Overexpression Of ATP-binding Cassette Transporters Research Articles

Abstract 7193: ABCB1-mediated chemotherapeutic drug resistance is reversed by a potent p53-MDM2 inhibitor NVP-HDM201

Abstract The failure of chemotherapy in cancer persists as a result of overexpression of various ATP-binding cassette (ABC) transporters. ABCB1 is one such transporter that actively pumps out chemotherapeutic drugs and it is one of the reasons for multidrug resistance (MDR). In the last few years, activation of p53 has been targeted as a therapeutic strategy in the treatment of cancers. Siremadlin (NVP-HDM201) is a potent and selective inhibitor of p53-MDM2 interaction. The objective of this study was to explore the ability of HDM201 to reverse tumor MDR attributable to overexpression of ABC transporters. According to our findings, HDM201 significantly increased the sensitivity of ABCB1-overexpressing cells to ABCB1 substrates, such as colchicine and doxorubicin. We found that HDM201 reverses ABCB1-mediated MDR by interacting with ABCB1 and blocking its function without affecting the expression and cellular localization of ABCB1. Moreover, HDM201 promoted the accumulation of paclitaxel in cell lines that overexpress ABCB1. At both high and low doses, HDM201 increased the ATPase activity of ABCB1. The computational support for the cross-reactivity of this p53 inhibitor with human ABCB1 comes from docking simulation results that align with the binding conformation of HDM201 within the vast cavity of the transmembrane region of ABCB1. In conclusion, by blocking the transport function of ABCB1, HDM201 can reverse ABCB1-mediated MDR in vitro. Citation Format: Harsh Patel, Chaoyun Cai, Zhuoxun Wu, Qiuxu Teng, Hanli Li, Zhe-Sheng Chen. ABCB1-mediated chemotherapeutic drug resistance is reversed by a potent p53-MDM2 inhibitor NVP-HDM201 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7193.

I-CBP112 declines overexpression of ATP-binding cassette transporters and sensitized drug-resistant MDA-MB-231 and A549 cell lines to chemotherapy drugs

Despite extensive efforts and ongoing progress in personalized anticancer approaches, chemotherapy remains the first line or the only treatment for some tumors that may develop resistance to chemotherapeutics in time due to inter alia overexpression of ATP-binding cassette transporters. Using clinically-relevant resistant models of triple negative breast cancer (MDA-MB-231; TNBC) as well as non-small cell lung cancer (A549; NSCLC), we tested the efficacy of I-CBP112 – CBP/EP300 bromodomain inhibitor to overcome drug resistance by declining ABC gene transcription. I-CBP112 significantly reduced ABCB1, ABCC1, ABCC2, ABCC3, ABCC5 and ABCG2 in all resistant lines, as well as ABCC10 in TNBC and ABCC4 in paclitaxel-resistant NSCLC, thereby increasing intracellular drug accumulation and cytotoxicity in 2D and 3D cultures. This was phenocopied only by the joint effect of ABC inhibitors such as tariquidar (ABCB1 – P-glycoprotein and ABCG2) and MK-571 (ABCC), whereas single inhibition of ABCB1/ABCG2 or ABCC proteins did not affect drug accumulation, thereby implying the need of simultaneous deficiency in activity of majority of drug pumps for enhanced drug retention. I-CBP112 failed to directly inhibit activity of ABCB1, ABCG2 and ABCC subfamily members at the same time. Importantly, I-CBP112 treated cancer cells polarized human macrophages into proinflammatory phenotypes. Moreover, I-CBP112 remained non-toxic to primary cell lines, nor did it enhance anticancer drug toxicity to blood-immune cells. In silico assay of ADMET properties confirmed the desired pharmacokinetic features of I-CBP112. The results suggest that the CBP/p300 inhibitor is a promising co-adjuvant to chemotherapy in drug-resistant cancer phenotypes, capable of decreasing ABC transporter expression.

ABC transporters affects tumor immune microenvironment to regulate cancer immunotherapy and multidrug resistance.

