ABCB1-overexpressing Cells Research Articles

BBA, a derivative of 23-hydroxybetulinic acid, potently reverses ABCB1-mediated drug resistance in vitro and in vivo.

23-O-(1,4'-Bipiperidine-1-carbonyl)betulinic acid (BBA), a synthetic derivative of 23-hydroxybetulinic acid (23-HBA), shows a reversal effect on multidrug resistance (MDR) in our preliminary screening. Overexpression of ATP-binding cassette (ABC) transporters such as ABCB1, ABCG2, and ABCC1 has been reported in recent studies to be a major factor contributing to MDR. Our study results showed that BBA enhanced the cytotoxicity of ABCB1 substrates and increased the accumulation of doxorubicin or rhodamine123 in ABCB1 overexpressing cells, but had no effect on non ABCB1 substrate, such as cisplatin; what's more, BBA slightly reversed ABCG2-mediated resistance to SN-38, but did not affect the ABCC1-mediated MDR. Further studies on the mechanism indicated that BBA did not alter the expression of ABCB1 at mRNA or protein levels, but affected the ABCB1 ATPase activity by stimulating the basal activity at lower concentrations and inhibiting the activity at higher concentrations. In addition, BBA inhibited the verapamil-stimulated ABCB1 ATPase activity and the photolabeling of ABCB1 with [(125)I] iodoarylazidoprazosin in a concentration-dependent manner, indicating that BBA directly interacts with ABCB1. The docking study confirmed this notion that BBA could bind to the drug binding site(s) on ABCB1, but its binding position was only partially overlapping with that of verapamil or iodoarylazidoprazosin. Importantly, BBA increased the inhibitory effect of paclitaxel in ABCB1 overexpressing KB-C2 cell xenografts in nude mice. Taken together, our findings suggest that BBA can reverse ABCB1-mediated MDR by inhibiting its efflux function of ABCB1, which supports the development of BBA as a novel potential MDR reversal agent used in the clinic.

HG-829 Is a Potent Noncompetitive Inhibitor of the ATP-Binding Cassette Multidrug Resistance Transporter ABCB1

Transmembrane drug export mediated by the ATP-binding cassette (ABC) transporter P-glycoprotein contributes to clinical resistance to antineoplastics. In this study, we identified the substituted quinoline HG-829 as a novel, noncompetitive, and potent P-glycoprotein inhibitor that overcomes in vitro and in vivo drug resistance. We found that nontoxic concentrations of HG-829 restored sensitivity to P-glycoprotein oncolytic substrates. In ABCB1-overexpressing cell lines, HG-829 significantly enhanced cytotoxicity to daunorubicin, paclitaxel, vinblastine, vincristine, and etoposide. Coadministration of HG-829 fully restored in vivo antitumor activity of daunorubicin in mice without added toxicity. Functional assays showed that HG-829 is not a Pgp substrate or competitive inhibitor of Pgp-mediated drug efflux but rather acts as a noncompetitive modulator of P-glycoprotein transport function. Taken together, our findings indicate that HG-829 is a potent, long-acting, and noncompetitive modulator of P-glycoprotein export function that may offer therapeutic promise for multidrug-resistant malignancies.

Crizotinib (PF‐02341066) reverses multidrug resistance in cancer cells by inhibiting the function of P‐glycoprotein

Besides targeting the well-known oncogenic c-Met, crizotinib is the first oral tyrosine kinase inhibitor inhibiting anaplastic lymphoma kinase (ALK) in clinical trials for the treatment of non-small cell lung cancer. Here, we assessed the possible reversal of multidrug resistance (MDR) by crizotinib in vitro and in vivo. 1-(4,5-Dimethylthiazol-2-yl)-3,5- diphenylformazan was used in vitro and xenografts in nude mice were used in vivo to investigate reversal of MDR by crizotinib. To understand the mechanisms for MDR reversal, the alterations of intracellular doxorubicin or rhodamine 123 accumulation, doxorubicin efflux, ABCB1 expression level, ATPase activity of ABCB1 and crizotinib-induced c-Met, Akt and ERK1/2 phosphorylation were examined. Crizotinib significantly enhanced the cytotoxicity of chemotherapeutic agents which are also ABCB1 substrates, in MDR cells with no effect found on sensitive cells in vitro and in vivo. Additionally, crizotinib significantly increased intracellular accumulation of rhodamine 123 and doxorubicin and inhibited the drug efflux in ABCB1-overexpressing MDR cells. Further studies showed that crizotinib enhanced the ATPase activity of ABCB1 in a concentration-dependent manner. However, expression of ABCB1 was not affected, and reversal of MDR by crizotinib was not related to the phosphorylation of c-Met, Akt or ERK1/2. Importantly, crizotinib significantly enhanced the effect of paclitaxel against KBv200 cell xenografts in nude mice. Crizotinib reversed ABCB1-mediated MDR by inhibiting ABCB1 transport function without affecting ABCB1 expression or blocking the Akt or ERK1/2 pathways. These findings are useful for planning combination chemotherapy of crizotinib with conventional chemotherapeutic drugs.

