Tivozanib Reverses Multidrug Resistance Mediated By ABCB1(P-glycoprotein) And ABCG2(BCRP)

BackgroundFor centuries, researchers searched for a cancer cure. Studies found out that one of the biggest impediments to realizing a successful therapy is the multidrug resistance (MDR) in cancer cells that cause cancer chemotherapy to fail. Overexpression of the ATP-binding cassette (ABC) transporters in cancer cells alters absorption, distribution, metabolism and excretion of various chemotherapy drugs. This results in MDR. Researchers have expended a lot of effort to find an effective inhibitor for ABC transporters. However, none have been approved clinically. This study shows that tivozanib (AV-951, KRN-951) reverse ABCB1and ABCG2(BCRP)-mediated multidrug resistance. Tivozanib is a potent inhibitor of vascular endothelial growth factor1,2and3receptors. Reversal stuies indicate that tivozanib can significantly reverse ABCB1-mediated resistance to paclitaxel, vinblastine and colchicine as well as ABCG2-mediated resistance to mitoxantrone, SN38and doxorubicin. Drug efflux assays showed that tivozanib increased the intracellular accumulation of substrates by inhibiting the ABCB1and ABCG2efflux activity. Furthermore, tivozanib at a higher concentration inhibited the ATPase activity of both ABCB1and ABCG2and inhibited the photolabeling of ABCB1or ABCG2. Our findings indicate a potential use for tivozanib as an adjuvant agent for chemotherapy by inhibiting the function of the ABC transporter and sensitizing the reaction of tumor cells to chemotheraputic drugs.Objective1. To test the cell toxicity of tivozanib.2. To detect whether tivozanib can reverse multidrug resistance (MDR) mediated by ATP-binding cassette (ABC) transporters. 3. To investigate the mechanism of reversal with tivozanib.Materials&methods1. Cell lines used in the following experiments include sensitive human oral squamous carcinoma (SCC) KB-3-1and MDR derivative KB-C2; Human Embryonic Kidney cells HEK293/pcDNA3.1, ABCB1trasnfectant cell line HEK/ABCB1, ABCG2trasnfectant cell lines ABCG2-482-R2, ABCG2-482-G2, ABCG2-482-T7.2. We use MTT assay to test the cell cytotoxicity of tivozanib in different cell lines. MTT assay was also used to determine the change of drug resistant fold to respective substrate antineoplastic agents in presence or absence of tivozanib.3. Western blotting was used to detect the change of expression of ABCB1and ABCG2protein levels in presence or absence of tivozanib in different cell lines.4. Accumulation assay were performed to measure the radioactivity of ABCB1substrate [3H]-paclitaxel and ABCG2substrate [3H]-mitoxantrone with or without tivozanib.5. Efflux assay was used to detect the the radioactivity of ABCB1substrate [3H]-paclitaxel and ABCG2substrate [3H]-mitoxantrone at different time point.6. We performed [125I]IAAP-photoaffinity labeling test to determine the affinity of ABCB1to [125I]IAAP and the affinity of ABCG2to [125I]IAAP after cointubation with different concentration of tivozanib.7. ATPase assay was done to test the ATPase activity of ABCB1and ABCG2in presence of various concentration of tivozanib.Results1. Tivozanib was relatively non-toxic (cell viability more than80%) at a concentration less than6μM in the KB-3-1and HEK293/pcDNA3.1parental cells, as well as their respective ABCB1and ABCG2overexpressing cell lines. 2. Tivozanib at non-toxic concentrations (2.5and5μM) in combination with antineoplastic agents inhibits MDR-mediated by ABCB1and ABCG2.3. Western blotting showed tivozanib does not alter ABCB1and/or ABCG2protein expression levels.4. Tivozanib inhibits the efflux function and increases the accumulation of ABCB1-and ABCG2-substrate antineoplastic drugs in ABCB1and ABCG2overexpressing cells, respectively.5.[125I]IAAP photolabeling studies demonstrate that tivozanib can bind to the same binding sites as IAAP in the transmembrane domains of ABCB1and ABCG2.6. Tivozanib at concentrations used in reversal studies (2.5and5μM) decrease ATPase activity in ABCB1as well as ABCG2transporter.ConclusionsWe conclude that tivozanib at non-toxic concentrations has previously unknown activity of reversing MDR mediated by ABCB1and ABCG2transporters. Tivozanib may be used as a new agent to overcome MDR in oral SCC.