Multidrug resistance (MDR) is the phenomenon in which cancer cells simultaneously develop resistance to a broad spectrum of structurally and mechanistically unrelated drugs. MDR severely hinders the effective treatment of cancer and is the major cause of chemotherapy failure. ATP-binding cassette (ABC) transporters are extensively expressed in various body tissues, and actively transport endogenous and exogenous substrates through biological membranes. Overexpression of ABC transporters is frequently observed in MDR cancer cells, which promotes efflux of chemotherapeutic drugs and reduces their intracellular accumulation. Increasing evidence suggests that ABC transporters regulate tumor immune microenvironment (TIME) by transporting various cytokines, thus controlling anti-tumor immunity and sensitivity to anticancer drugs. On the other hand, the expression of various ABC transporters is regulated by cytokines and other immune signaling molecules. Targeted inhibition of ABC transporter expression or function can enhance the efficacy of immune checkpoint inhibitors by promoting anticancer immune microenvironment. This review provides an update on the recent research progress in this field.

The role of microRNAs on doxorubicin drug resistance in breast cancer.

Resistance to chemotherapeutic drugs is a serious challenge for effective therapy of cancers. Doxorubicin is a drug which is typically used for breast cancer treatment. Several mechanisms are involved in resistance to doxorubicin including overexpression of ATP-binding cassette (ABC) transporters, altering apoptosis, autophagy and cell cycle arrest. In this review, we focus on the potential effects of microRNAs on doxorubicin resistance in breast cancer. Literature review focusing on the 'microRNAs and doxorubicin drug resistance in breast cancer' was conducted comprehensively. The search was performed in PubMed, Scopus, Google and Google Scholar databases and reference lists of relevant articles were also included. MicroRNAs play essential role in resistance of breast cancer to doxorubicin by affecting several key cellular pathways, including overexpression of ABC transporters, altering apoptosis, autophagy and cell signaling pathways, cell cycle arrest, epithelial to mesenchymal transition (EMT) and cancer stem cells (CSCs). Cancer treatment methods are moving from conventional therapies to targeted therapies such as using microRNAs. MiRNAs can act as regulatory molecules to overcome breast cancer doxorubicin resistance by controlling the expression levels of genes involved in different cellular pathways. Thus, exact elucidation of their role in different cellular processes can help overcome the breast cancer development and drug resistance.

1,2,4-Triazole-based anticonvulsant agents with additional ROS scavenging activity are effective in a model of pharmacoresistant epilepsy

There are numerous studies supporting the contribution of oxidative stress to the pathogenesis of epilepsy. Prolonged oxidative stress is associated with the overexpression of ATP-binding cassette transporters, which results in antiepileptic drugs resistance. During our studies, three 1,2,4-triazole-3-thione derivatives were evaluated for the antioxidant activity and anticonvulsant effect in the 6 Hz model of pharmacoresistant epilepsy. The investigated compounds exhibited 2-3 times more potent anticonvulsant activity than valproic acid in 6 Hz test in mice, which is well-established preclinical model of pharmacoresistant epilepsy. The antioxidant/ROS scavenging activity was confirmed in both single-electron transfer-based methods (DPPH and CUPRAC) and during flow cytometric analysis of total ROS activity in U-87 MG cells. Based on the enzymatic studies on human carbonic anhydrases (CAs), acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), one can assume that the herein investigated drug candidates will not impair the cognitive processes mediated by CAs and will have minimal off-target cholinergic effects.

Enhanced acid-stress tolerance in Lactococcus lactis NZ9000 by overexpression of ABC transporters

BackgroundMicrobial cell factories are widely used in the production of acidic products such as organic acids and amino acids. However, the metabolic activity of microbial cells and their production efficiency are severely inhibited with the accumulation of intracellular acidic metabolites. Therefore, it remains a key issue to enhance the acid tolerance of microbial cells. In this study, we investigated the effects of four ATP-binding cassette (ABC) transporters on acid stress tolerance in Lactococcus lactis.ResultsOverexpressing the rbsA, rbsB, msmK, and dppA genes exhibited 5.8-, 12.2-, 213.7-, and 5.2-fold higher survival rates than the control strain, respectively, after acid shock for 3 h at pH 4.0. Subsequently, transcriptional profile alterations in recombinant strains were analyzed during acid stress. The differentially expressed genes associated with cold-shock proteins (csp), fatty acid biosynthesis (fabH), and coenzyme A biosynthesis (coaD) were up-regulated in the four recombinant strains during acid stress. Additionally, some genes were differentially expressed in specific recombinant strains. For example, in L. lactis (RbsB), genes involved in the pyrimidine biosynthetic pathway (pyrCBDEK) and glycine or betaine transport process (busAA and busAB) were up-regulated during acid stress, and the argG genes showed up-regulations in L. lactis (MsmK). Finally, we found that overexpression of the ABC transporters RbsB and MsmK increased intracellular ATP concentrations to protect cells against acidic damage in the initial stage of acid stress. Furthermore, L. lactis (MsmK) consistently maintained elevated ATP concentrations under acid stress.ConclusionsThis study elucidates the common and specific mechanisms underlying improved acid tolerance by manipulating ABC transporters and provides a further understanding of the role of ABC transporters in acid-stress tolerance.