Enhancing Chemosensitivity in ABCB1- and ABCG2-Overexpressing Cells and Cancer Stem-like Cells by An Aurora Kinase Inhibitor CCT129202

Imidazopyridine CCT129202 is an inhibitor of Aurora kinase activity and displays a favorable antineoplastic effect in preclinical studies. Here, we investigated the enhanced effect of CCT129202 on the cytotoxicity of chemotherapeutic drugs in multidrug resistant (MDR) cells with overexpression of ATP-binding cassette (ABC) transporters and cancer stem-like cells. CCT129202 of more than 90% cell survival concentration significantly enhanced the cytotoxicity of substrate drugs and increased the intracellular accumulations of doxorubicin and rhodamine 123 in ABCB1 and ABCG2 overexpressing cells, while no effect was found on parental sensitive cells. Interestingly, CCT129202 also potentiated the sensitivity of cancer stem-like cells to doxorubicin. Importantly, CCT129202 increased the inhibitory effect of vincristine and paclitaxel on ABCB1 overexpressing KBv200 cell xenografts in nude mice and human esophageal cancer tissue overexpressing ABCB1 ex vivo, respectively. Furthermore, the ATPase activity of ABCB1 was inhibited by CCT129202. Homology modeling predicted the binding conformation of CCT129202 within the large hydrophobic cavity of ABCB1. On the other hand, CCT129202 neither apparently altered the expression levels of ABCB1 and ABCG2 nor inhibited the activity of Aurora kinases in MDR cells under the concentration of reversal MDR. In conclusion, CCT129202 significantly reversed ABCB1- and ABCG2-mediated MDR in vitro, in vivo and ex vivo by inhibiting the function of their transporters and enhanced the eradication of cancer stem-like cells by chemotherapeutic agents. CCT129202 may be a candidate as MDR reversal agent for antineoplastic combination therapy and merits further clinical investigation.

Abstract 1443: Pharmacokinetics and pharmacogenetics of Actinomycin D in an animal model and pediatric patient population

Abstract ATP binding cassette (ABC) transporters, such as ABCB1 (MDR1/P-gp), are expressed throughout the body to protect against exogenous toxins. ABC transporters control removal from the body of anti-cancer drugs such as doxorubicin and etoposide via biliary or renal excretion. Actinomycin D (Act D) has been used successfully to treat adult and childhood cancers for over 30 years. It continues to play a key role in the treatment of Wilms tumour patients, with 5-year survival rates >80% currently observed. Although Act D is generally well tolerated, severe hepatic toxicity has been associated with treatment. It has previously been demonstrated that Act D exposure is associated with a large degree of inter-patient variability in children with cancer. As common single nucleotide polymorphisms (SNPs) in ABC transporters have previously been shown to alter clinical exposure to various anti-cancer agents, we have carried out translational and clinical studies to investigate the transport of Act D using well established cell line and animal models. Growth inhibition (GI) and intracellular accumulation studies, over a range of Act D concentrations (0-10µM), were initially carried out to determine the role of ABC transporter expression in MDCKII-WT and ABCB1, ABCC1, ABCC2 and ABCG2 over-expressing cell lines. To assess the importance of ABCB1 and ABCC2 transporter expression in vivo, Act D pharmacokinetics and exposure were determined in Abcb1a/1b and Abcc2 knockout mice over 6 hours. In addition, liver, kidney and brain tissue samples were analysed to assess Act D tissue accumulation. Further pharmacokinetic sampling has also been carried out from children receiving Act D as part of their standard treatment, with analysis using a validated LC/MS method. Blood samples were obtained from all patients for pharmacogenetic analysis. Following treatment with Act D, a 59-fold decrease in Act D sensitivity was observed in ABCB1 over-expressing cells (GI50: 745nM), a 2-fold decrease in ABCC1 (GI50: 25.7nM) and a 3-fold decrease (GI50: 40.4nM) in ABCC2, as compared to the parental cell line (GI50: 12.7nM). Intracellular accumulation of Act D was lower in ABCB1 cells compared to the parental cell line, following incubation with Act D for 6 hours. This effect was abrogated by use of the ABCB1 inhibitor verapamil. In vivo pharmacokinetic analysis revealed a 64% higher AUC0-6h in mice with no Abcb1a/1b expression and a 22% lower AUC0-6h in Abcc2-/- mice compared to WT. Pharmacokinetic analysis of samples obtained from 71 patients indicated a large inter-patient variability in Act D exposure (AUC0-24h range: 1.7 - 19.4 µg/ml.min). Ongoing investigations into pharmacogenetic variation in ABCB1 and ABCC2 tagSNPs in these patients provide novel preliminary data relating to links between ABCB1 haplotypes and Act D exposure in children with cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1443. doi:1538-7445.AM2012-1443

Abstract 773: Crizotinib (PF-02341066) reverses multidrug resistance in cancer cells by inhibiting the function of P-glycoprotein

