexpressed cells，which means that sorafenib may be specific to BCRP. Sorafenib significantincreased accumulation of mitoxantrone in HEK293/BCRP but had little effects on HEK293/Vec,indicating that the effect of Sorafenib on mitoxantrone accumulation is likely via inhibiting BCRP. We alsofound that Sorafenib has no effect on the effluex activity of P-gp and MRP1. BCRP ATPase activity couldbe stimulated by sorafenib, thus sorafenib may serve as a substrate of BCRP. But the binding site ofSorafenib seems to be different with that of MX and ADR, for the kinetics assay showed that sorafenib was non-competitive inhibitor of mitoxantr and adriamycin efflux. Sorafenib could also dramatically decreasethe expression of BCRP in both concentration-dependent and time-relied manners，and had little influenceon P-gp and MRP1expression. Furthermore, we found that sorafenib decrease BCRP protein level in thelysosome by inducing BCRP conformational changes. However, we found that sorafenib under reversalcondition had no influence on the total and phosphorylated forms of AKT and ERK1/2, and no significantchanges in BCRP mRNA level.(3) Vatalanib significantly enhanced the cytotoxicity of BCRP substrateanticancer drugs including topotecan and doxorubicin to BCRP-overexpressing HEK293/BCRP cells in aconcentration-dependent manner, and significantly reversed BCRP-mediated MDR at a non-toxicconcentration of5μM，but did not effect the sensitivity of parental cell HEK293/VEC to mitoxantrone,topotecan and doxorubicin. Vatalanib did not have such reversal affect on P-gp and MRP1overexpressedcells. Vatalanib did not alter the intracellular accumulation of MX in HEK293/BCRP, and had no influenceon the BCRP-mediated drug efflux. The ATPase assay indicated that Vatalanib may serve as a substrate ofBCRP. Vatalanib dramatically suppress both the protein and mRNA expression level of BCRP inconcentration-and time-dependent manners，but had little influence on P-gp and MRP1expression.Treatment with Vatalanib under reversal condition had no influence on the total and phosphorylated formsof AKT and ERK1/2in resistant cells.Conclusion:(1) Midostaurin reverses P-gp-mediated multidrug resistance by inhibiting the effluxfunction of P-gp, not likely to be a substrate of P-gp. Midostaurin posses most of the features of thethird-generation P-gp inhibitors，it could be considered as a lead compound for furthe reversal agentdevelopment.(2) Sorafenib could significantly reverse drug resistance to MX, TOPT, ADR in BCRPoverexpressed cells, but little effect on P-gp and MRP1overexpressed cells. Thus, Sorafenib could beconsidered as a good candidate for cancer combinational therapy to overcome BCRP-mediated MDR in clinic. Sorafenib could also be useful for further development of novel dual-acting BCRP inhibitors, whichhave both inhibiting effluex function and inducing degradation. However, as sorafenib has been shown tobe recognized and effluxed by BCRP, the MDR-ABC transporters might be closely to the emergingresistance to sorafenib in clinic.(3) Vatalanib significantly reverses BCRP-mediated MDR withspecificity，and may be used for combinational therapy whith BCRP substrate chemotherapeutics toconquer MDR mediated by the BCRP. Since vatalanib was showed to be substrate of BCRP, the transportermight be closely relationship with the resistance possiblely occur to vatalanib in clinic. Vatalanib reversedMDR by decreaseing BCRP mRNA expression，which seems to has unique reversal mechanism comparedwith all the reported BCRP reversal agents, and may be develop novel reversal agent and conquer MDRmediated by ABC transporter.