Using LC-MS/MS-based targeted proteomics to monitor the pattern of ABC transporters expression in the development of drug resistance.

PurposeThe overexpression of ATP-binding cassette transporters (ABC transporters), mainly including permeability glycoproteins (P-gp), multidrug resistance (MDR)-related protein 1 (MRP1), and breast cancer resistance proteins (BCRP), is one of the main reasons for the development of MDR which directly leads to chemotherapy failure. However, most of the currently used detection methods in MDR-related studies are qualitative or semiquantitative, but not quantitative. As a result, the measurement criteria of different experiments are not unified. Moreover, there are many contradictory results of the studies of the induction effect of drugs on ABC transporters. So, it is necessary to establish a quantitative assay for the quantification of P-gp, MRP1, and BCRP to study the mechanism of drug resistance.MethodsIn this paper, a novel and advanced liquid chromatography/mass spectrometry (MS)/MS-based targeted proteomics method for the quantification of P-gp, MRP1, and BCRP was developed and validated. Then, the cell lines MCF-7, HepG-2, and SMMC-7721 were, respectively, induced by different concentrations of doxorubicin (adriamycin [ADM]), mitoxantrone (MX), and methotrexate (MTX), to establish resistance cell lines. The method established was used to quantify the expression of P-gp, MRP1, and BCRP.ResultsThe result showed that the induction effects of drugs on protein were relatively stable and selective. ADM, MX, and MTX could induce overexpression of P-gp, MRP1, and BCRP. And, the induction effect of different drugs on proteins was selective. The pattern of overexpression of ABC transporters in the three types of resistance cell lines was different.ConclusionDuring the development of drug resistance, the cell type and patch, but not drug type, were the most important determinant factors of the overexpression level of ABC transporters in resistance cell lines. This study provides a good foundation for understanding the development of drug resistance in cell lines and can be used to explain the contradictory results in other published studies as described above.

ABC Transporters in Cancer Stem Cells: Beyond Chemoresistance.

The efficacy of chemotherapy is one of the main challenges in cancer treatment and one of the major obstacles to overcome in achieving lasting remission and a definitive cure in patients with cancer is the emergence of cancer resistance. Indeed, drug resistance is ultimately accountable for poor treatment outcomes and tumour relapse. There are various molecular mechanisms involved in multidrug resistance, such as the change in the activity of membrane transporters primarily belonging to the ATP binding cassette (ABC) transporter family. In addition, it has been proposed that this common feature could be attributed to a subpopulation of slow-cycling cancer stem cells (CSCs), endowed with enhanced tumorigenic potential and multidrug resistance. CSCs are characterized by the overexpression of specific surface markers that vary in different cancer cell types. Overexpression of ABC transporters has been reported in several cancers and more predominantly in CSCs. While the major focus on the role played by ABC transporters in cancer is polarized by their involvement in chemoresistance, emerging evidence supports a more active role of these proteins, in which they release specific bioactive molecules in the extracellular milieu. This review will outline our current understanding of the role played by ABC transporters in CSCs, how their expression is regulated and how they support the malignant metabolic phenotype. To summarize, we suggest that the increased expression of ABC transporters in CSCs may have precise functional roles and provide the opportunity to target, particularly these cells, by using specific ABC transporter inhibitors.