Abstract Background and purpose: Besides targeting the well-known oncogenic c-Met, crizotinib is the first oral tyrosine kinase inhibitor (TKI) inhibiting anaplastic lymphoma kinase (ALK) in clinical trials for the treatment of non-small cell lung cancer (NSCLC). In this study, we investigated the potential multidrug resistance (MDR) reversal by crizotinib in vitro and in vivo. Experimental approach: MTT methods were used in vitro and the xenografts in nude mice were used in vivo to investigate crizotinib reversal of MDR. To understand the mechanisms for MDR reversal, the alterations of intracellular doxorubicin(Dox) or rhodamine 123 accumulation, Dox effluxion, ABCB1 expression level, ATPase activity of ABCB1, and crizotinib-induced c-Met, Akt and Erk1/2 phosphorylation were examined. Key results: Crizotinib significantly enhanced the cytotoxicity of ABCB1 substrate chemotherapeutic agents in MDR cells, with no effect found on sensitive cells in vitro and in vivo. In addition, crizotinib significantly increased the intracellular accumulation of rhodamine 123 and doxorubicin and inhibited the drug efflux in ABCB1-overexpressing MDR cells. Further studies showed that crizotinib enhanced the ATPase activity of ABCB1 in concentration-dependent manner. However, the expressions of ABCB1 were not affected and the reversal activity of crizotinib was not related to the phosphorylation of c-Met, Akt or Erk1/2. Importantly, crizotinib significantly enhanced the effect of paclitaxel against the KBv200 cell xenografts in nude mice. Conclusions and implications: Crizotinib was found to reverse ABCB1-mediated MDR by inhibiting ABCB1 transport function without involvement of ABCB1 expression or blockade of the Akt or Erk1/2 pathways. These findings is useful for combination chemotherapy of crizotinib with conventional chemotherapeutic drugs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 773. doi:1538-7445.AM2012-773

Neratinib Reverses ATP-Binding Cassette B1-Mediated Chemotherapeutic Drug Resistance In Vitro, In Vivo, and Ex Vivo

Neratinib, an irreversible inhibitor of epidermal growth factor receptor and human epidermal receptor 2, is in phase III clinical trials for patients with human epidermal receptor 2-positive, locally advanced or metastatic breast cancer. The objective of this study was to explore the ability of neratinib to reverse tumor multidrug resistance attributable to overexpression of ATP-binding cassette (ABC) transporters. Our results showed that neratinib remarkably enhanced the sensitivity of ABCB1-overexpressing cells to ABCB1 substrates. It is noteworthy that neratinib augmented the effect of chemotherapeutic agents in inhibiting the growth of ABCB1-overexpressing primary leukemia blasts and KBv200 cell xenografts in nude mice. Furthermore, neratinib increased doxorubicin accumulation in ABCB1-overexpressing cell lines and Rhodamine 123 accumulation in ABCB1-overexpressing cell lines and primary leukemia blasts. Neratinib stimulated the ATPase activity of ABCB1 at low concentrations but inhibited it at high concentrations. Likewise, neratinib inhibited the photolabeling of ABCB1 with [(125)I]iodoarylazidoprazosin in a concentration-dependent manner (IC(50) = 0.24 μM). Neither the expression of ABCB1 at the mRNA and protein levels nor the phosphorylation of Akt was affected by neratinib at reversal concentrations. Docking simulation results were consistent with the binding conformation of neratinib within the large cavity of the transmembrane region of ABCB1, which provides computational support for the cross-reactivity of tyrosine kinase inhibitors with human ABCB1. In conclusion, neratinib can reverse ABCB1-mediated multidrug resistance in vitro, ex vivo, and in vivo by inhibiting its transport function.

Apatinib (YN968D1) enhances the efficacy of conventional chemotherapeutical drugs in side population cells and ABCB1-overexpressing leukemia cells

P-glycoprotein (P-gp, ABCB1) overexpression and enrichment of stem-like cells are linked to poor prognosis in tumor patients. In this study, we investigated the effect of apatinib, an oral multi-targeted tyrosine kinase inhibitor (TKI) on enhancing the efficacy of conventional anticancer drugs in side population (SP) cells and ABCB1-overexpressing leukemia cells in vitro, in vivo and ex vivo. Our results showed that apatinib significantly enhanced the cytotoxicity and cell apoptosis induced by doxorubicin in SP cells sorted from K562 cells. Furthermore, apatinib also strongly reversed multidrug resistance (MDR) in K562/ADR cells, and the primary leukemia blasts overexpressing ABCB1 while showed no synergistic interactions with chemotherapeutic agents in MRP1-, MRP4-, MRP7- and LRP-overexpressing cells. Apatinib treatment markedly increased the intracellular accumulation of doxorubicin and rhodamine 123 in K562/ADR cells and the accumulation of rhodamine 123 in the primary leukemia blasts with ABCB1 overexpression. Apatinib stimulated the ATPase activity of P-gp in a dose-dependent manner but did not alter the expression of ABCB1 at both mRNA and protein levels. The phosphorylation level of AKT and ERK1/2 remained unchanged after apatinib treatment in both sensitive and MDR cells. Importantly, apatinib significantly enhanced the antitumor activity of doxorubicin in nude mice bearing K562/ADR xenografts. Taken together, our results suggest that apatinib could target to SP cells and ABCB1-overexpressing leukemia cells to enhance the efficacy of chemotherapeutic drugs. These findings should be useful for the combination of apatinib and chemotherapeutic agents in the clinic.

