BIRB796, The Inhibitor Of P38Mitogen-Activated Protein Kinase, Reverses ABCB1Induced Multidrug Resistance

Objective:ATP-binding cassette(ABC) transporters is one of the key reason of multidrug resistance(MDR). Till now, P-glycoprotein (P-gp/MDR1/ABCB1),multidrug resistance associated-proteins (MRPs/ABCCs) and breast cancer resistance protein (BCRP/MXR/ABCG2) are the major member of ABC transporters leading to multidrug resistance. p38mitogen-activated protein kinases(MAPK)signal pathway, is the important way of MAPK, which is involed in inflamation, proliferation, apoptosis and other physiological processes. Till now, the inhibitors of p38MAPK were found that they could enhance the treatment of chemotherapy in some cancer diseases. However, the mechanism is obscure. Therefore, we sellect the new generation of p38MAPK inhibitor,BIRB796, which has entered the phase III clinical trail,to explore whether it could reverse the ABC transporters induced multidrug resistance. Methods:The MTT assay was used to access cytotoxicity. The intracellular accumulation of doxorubicin and rhodamine123were analysed by flow cytometry. The KBV200cell xenograft model was established to explore the efficacy of BIRB796in vivo to reverse ABCB1-mediated MDR. Western blotting was used to determine the protein expression. Reverse transcription-PCR and Real-time PCR was used to assess the expression of ABCB1at mRNA level. The verapamil-stimulated ABCB1ATPase activity was estimated by Pgp-GloTM assay systems. RNA interference was used to observe the effect of p38MAPK silence on the survival of KBV200cells. Docking simulation was used to analyses the bingding site of BIRB796to the homology model of human ABCB1at the molecular level. Results:Our results showed that BIRB796could reverse ABCB1-mediated MDR in both the drug selected and transfected ABCB1-overexpressing cell models, but did not enhance the efficacy of substrate-chemotherapeutical agents in ABCC1or ABCG2overexpression cells and their parental sensitive cells. Furthermore, BIRB796increased the intracellular accumulation of the ABCB1substrates, such as rhodamine123and doxorubicin. Moreover, BIRB796 bidirectionally mediated the ATPase activity of ABCB1, stimulating at low concentration, inhibiting at high concentration. However, BIRB796did not alter the expression of ABCB1both at protein and mRNA level. The down-regulation of p38by siRNA neither affected the expression of ABCB1nor the cytotoxic effect of paclitaxel on KBV200. The binding model of BIRB796within the large cavity of the transmembrane region of ABCB1may form the basis for future lead optimization studies. Importantly, BIRB796also enhanced the effect of paclitaxel on the inhibition of growth of the ABCBl-overexpressing KBV200cell xenografts in nude mice. Conclusion:1. BIRB796enhances the efficacy of chemotherapeutic agent in ABCB1overexpressing cells, which by increase the concentration of chemotherapeutic agent (the substrate of ABCB1) in tumor cells.2. BIRB796does not reverse the resistance of ABCC1and ABCG2induced multidrug resistance.3. BIRB796reverses ABCB1-mediated MDR in the Nude Mouse Xenograft Model, but does not enhance the toxicity of paclitaxel in nude mouse.4. BIRB796does not affect the expression of ABCB1both at protein and mRNA level.5. BIRB796bidirectionally mediates the ATPase activity of ABCB1, inhibiting the efflux function of ABCB1.6. Down-regulation of p38does not affect the expression of ABCB1and the cytotoxic effect of chemotherapeutic agents in drug selected cells.7. BIRB796docking analysis with human ABCB1homology model reveals the potential molecular interation; and the hydrophobic interactions contribute most to the binding affinity of BIRB796towards ABCB1drug-binding site-1.