Abstract 5045: Overexpression of ABC transporters through HGF/c-MET activation results in anti-cancer drug resistance in SCLC

Abstract Upregulation of hepatocyte growth factor (HGF)/c-MET pathway causes drug resistance to anti-cancer agents such as epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors in non-small lung cancer (NSCLC) with EGFR mutation and cytotoxic agents in small cell lung cancer (SCLC), but the roles and mechanisms of c-MET in drug-resistant cancer cells are not clarified. We studied to reveal the role of c-MET overexpression in the resistant lung cancer cell lines and to demonstrate whether MET-inhibition could restore drug sensitivity in anti-cancer drug resistant cell lines via ABC transporters down-regulation. In this study we used the 7-ethyl-10-hydroxycamptothesin (SN-38)-resistant cell line (PC-6/SN-38) that was derived from the human SCLC cell line PC-6. We compared the expression levels of the purposed genes by quantitative real-time PCR (qRT-PCR) and western blotting (WB). MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) assay was used to measure cell viability of the resistant cells compared with the parental cells. Inhibition of c-MET activation was performed by c-MET inhibitors, PHA665752 and Crizotinib or small interfering RNA against c-MET. We found MET inhibitors (4 or 8μM) reduced cell growth and restored drug sensitivity (2μM) of resistant cells, PC-6/SN38, compared to parental cells. To reveal the mechanisms of c-MET in drug resistance we examined ATP-binding cassette (ABC) transporters expression in PC-6/SN-38 compared to the parental cells by qRT-PCR, and found the mRNA level of ABCG2, which effluxes SN-38 as substrate, in PC-6/SN-38 cells was extremely increased and another ABC transporters that do not efflux SN-38 were not. Recently we reported PC-6/SN-38 cells had c-MET overexpression, and c-MET inhibition in resistant cells such PC-6/SN-38 resulted in restoration drug resistance. Based on this report we examined c-MET inhibition by c-MET inhibitor and small interfering RNA against c-MET in PC-6/SN38, and qRT-PCR and WB were performed to investigate alteration of ABCG2 expression, and found both two c-MET inhibition reduced ABCG2 expression. In addition, to demonstrate c-MET upregulation in resistant cells is addicted to the ligand HGF, PC-6 cells were incubated with HGF treated for 3 weeks and made PC-6/HGF, then continued to incubate with HGF and SN-38 was added two times in a week. Although c-MET expression is equal in PC-6 and PC-6/HGF, after SN-38 treatment c-MET was overexpressed. Furthermore, serum HGF level in SCLC patients correlated with clinical course. We demonstrated upregulation of ABC transporters via HGF/c-MET signaling activation might be one of the mechanisms of acquired resistance to cytotoxic anticancer agents in lung cancer cells. Decreased ABC transporters expression through inhibition of c-MET activation might overcome drug resistance to cytotoxic agents. Citation Format: Yoshitaka Yagi, Hiroaki Ozasa, Takahiro Tsuji, Yuichi Sakamori, Takeshi Nomizo, Hiroki Nagai, Young Hak Kim, Ken Maeno, Tetsuya Oguri, Michiaki Mishima. Overexpression of ABC transporters through HGF/c-MET activation results in anti-cancer drug resistance in SCLC. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5045.

Fabrication of doxorubicin nanoparticles by controlled antisolvent precipitation for enhanced intracellular delivery

Over-expression of ATP-binding cassette transporters is one of the most important mechanisms responsible for multidrug resistance. Here, we aimed to develop a stable polymeric nanoparticle system by flash nanoprecipitation (FNP) for enhanced anticancer drug delivery into drug resistant cancer cells. As an antisolvent precipitation process, FNP works best for highly lipophilic solutes (logP>6). Thus we also aimed to evaluate the applicability of FNP to drugs with relatively low lipophilicity (logP=1–2). To this end, doxorubicin (DOX), an anthracycline anticancer agent and a P-gp substrate with a logP of 1.3, was selected as a model drug for the assessment. DOX was successfully incorporated into the amphiphilic diblock copolymer, polyethylene glycol-b-polylactic acid (PEG-b-PLA), by FNP using a four-stream multi-inlet vortex mixer. Optimization of key processing parameters and co-formulation with the co-stabilizer, polyvinylpyrrolidone, yielded highly stable, roughly spherical DOX-loaded PEG-b-PLA nanoparticles (DOX.NP) with mean particle size below 100nm, drug loading up to 14%, and drug encapsulation efficiency up to 49%. DOX.NP exhibited a pH-dependent drug release profile with higher cumulative release rate at acidic pHs. Surface analysis of DOX.NP by XPS revealed an absence of DOX on the particle surface, indicative of complete drug encapsulation. While there were no significant differences in cytotoxic effect on P-gp over-expressing LCC6/MDR cell line between DOX.NP and free DOX in buffered aqueous media, DOX.NP exhibited a considerably higher cellular uptake and intracellular retention after efflux. The apparent lack of cytotoxicity enhancement with DOX.NP may be attributable to its slow DOX release inside the cells.