The Phosphodiesterase-5 Inhibitor Vardenafil Is a Potent Inhibitor of ABCB1/P-Glycoprotein Transporter

One of the major causes of chemotherapy failure in cancer treatment is multidrug resistance (MDR) which is mediated by the ABCB1/P-glycoprotein. Previously, through the use of an extensive screening process, we found that vardenafil, a phosphodiesterase 5 (PDE-5) inhibitor significantly reverses MDR in ABCB1 overexpressing cancer cells, and its efficacy was greater than that of tadalafil, another PDE-5 inhibitor. The present study was designed to determine the reversal mechanisms of vardenafil and tadalafil on ABC transporters-mediated MDR. Vardenafil or tadalafil alone, at concentrations up to 20 µM, had no significant toxic effects on any of the cell lines used in this study, regardless of their membrane transporter status. However, vardenafil when used in combination with anticancer substrates of ABCB1, significantly potentiated their cytotoxicity in ABCB1 overexpressing cells in a concentration-dependent manner, and this effect was greater than that of tadalafil. The sensitivity of the parenteral cell lines to cytotoxic anticancer drugs was not significantly altered by vardenafil. The differential effects of vardenafil and tadalafil appear to be specific for the ABCB1 transporter as both vardenafil and tadalafil had no significant effect on the reversal of drug resistance conferred by ABCC1 (MRP1) and ABCG2 (BCRP) transporters. Vardenafil significantly increased the intracellular accumulation of [3H]-paclitaxel in the ABCB1 overexpressing KB-C2 cells. In addition, vardenafil significantly stimulated the ATPase activity of ABCB1 and inhibited the photolabeling of ABCB1 with [125I]-IAAP. Furthermore, Western blot analysis indicated the incubation of cells with either vardenafil or tadalafil for 72 h did not alter ABCB1 protein expression. Overall, our results suggest that vardenafil reverses ABCB1-mediated MDR by directly blocking the drug efflux function of ABCB1.

Abstract 1739: Reversal of ABCB1- and ABCG2-mediated drug resistance by sildenafil

Abstract Sildenafil is a selective inhibitor of type 5 cGMP-specific phosphodiesterase and is clinically used to treat erectile dysfunction and pulmonary arterial hypertension. Overexpression of ABC transporters such as ABCB1, ABCC1 and ABCG2 is one of the main causes of the development of resistance in cancer cells to chemotherapeutic drugs. However, the effect of sildenafil on these ABC transporters is still unknown. In the present study, we found that sildenafil had differential effects on these transporters. In ABCB1-overexpressing cells, non-toxic doses of sildenafil were found to inhibit resistance to ABCB1 substrate anticancer drugs, as well as significantly increase intracellular accumulation of [3H]-paclitaxel. Similarly, in ABCG2-overexpressing cells, sildenafil partially reversed resistance to ABCG2 substrate anticancer drugs and increased intracellular accumulation of [3H]-mitoxantrone as well as the fluorescent compound BODIPY-prazosin. Sildenafil also moderately inhibited the transport of [3H]-E217βG and [3H]-methotrexate by ABCG2. Sildenafil significantly stimulated ABCB1 ATPase activity but it only slightly stimulated ABCG2 ATPase activity. However, it inhibited photolabeling of both ABCB1 and ABCG2 with [125I]-IAAP. Additionally, sildenafil did not alter the sensitivity of parental, ABCB1- or ABCG2-overexpressing cells to non-ABCB1 and non-ABCG2 substrate drug and had no effect on the function of ABCC1. We also modeled the binding of sildenafil within the large cavity of the transmembrane region of homology model of ABCB1 based on the mouse Abcb1a structure. Overall, we conclude that sildenafil can inhibit the function of ABCB1 and ABCG2, with a stronger effect on ABCB1. These findings suggest that it may be possible to develop potent MDR reversal agents from clinically approved drugs with known side effects and drug interactions. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1739. doi:10.1158/1538-7445.AM2011-1739

Abstract 5447: Investigating the relationship between pharmacogenetic variation in ABCB1 and ABCC2 tagSNPs and actinomycin D pharmacokinetics in children