Semi-synthetic ocotillol analogues as selective ABCB1-mediated drug resistance reversal agents.

Overexpression of ATP-Binding Cassette transporters leads to multidrug resistance in cancer cells and results in the failure of chemotherapy. In this in-vitro study, we investigated whether or not (20S, 24R/S)-epoxy-12β, 25-dihydroxy-dommarane-3β-amine (ORA and OSA), a pair of semi-synthetic ocotillol analogue epimers, could inhibit the ABCB1 transporter. ORA (1 μM and 3 μM) significantly reversed the resistance to paclitaxel and vincristine in ABCB1-overexpressing SW620/Ad300 and HEK/ABCB1 cells, whereas OSA had no significant effects. In addition, ORA (3 μM) significantly increased the intracellular accumulation of [3H]-paclitaxel by suppressing the efflux function of ABCB1. Meanwhile, both ORA (3 μM) and OSA (3 μM) did not significantly alter the expression level or the subcellular location of ABCB1 protein. Moreover, the ABCB1 ATPase study suggested that ORA had a stronger stimulatory effect on the ATPase activity than OSA. ORA also exhibited a higher docking score as compared with OSA inside transmembrane domain of ABCB1. Overall, we concluded that ORA reverse ABCB1-mediated MDR by competitively inhibiting the ABCB1 drug efflux function.

Inhibition of Glutathione Peroxidase Mediates the Collateral Sensitivity of Multidrug-resistant Cells to Tiopronin

Multidrug resistance (MDR) is a major obstacle to the successful chemotherapy of cancer. MDR is often the result of overexpression of ATP-binding cassette transporters following chemotherapy. A common ATP-binding cassette transporter that is overexpressed in MDR cancer cells is P-glycoprotein, which actively effluxes drugs against a concentration gradient, producing an MDR phenotype. Collateral sensitivity (CS), a phenomenon of drug hypersensitivity, is defined as the ability of certain compounds to selectively target MDR cells, but not the drug-sensitive parent cells from which they were derived. The drug tiopronin has been previously shown to elicit CS. However, unlike other CS agents, the mechanism of action was not dependent on the expression of P-glycoprotein in MDR cells. We have determined that the CS activity of tiopronin is mediated by the generation of reactive oxygen species (ROS) and that CS can be reversed by a variety of ROS-scavenging compounds. Specifically, selective toxicity of tiopronin toward MDR cells is achieved by inhibition of glutathione peroxidase (GPx), and the mode of inhibition of GPx1 by tiopronin is shown in this report. Why MDR cells are particularly sensitive to ROS is discussed, as is the difficulty in exploiting this hypersensitivity to tiopronin in the clinic.

Overexpression of multidrug resistance-associated protein 2 in the brain of pentylenetetrazole-kindled rats

Clinical studies and animal models have shown that pharmacoresistant epilepsy is partly due to the overexpression of ATP-binding cassette transporters at the brain. The purposes of the study were to investigate the function and expression of multidrug resistance-associated protein 2 (Mrp2) in the brain of pentylenetetrazole (PTZ)-kindled rats, and the effect of the altered Mrp2 function and expression on phenytoin (PHT) distribution in the brain. Kindled rats were developed by sub-convulsive dose of PTZ (33mg/kg, every day, intraperitoneal (i.p.)) for 28days. Mrp2 expression and function were measured by western blot and bromosulfophthalein (BSP) distribution in the brain. PHT concentrations in the brain of PTZ-kindled rats were measured alone or with co-administration of probenecid (50mg/kg). Further experiment was designed to investigate whether PHT treatment prevented the up-regulated brain Mrp2 expression and function induced by PTZ-kindling. The results showed that PTZ-kindling resulted in an increase of Mrp2 level in the hippocampus and cortex of rats, accompanied by significant decreases in the brain-to-plasma concentration ratio of BSP. PTZ-kindling also decreased PHT levels in the hippocampus and cortex without altering PHT concentrations in plasma, resulting in a lower brain-to-plasma concentration ratio of PHT. Co-administration of probenecid increased the brain-to-plasma ratio of BSP and PHT in the brain of both normal and PTZ-kindled rats. A 14-day PHT treatment prevented the up-regulation of Mrp2 expression and function induced by PTZ-kindling, accompanied by increases of PHT concentrations in the brain and good anticonvulsive effects. The present study demonstrated that chronic PTZ-kindling increased Mrp2 expression and function in the rat brain, and the up-regulation partly came from epileptic seizure.