Abstract Actinomycin D (Act D) has been used successfully to treat adult and childhood cancers for over 30 years. It continues to play a key role in the treatment of Wilms tumour patients, with 5-year survival rates >80% currently observed. While treatment is generally well tolerated, veno-occlusive disease (VOD) is a potentially life-threatening side effect observed in 1.7-13.5% of patients. Dose-intensity of Act D treatment has been highlighted as a possible risk-factor for VOD in paediatric patient populations. ATP binding cassette (ABC) transporters are expressed throughout the body to protect against exogenous compounds. In the liver and kidney, ABC transporters facilitate the removal of compounds via biliary or renal excretion and have previously been shown to play a major role in the transport of anti-cancer agents such as doxorubicin and irinotecan. Common single nucleotide polymorphisms (SNPs) for these transporters have been shown to alter clinical exposure to various anti-cancer agents. We have therefore carried out translational and clinical studies focusing on Act D pharmacokinetics in Wilms tumour patients and pharmacogenetic variation in key ABC transporters. Initial experiments were carried out to determine the potential importance of ABC transporter expression on the in vitro activity and transport of Act D, using MDCKII-WT alongside ABCB1, ABCG2 and ABCC2 over-expressing cell lines. Growth inhibition (GI) and intracellular accumulation experiments were carried out over a range of Act D concentrations (0-10µM). Pharmacokinetic analysis of samples obtained from patients receiving Act D was carried out using a validated LC/MS method and blood samples were obtained from all patients for pharmacogenetic analysis. Finally, the International HapMap database was used to determine tagSNPs for ABCB1 and ABCC2 with minor allele frequencies of greater than 0.1 in a Caucasian population, allowing the link between ABCB1 and ABCC2 haplotypes and Act D exposure to be investigated. GI experiments indicated a 40-fold decrease in Act D sensitivity in cells over-expressing ABCB1 (GI50: 0.85µM) and a 3-fold decrease in cells over-expressing ABCC2 (GI50: 0.06µM) compared to the control cell line (GI50: 0.02µM). Higher intracellular levels of Act D (normalized per mg protein) were observed in the WT parental cell line (650nM) as compared to those over-expressing ABCB1 (215nM), following a 6h incubation with 2µM Act D. This effect was abrogated by use of the ABCB1 inhibitor verapamil. Pharmacokinetic analysis of samples obtained from 56 patients indicated a large inter-patient variability in Act D exposure (AUC0-24h range: 1.6 – 11.4 µg/ml.min). Ongoing investigations into pharmacogenetic variation in ABCB1 and ABCC2 tagSNPs in these patients provide novel preliminary data relating to links between ABCB1 and ABCC2 haplotypes and Act D exposure in children with cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5447. doi:10.1158/1538-7445.AM2011-5447

Sildenafil Reverses ABCB1- and ABCG2-Mediated Chemotherapeutic Drug Resistance

Sildenafil is a potent and selective inhibitor of the type 5 cGMP (cyclic guanosine 3',5'-monophosphate)-specific phosphodiesterase that is used clinically to treat erectile dysfunction and pulmonary arterial hypertension. Here, we report that sildenafil has differential effects on cell surface ABC transporters such as ABCB1, ABCC1, and ABCG2 that modulate intracompartmental and intracellular concentrations of chemotherapeutic drugs. In ABCB1-overexpressing cells, nontoxic doses of sildenafil inhibited resistance and increased the effective intracellular concentration of ABCB1 substrate drugs such as paclitaxel. Similarly, in ABCG2-overexpressing cells, sildenafil inhibited resistance to ABCG2 substrate anticancer drugs, for example, increasing the effective intracellular concentration of mitoxantrone or the fluorescent compound BODIPY-prazosin. Sildenafil also moderately inhibited the transport of E(2)17βG and methotrexate by the ABCG2 transporter. Mechanistic investigations revealed that sildenafil stimulated ABCB1 ATPase activity and inhibited photolabeling of ABCB1 with [(125)I]-iodoarylazidoprazosin (IAAP), whereas it only slightly stimulated ABCG2 ATPase activity and inhibited photolabeling of ABCG2 with [(125)I]-IAAP. In contrast, sildenafil did not alter the sensitivity of parental, ABCB1-, or ABCG2-overexpressing cells to non-ABCB1 and non-ABCG2 substrate drugs, nor did sildenafil affect the function of another ABC drug transporter, ABCC1. Homology modeling predicted the binding conformation of sildenafil within the large cavity of the transmembrane region of ABCB1. Overall, we found that sildenafil inhibits the transporter function of ABCB1 and ABCG2, with a stronger effect on ABCB1. Our findings suggest a possible strategy to enhance the distribution and potentially the activity of anticancer drugs by jointly using a clinically approved drug with known side effects and drug-drug interactions.

Effect of BIBF 1120 on reversal of ABCB1-mediated multidrug resistance

The overexpression of ATP-binding cassette (ABC) transporters is one of the main causes of multi-drug resistance (MDR) which represents a major obstacle to the success of cancer chemotherapy. In this study, we examined the effect of BIBF 1120, an inhibitor of vascular endothelial growth factor receptors (VEGFRs), platelet-derived growth factor receptors (PDGFRs) and fibroblast growth factor receptors (FGFRs) tyrosine kinases, on the reversal of multidrug resistance in vitro. The doxorubicin and rhodamine 123 retention assay was performed by flowcytometry. Western blot were employed to identify ABCB1 expression level and the effect of BIBF 1120 on the blockade of Akt and ERK1/2 phosphorylation. The expression of mdr1 mRNA was determined by RT-PCR analysis. The ATPase activity of ABCB1 was investigated using Pgp-Glo™ assay systems. BIBF 1120 significantly enhanced the cytotoxicity of doxorubicin and paclitaxel and increased the accumulation of ABCB1 substrates in ABCB1-overexpressing cancer cells, whereas it had no effect on the parental cells. On the other hand, BIBF 1120 did not alter the cytotoxicity of non-ABCB1 substrates and was unable to reverse ABCC1 or ABCG2-mediated MDR. Furthermore, BIBF 1120 inhibited the ATPase activity of ABCB1 in a concentration-dependent manner. However, no detectable alteration on the expression level of mdr1 mRNA or ABCB1 protein was identified in ABCB1-overexpressing cancer cells by different treatments of BIBF 1120. Interestly, total and phosphorylated forms of AKT and ERK1/2 were not inhibited by BIBF 1120 at the reversal concentrations. Our results suggest that BIBF 1120 is capable of overcoming ABCB1-mediated drug resistance by inhibiting ABCB1 function, which may have clinical significance for BIBF 1120 combinational treatment of certain resistant cancers.

Sensitization of ABCB1 overexpressing cells to chemotherapeutic agents by FG020326 via binding to ABCB1 and inhibiting its function

The effectiveness of chemotherapeutic treatment is usually limited by the overexpression of adenosine triphosphate binding cassette (ABC) transporters, which mediate multidrug resistance (MDR) by acting as efflux pumps to remove chemotherapeutic agents from MDR cancer cells. Thus, the inhibition of ABC transporters may represent a promising strategy to reverse MDR. This study was to characterize the actions of FG020326, a newly synthesized triaryl-substituted imidazole derivative, to reverse MDR in vitro and in vivo. FG020326 significantly potentiated the cytotoxicity of paclitaxel, doxorubicin, and vincristine in the ABCB1 (P-glycoprotein, P-gp) overexpressing cells KBv200 and MCF-7/adr, but not in the ABCB1 negative parental cell lines KB and MCF-7. However, FG020326 did not alter the cytotoxicity of the aforementioned drugs in ABCC1 (MRP1), ABCC4 (MRP4), ABCG2 (BCRP) and LRP overexpressing cell lines, KB-CV60, NIH3T3/MRP4-2, S1-M1-80 and SW1573/2R120, respectively. FG020326, following p.o. administration, was present in concentrations sufficient for reversal of MDR in mice. The co-administration of FG020326 with paclitaxel or vincristine significantly enhanced the antitumor activity of these drugs without significantly increasing toxicity in the mice bearing the KBv200 cell xenografts. In addition, FG020326, at concentrations that reversed MDR, did not significantly affect the activity of CYP3A4 or alter the pharmacokinetic profile of paclitaxel after co-administration with paclitaxel. FG020326 produced a significant concentration-dependent displacement of [ 3H]azidopine and inhibition of efflux of drug from cells. Furthermore, FG020326 was co-localized with ABCB1 in cell membranes. Hence, FG020326 is characterized as a third generation MDR modulator that holds great promise for the treatment of cancer patients with ABCB1-mediated MDR.

Bullatacin Triggered ABCB1‐Overexpressing Cell Apoptosis via the Mitochondrial‐Dependent Pathway

This paper was to explore bullatacin-mediated multidrug-resistant cell apoptosis at extremely low concentration. To investigate its precise mechanisms, the pathway of cell apoptosis induced by bullatacin was examined. Bullatacin causes an upregulation of ROS and a downregulation of ΔΨm in a concentration-dependent manner in ABCB1-overexpressing KBv200 cells. In addition, cleavers of caspase-9, caspase-3, and PARP were observed following the release of cytochrome c from mitochondria after bullatacin treatment. However, neither cleavage of caspase-8 nor change of expression level of bcl-2, bax and Fas was observed by the same treatment. Pretreating KBv200 cells with N-acetylcysteine, an antioxidant modulator, resulted in a significant reduction of ROS generation and cell apoptosis induced by bullatacin. Bullatacin-induced apoptosis was antagonized by z-LEHD-fmk, a caspase-9 inhibitor, but not by z-IETD-fmk, a caspase-8 inhibitor. These implied that apoptosis of KBv200 cells induced by bullatacin was associated with the mitochondria-dependent pathway that was limited to activation of apical caspase-9.