Roles of Sildenafil in Enhancing Drug Sensitivity in Cancer

The phenomenon of multidrug resistance (MDR) has decreased the hope for successful cancer chemotherapy. The ATP-binding cassette (ABC) transporter superfamily is the largest transmembrane family. The overexpression of ABC transporters is a major determinant of MDR in cancer cells both in vitro and in vivo. Unfortunately, until recently, most of the strategies used to surmount ABC-transporter-mediated MDR have had limited success. An ideal modulator of MDR would be one that has a low liability to induce toxicity and alter the pharmacokinetic profile of antineoplastic drugs. Sildenafil, an inhibitor of cGMP-specific phosphodiesterase type 5, was found to significantly reverse ABC-transporter-mediated MDR. Our results indicate that sildenafil has differential inhibitory effects on ABC transporters: It significantly decreases the efflux activity of ABCB1 and ABCG2, but has no significant effects on ABCC1. Emerging evidence indicates that sildenafil and other phosphodiesterase type 5 inhibitors may enhance the sensitivity of certain types of cancer to standard chemotherapeutic drugs.

The role of Brugia malayi ATP-binding cassette (ABC) transporters in potentiating drug sensitivity

ATP binding cassette (ABC) systems are a diverse group of proteins that have been identified in every organism, from bacteria to humans. Analysis of nematode genomes indicates that the number and arrangement of ABC systems are similar to other organisms, with the majority being ABC transporters. There are few functional studies of ABC transporters in parasitic nematodes; most reports have been on their identification or use as genetic markers to monitor drug resistance. In eukaryotes, some ABC transporters function in tissue defense by actively removing drugs, thus preventing their accumulation. The overexpression of ABC transporters that function as efflux pumps, such as P-glycoprotein (PGP) and the multidrug resistance associated protein (MRP) are known to confer resistance. Drug sensitivity can be restored by administration of PGP interfering or MDR reversal agents. The objective of this study was to determine if ABC systems in filarioid nematodes function similarly to those of other organisms. The relative expression of 33 ABC systems identified in Brugia malayi was quantified following exogenous exposure to the commonly used drug ivermectin (IVM). Following exposure of adults and microfilariae to IVM, there was a significant increase in the transcriptional profiles of a number of ABC systems, mostly within the PGP and MRP subgroups. Coadministration of PGP-interfering and MDR-reversal agents with IVM potentiated sensitivity to the drug in adults and microfilariae. The results suggest that B. malayi ABC transporters function similarly to those in other organisms and are a factor in determining drug sensitivity.

Theoretical optimization of Irinotecan-based anticancer strategies in the case of drug-induced efflux

The anticancer drug Irinotecan (CPT11) is known to trigger the induction of ATP-Binding Cassette (ABC) transporters, responsible for the efflux of the drug and its metabolites outside of the cells. The drug-modulated overexpression of those transporters prevents its accumulation in the intracellular medium, therefore decreasing its efficacy. A critical clinical concern lies in the design of CPT11-based therapeutic strategies which eradicate a maximum number of cancer cells despite their ability to become resistant. In order to address this issue, we supplemented an existing mathematical model of CPT11 molecular pharmacokinetics–pharmacodynamics (PK–PD) with a new model of CPT11-induced overexpression of ABC transporters. We then theoretically optimized exposure to CPT11 given as a single agent or combined either with ABC transporter inhibitors, or with inhibitors of nuclear factors whose activation is responsible for transporter overexpression. We firstly considered a cancer cell population endowed with the ability of inducing their transporters. For any drug combination, we concluded that the highest concentration of CPT11 should be administered in order to kill a maximum number of cancer cells, despite the triggering of resistance. We then considered a population of healthy cells which were assumed to be identical to cancer cells except that they were not able to become resistant. Optimal schemes were defined as the ones which maximized DNA damage in cancer cells under the constraint of DNA damage in healthy cells not exceeding a tolerability threshold. The optimal therapeutic strategy consisted in combining CPT11 with ABC transporter inhibitors as it achieved a complete reversal of resistance by means of the lowest concentrations of CPT11.

Prolonged Drug Selection of Breast Cancer Cells and Enrichment of Cancer Stem Cell Characteristics

Cancer stem cells are presumed to have virtually unlimited proliferative and self-renewal abilities and to be highly resistant to chemotherapy, a feature that is associated with overexpression of ATP-binding cassette transporters. We investigated whether prolonged continuous selection of cells for drug resistance enriches cultures for cancer stem-like cells. Cancer stem cells were defined as CD44+/CD24⁻ cells that could self-renew (ie, generate cells with the tumorigenic CD44+/CD24⁻ phenotype), differentiate, invade, and form tumors in vivo. We used doxorubicin-selected MCF-7/ADR cells, weakly tumorigenic parental MCF-7 cells, and MCF-7/MDR, an MCF-7 subline with forced expression of ABCB1 protein. Cells were examined for cell surface markers and side-population fractions by microarray and flow cytometry, with in vitro invasion assays, and for ability to form mammospheres. Xenograft tumors were generated in mice to examine tumorigenicity (n = 52). The mRNA expression of multidrug resistance genes was examined in putative cancer stem cells and pathway analysis of statistically significantly differentially expressed genes was performed. All statistical tests were two-sided. Pathway analysis showed that MCF-7/ADR cells express mRNAs from ABCB1 and other genes also found in breast cancer stem cells (eg, CD44, TGFB1, and SNAI1). MCF-7/ADR cells were highly invasive, formed mammospheres, and were tumorigenic in mice. In contrast to parental MCF-7 cells, more than 30% of MCF-7/ADR cells had a CD44+/CD24⁻ phenotype, could self-renew, and differentiate (ie, produce CD44+/CD24⁻ and CD44+/CD24+ cells) and overexpressed various multidrug resistance-linked genes (including ABCB1, CCNE1, and MMP9). MCF-7/ADR cells were statistically significantly more invasive in Matrigel than parental MCF-7 cells (MCF-7 cells = 0.82 cell per field and MCF-7/ADR = 7.51 cells per field, difference = 6.69 cells per field, 95% confidence interval = 4.82 to 8.55 cells per field, P < .001). No enrichment in the CD44+/CD24⁻ or CD133+ population was detected in MCF-7/MDR. The cell population with cancer stem cell characteristics increased after prolonged continuous selection for doxorubicin resistance.

Abstract 4323: ATP binding cassette transporters modulate both D-luciferin- and coelenterazine-based bioluminescence imaging

Abstract Bioluminescence imaging (BLI) of luciferase reporters provides a relatively simple, cost-effective, and extremely sensitive means to image fundamental biological processes in cancer research, including tumor growth, angiogenesis and metastasis. Translocation of D-luciferin (the substrate for Click Beetle Red (cLuc) and Firefly (fLuc) luciferase), and coelenterazine (the substrate for Renilla (rLuc), and Gaussia (gLuc) luciferase), across the cellular membrane could affect BLI intensity in vivo. ATP-binding cassette (ABC) transporters are membrane-bound efflux pumps utilizing the energy from ATP hydrolysis. They are implicated in multidrug resistant phenotypes of tumor cells and may be cancer stem cell markers. To investigate the effect of ABC transporters on luciferase-based BLI, we generated HEK-293 reporter cells with selective overexpression of different ABC transporters (ABCB1, ABCC1 and ABCG2) and different luciferases (cLuc, fLuc, rLuc and gLuc). In vitro studies showed that BLI signal intensity of intact cells compared to cell lysates decreased 89%, 77% and 75% when ABCG2 was overexpressed in HEK-293 cLuc, fLuc and rLuc reporter cells, respectively. No significant BLI change was observed in HEK-293 gLuc reporter cells with high expression of ABCG2. Overexpression of ABCB1 reduced BLI intensity only in HEK-293 rLuc reporter cells by 51%. ABCC1 overexpression did not affect BLI intensity in any of the HEK-293 reporter cells. These data indicate that ABCG2 affects BLI signal intensity generated from both D-luciferin and coelenterazine, and that ABCB1 affects coelenterazine (but not D-luciferin) signal intensity. Selective ABC transporter inhibitors, Reversin 121 against ABCB1, MK-571 against ABCC1, and fumitremorgin C (FTC) against ABCG2, were applied in HEK-293 reporter cells with ABC transporter overexpression. Compared with vehicle treatment, FTC increased BLI intensity more than 2-fold in HEK-293 cLuc, fLuc and rLuc reporter cells; Reversin 121 elevated BLI intensity 2.1 fold only in HEK-293 rLuc reporter cells. BLI of xenografts derived from HEK-293 reporter cells with overexpression of ABC transporters confirmed the results of inhibitor treatment in vivo. These findings demonstrate that ABC transporters can significantly modulate intracellular delivery of luciferase-based substrates, and that endogenous expression of ABC transporters can significantly affect BLI readout, both in vitro and in vivo. In this context, the BLI-based assay can provide a high throughout platform to screen the novel ABC transporter inhibitors for the improvement of antitumor therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4323.