Inhibiting the function of ABCB1 and ABCG2 by the EGFR tyrosine kinase inhibitor AG1478

The tyrphostin 4-(3-chloroanilino)-6,7-dimethoxyquinazoline (AG1478) is a potent and specific EGFR tyrosine kinase inhibitor (TKI); its promising pre-clinical results have led to clinical trials. Overexpression of ATP-binding cassette (ABC) transporters such as ABCB1, ABCC1 and ABCG2 is one of the main causes of multidrug resistance (MDR) and usually results in the failure of cancer chemotherapy. However, the interaction of AG1478 with these ABC transporters is still unclear. In the present study, we have investigated this interaction and found that AG1478 has differential effects on these transporters. In ABCB1-overexpressing cells, non-toxic doses of AG1478 were found to partially inhibit resistance to ABCB1 substrate anticancer drugs as well as increase intracellular accumulation of [ 3H]-paclitaxel. Similarly, in ABCG2-overexpressing cells, AG1478 significantly reversed resistance to ABCG2 substrate anticancer drugs and increased intracellular accumulation of [ 3H]-mitoxantrone as well as fluorescent compound BODIPY-prazosin. AG1478 also profoundly inhibited the transport of [ 3H]-E 217βG and [ 3H]-methotrexate by ABCG2. We also found that AG1478 slightly stimulated ABCB1 ATPase activity and significantly stimulated ABCG2 ATPase activity. Interestingly, AG1478 did not inhibit the photolabeling of ABCB1 or ABCG2 with [ 125I]-iodoarylazidoprazosin. Additionally, AG1478 did not alter the sensitivity of parental, ABCB1- or ABCG2-overexpressing cells to non-ABCB1 and non-ABCG2 substrate drug and had no effect on the function of ABCC1. Overall, we conclude that AG1478 is able to inhibit the function of ABCB1 and ABCG2, with a more pronounced effect on ABCG2. Our findings provide valuable contributions to the development of safer and more effective EGFR TKIs for use as anticancer agents in the clinic.

Lapatinib (Tykerb, GW572016) Reverses Multidrug Resistance in Cancer Cells by Inhibiting the Activity of ATP-Binding Cassette Subfamily B Member 1 and G Member 2

Lapatinib is active at the ATP-binding site of tyrosine kinases that are associated with the human epidermal growth factor receptor (Her-1 or ErbB1) and Her-2. It is conceivable that lapatinib may inhibit the function of ATP-binding cassette (ABC) transporters by binding to their ATP-binding sites. The aim of this study was to investigate the ability of lapatinib to reverse tumor multidrug resistance (MDR) due to overexpression of ABC subfamily B member 1 (ABCB1) and ABC subfamily G member 2 (ABCG2) transporters. Our results showed that lapatinib significantly enhanced the sensitivity to ABCB1 or ABCG2 substrates in cells expressing these transporters, although a small synergetic effect was observed in combining lapatinib and conventional chemotherapeutic agents in parental sensitive MCF-7 or S1 cells. Lapatinib alone, however, did not significantly alter the sensitivity of non-ABCB1 or non-ABCG2 substrates in sensitive and resistant cells. Additionally, lapatinib significantly increased the accumulation of doxorubicin or mitoxantrone in ABCB1- or ABCG2-overexpressing cells and inhibited the transport of methotrexate and E(2)17betaG by ABCG2. Furthermore, lapatinib stimulated the ATPase activity of both ABCB1 and ABCG2 and inhibited the photolabeling of ABCB1 or ABCG2 with [(125)I]iodoarylazidoprazosin in a concentration-dependent manner. However, lapatinib did not affect the expression of these transporters at mRNA or protein levels. Importantly, lapatinib also strongly enhanced the effect of paclitaxel on the inhibition of growth of the ABCB1-overexpressing KBv200 cell xenografts in nude mice. Overall, we conclude that lapatinib reverses ABCB1- and ABCG2-mediated MDR by directly inhibiting their transport function. These findings may be useful for cancer combinational therapy with lapatinib in the clinic.

Dasatinib Cellular Uptake and Efflux in Chronic Myeloid Leukemia Cells: Therapeutic Implications

The organic cation transporter OCT-1 mediates active transport of imatinib. We recently showed that low OCT-1 activity is a major contributor to suboptimal response in chronic myeloid leukemia (CML) patients treated with imatinib. The relevance of OCT-1 activity and efflux pumps in determining intracellular uptake and retention (IUR) of dasatinib was assessed. The effect of OCT inhibitors on [14C]dasatinib and [14C]imatinib IUR was compared using peripheral blood mononuclear cells from newly diagnosed CML patients. The role of efflux transporters was studied using ABCB1- and ABCG2-overexpressing cell lines and relevant inhibitors. Unlike imatinib, there was no significant difference in the dasatinib IUR at 37 degrees C and 4 degrees C (P = 0.8), and OCT-1 inhibitors including prazosin did not reduce dasatinib IUR significantly. In CML mononuclear cells, prazosin inhibitable IUR was significantly higher for imatinib than dasatinib (6.38 versus 1.48 ng/200,000 cells; P = 0.002; n = 11). Patients with high OCT-1 activity based on their imatinib uptake had IC50(dasatinib) values equivalent to patients with low OCT-1 activity. Dasatinib IUR was significantly lower in ABCB1-overexpressing cell lines compared with parental cell lines (P < 0.05). PSC833 (ABCB1 inhibitor) significantly increased the dasatinib IUR (P < 0.05) and reduced IC50(dasatinib) (from 100 to 8 nmol/L) in K562-DOX cell line. The ABCG2 inhibitor Ko143 significantly increased dasatinib IUR in ABCG2-overexpressing cell lines and reduced IC(50)(dasatinib). Unlike imatinib, dasatinib cellular uptake is not significantly affected by OCT-1 activity, so that expression and function of OCT-1 is unlikely to affect response to dasatinib. Dasatinib is a substrate of both efflux proteins, ABCB1 and ABCG2.

In Contrast to Imatinib, OCT-1 Mediated Influx Has Minimal Impact on Cellular Uptake of Dasatinib in CML Patients at Diagnosis.

Molecular response to imatinib in newly diagnosed CML patients correlates with intrinsic sensitivity to imatinib (IC50imatinib) which is related to imatinib intracellular uptake and retention (IUR). Imatinib cellular uptake is OCT-1 dependent and low OCT-1 activity in newly diagnosed CML patients is a major contributor to suboptimal response to imatinib. The cellular transport pathways for dasatinib, a more potent BCR-ABL kinase inhibitor, are yet to be defined. We have assessed dasatinib cellular transport pathways using mononuclear cells (MNCs) from newly diagnosed CML patients and cell lines. Dasatinib IUR was determined with 14C-labelled dasatinib (Bristol-Myers Squibb) at 4°C and 37°C, with/without influx/efflux inhibitors. There was no significant difference in dasatinib IUR at 37°C and 4°C in KU812 cell line (27 ng vs. 26 ng) and MNCs of CML patients (17 ng vs. 17 ng, n=10), indicating cellular uptake was not temperature dependent and most likely passive. Prazosin and progesterone (OCT-1 and OCT-3 inhibitors) did not significantly reduce IUR and did not reduce inter-patient variation in dasatinib IUR in contrast to imatinib IUR (P=0.45 and P=0.93 respectively, Fig. 1). Procainamide (OCT-1 and OCT-2 inhibitor), nicotinamide (OCT-2) and corticosterone (OCT-3 inhibitor) reduced dasatinib IUR but this did not reach significance (P=0.42, 0.74, 0.79 respectively). These data suggest that dasatinib uptake is not OCT dependent. Using ABCB1 overexpressing cell lines, we found that dasatinib IUR is lower in K562-Dox (P<0.001) and VBL-100 (P=0.05) compared to parental cell lines (K562 and CCRF-CEM respectively). PSC-833 (ABCB1 inhibitor, Novartis) significantly increased dasatinib IUR (P<0.0001), reduced IC50dasatinib in K562-Dox cell line (100 nM vs. 16 nM) and increased IUR in VBL-100 (P=0.01) suggesting ABCB1 actively efflux dasatinib. Median IC50dasatinib in 18 newly diagnosed CML patients was 2.20 nM (± 0.8), which is significantly below median plasma concentration achieved (100–200 nM). Interpatient variability in IC50dasatinib and IUR is therefore unlikely to be clinically significant for newly diagnosed CML patients receiving dasatinib. In conclusion1.dasatinib cellular uptake is predominantly passive and may be OCT independent.2.dasatinib is effluxed actively by ABCB1 in cell lines over-expressing these proteins.3.Inter-patient variation in dasatinib intracellular uptake and intrinsic sensitivity may not be clinically significant in newly diagnosed CML patients receiving dasatinib, in contrast to recipients of imatinib. [Display omitted]

3536 POSTER A phase II study of induction chemotherapy with gemcitabine plus S-1 (GS) followed by chemoradiation for locally advanced pancreatic cancer (LAPC)

Cancer microenvironment is characterized by significantly lower oxygen concentration. This hypoxic condition is known to reduce drug responsiveness to cancer chemotherapy via multiple mechanisms, among which the upregulation of the ATP-binding cassette (ABC) efflux transporters confers resistance to a wide variety of structurally unrelated anticancer drugs. Vatalanib (PTK787/ZK22584) is a multitargeted tyrosine kinase inhibitor for all isoforms of VEGFR, PDGFR and c-Kit, which exhibit potent anticancer activity in vitro and in vivo. We investigated the potentiation effect of vatalanib on the anticancer activity of conventional cytotoxic drugs in colon cancer cell lines under both normoxic and hypoxic conditions. Mechanistically, vatalanib was found to inhibit ABCG2 and ABCB1 efflux activity, presumably by acting as a competitive inhibitor and interfering with their ATPase activity. Under hypoxic growth condition, ABCG2 and ABCB1-overexpressing cells sorted out by FACS technique as side population (SP) were found to be significantly more responsive to SN-38 (ABCG2 and ABCB1 substrate anticancer drug) in the presence of vatalanib. The anchorage independent soft agar colony formation capacity of the SP cells was remarkably reduced upon treatment with a combination of SN-38 and vatalanib, compared to SN-38 alone. However, vatalanib, at concentrations that produced the circumvention of the transporters-mediated resistance, did not appreciably alter ABCG2/ABCB1 mRNA or protein expression levels or the phosphorylation of Akt and extracellular signal-regulated kinase (ERK1/2). Our study thus advocates the further investigation of vatalanib for use in combination chemotherapy to eradicate drug-resistant cancer cells under hypoxia.