Nilotinib (AMN107, Tasigna ®) reverses multidrug resistance by inhibiting the activity of the ABCB1/Pgp and ABCG2/BCRP/MXR transporters

Nilotinib, a BCR-Abl tyrosine kinase inhibitor (TKI), was developed to surmount resistance or intolerance to imatinib in patients with Philadelphia positive chronic myelogenous leukemia. Recently, it was shown that several human multidrug resistance (MDR) ATP-binding cassette (ABC) proteins could be modulated by specific TKIs. MDR can produce cancer chemotherapy failure, typically due to overexpression of ABC transporters, which are involved in the extrusion of therapeutic drugs. Here, we report for the first time that nilotinib potentiates the cytotoxicity of widely used therapeutic substrates of ABCG2, such as mitoxantrone, doxorubicin, and ABCB1 substrates including colchicine, vincristine, and paclitaxel. Nilotinib also significantly enhances the accumulation of paclitaxel in cell lines overexpressing ABCB1. Similarly, nilotinib significantly increases the intracellular accumulation of mitoxantrone in cells transfected with ABCG2. Furthermore, nilotinib produces a concentration-dependent inhibition of the ABCG2-mediated transport of methotrexate (MTX), as well as E 217βG a physiological substrate of ABCG2. Uptake studies in membrane vesicles overexpressing ABCG2 have indicated that nilotinib inhibits ABCG2 similar to other established TKIs as well as fumitremorgin C. Nilotinib is a potent competitive inhibitor of MTX transport by ABCG2 with a K i value of 0.69 ± 0.083 μM as demonstrated by kinetic analysis of nilotinib. Overall, our results indicate that nilotinib could reverse ABCB1- and ABCG2-mediated MDR by blocking the efflux function of these transporters. These findings may be used to guide the design of present and future clinical trials with nilotinib, elucidating potential pharmacokinetic interactions. Also, these findings may be useful in clinical practice for cancer combination therapy with nilotinib.

Expression of the multidrug resistance proteins MRP2 and MRP3 in human cholangiocellular carcinomas

Cholangiocellular carcinomas and gallbladder carcinomas are highly aggressive tumours with a poor prognosis and are generally regarded as chemoresistant tumours. Overexpression of ATP-binding cassette transporters of the multidrug resistance protein (MDR) and multidrug resistance-related protein (MRP) family in cancer cells is a major cause for the multidrug resistance phenotype in vitro and in vivo. To further define the role of MRP family members in biliary tract cancer, we studied the expression and localization of MRP2 and MRP3 in cholangiocellular carcinomas and gallbladder carcinomas. The expression and cellular localization of the multidrug resistance proteins MRP2 and MRP3 in human cholangiocellular carcinomas and gallbladder carcinomas were analysed by immunohistochemistry using isoform-specific antibodies. Expression of MRP isoforms was studied in vitro in Mz-ChA-1 cells derived from gallbladder adenocarcinoma by reverse transcription-polymerase chain reaction (RT-PCR), immunoblotting and immunofluorescence microscopy. Mz-ChA-1 cells constitutively expressed MDR P-glycoproteins, MRP1, MRP2 and MRP3 by RT-PCR, immunoblotting and immunofluorescence microscopy. MRP2 and MRP3 are expressed in the respective apical and basolateral membrane domains. MRP3 was the predominant MRP isoform in gallbladder carcinomas (93%) and cholangiocellular carcinomas (57%), whereas MRP2 expression was detected in only 29% of gallbladder carcinomas and was undetectable in cholangiocellular carcinomas. Our findings suggest that the intrinsic multidrug resistance of cholangiocellular and gallbladder carcinomas seems to be independent of MRP2 expression while the expression of MRP3 may contribute to the MDR phenotype.