ABC Transporters and Cancer Drug Resistance

Role of ABC Transporters and Multi-Drug Resistance Reversal Using RNA Interference (RNAi)*

Chemotherapy is the treatment of choice against many types of cancer. However, over time chemotherapeutic drugs can become less effective due to the development of resistance that involves a group of membrane proteins. These multi-drug transporters expel cytotoxic molecules from the cell, thus keeping intracellular drug concentrations below the cell-killing threshold. These transporters belong to the superfamily of ATP Binding Cassette (ABC) proteins that are present in all organisms from bacteria to humans. The transport activity of ABC proteins has an important effect on the efficacy of pharmaceuticals by modulating the absorption, distribution, and excretion of these xenobiotics.

ABC transporter proteins are located in the plasma membrane of cells and in the membranes of cellular organelles where they mediate the transport of various substrate molecules. These substrates exhibit a wide variety of chemical structures. Most ABC proteins are active transporters, which utilize the energy generated by ATP hydrolysis; however, some ABC transporters form transmembrane channels.

Cancer Multi-Drug Resistance - The Players

Numerous clinical data revealed that the multi-drug resistance phenotype in tumors is associated with the overexpression of certain ABC transporters, termed multi-drug resistance (MDR) proteins. P-glycoprotein (P-gp, MDR1, ABCB1) was the first discovered ABC transporter1-3 and is likely to be responsible for the most widely observed mechanism in clinical multi-drug resistance.4-7 Soon after the cloning and characterization of MDR1, it became evident that other efflux pumps also play significant roles in transport- associated drug resistance. Two other ABC transporters have definitively demonstrated participation in the multi-drug resistance of tumors: the multi-drug resistance protein 1 (MRP1, ABCC1), and the mitoxantrone resistance protein (MXR/BCRP, ABCG2).7-11

Basic Mechanism of Cancer Multi-Drug Resistance and Substrate Specifi city of MDR-ABC Transporters

The generally accepted mechanism of multi-drug resistance is that the MDR proteins actively expel the cytotoxic drugs from cells, maintaining the drug concentration within the cells below the toxic level. The drug efflux mediated by these primary active transporters is driven by the energy of ATP hydrolysis. Tumors with MDR protein overexpression (e.g., hepatomas, lung or colon carcinomas) often show primary (or intrinsic) resistance to chemotherapy treatment. In addition, chemotherapy itself may induce the overexpression of these proteins, resulting in the multi-drug resistant clones becoming less sensitive to treatment (secondary drug resistance).12-14

The most intriguing characteristic distinguishing the MDR proteins from other mammalian transporters is their broad substrate specificity. Unlike other selective (classical) transport proteins, multidrug transporters recognize and handle a large number of structurally diverse, mainly hydrophobic compounds, which explains cross-resistance to several chemically unrelated compounds, a characteristic feature of the multi-drug resistance phenotype.4-7 In addition to their overlapping substrate specificity, each transporter can handle unique compounds. The following table of MDR-substrate anticancer agents provides a selection of anticancer agents available in our catalog and identifies the key ABC transporter(s) responsible for each agent’s cellular efflux.

Circumvention of Cancer Multi-Drug Resistance

Prevention of clinical multi-drug resistance should significantly improve therapeutic response in a large number of cancer patients. The initial search for pharmacological modulators of MDR transporters yielded two generations of compounds having poor clinical response profiles. Therefore, there has been a shift to structure-based drug design to synthesize modulator compounds characterized by a high affinity to MDR transporters.15,16 Additionally, research that utilizes siRNA and shRNA-mediated RNAi-based gene silencing methodology has recently delivered promising results.

MDR Modifying Agents

MDR modifying agents, which competitively or non-competitively inhibit the MDR proteins, may increase the cytotoxic action of multi-drug resistant related drugs by preventing the active efflux of these drugs from the target cells. The co-application of an “MDR-modulating” compound in combination with chemotherapy would be expected to significantly improve the cancer cure rate. The first-generation modulators consisted of compounds that were already in clinical use. Calcium channel blockers, quinine derivatives, calmodulin inhibitors, and the immunosuppressive agent cyclosporin A, were all shown to interact with the MDR transporters in vitro and in vivo. These modulators were not specifically developed for MDR protein inhibition, and their inherent low affinity for MDR transporters resulted in a high toxicity profile and were never shown to inhibit P-gp in patients.15-18

Most of the second-generation modulators were derivatives of the first-generation compounds that retained MDR modulatory effects, but with reduced activity toward other physiological targets. Prominent examples of this group are R-verapamil, biricodar (VX- 710), and valspodar (PSC-833). These modulators were shown to inhibit P-gp in patients, but further study revealed significant pharmokinetic interaction with several anticancer drugs, which delayed excretion of the anticancer agent, resulting in toxicity requiring reduction of anticancer drug doses.15-18

The third-generation MDR modulators are designed to interact with specific MDR transporters7, 11-13, 19, 20 with high affinity and with efficacy at nanomolar concentrations. Development of this class of MDR modulators employed combinatorial chemistry to produce potent and selective inhibitors. Examples are the small hydrophobic peptide derivatives named reversins, which were shown to have a strong inhibitory effect on P-gp/MDR1-mediated drug efflux without any toxic effect in the control cells.21 Ongoing clinical trials using third-generation MDR modulators for specific cancer types include: elacridar (GF120918), tariquidar (XR9576), zosuquidar (LY335979), laniquidar (R101933), and ONT-093. Still the shortcomings of earlier generation modulators continue to exist.17, 22 Other approaches to prevent the expression or function of multi-drug transporters are being considered, including the use of MDR protein targeted antibodies, the use of carriers that deliver these drugs selectively to tumor tissues, and the use of RNA interference.

Multi-Drug Resistance Reversal Using RNAi

The stable reversal of MDR protein-mediated drug efflux by RNAi technology has been demonstrated in vitro for MDR1, MXR, MRP2, and MDR3. One of the early multi-drug resistance studies using RNAi technology reported a complete suppression of MDR1 expression on the mRNA and protein level in human gastric carcinoma cells.23 A subsequent study further demonstrated inhibition of both MDR1 and MDR3 expression in conjunction with the reversal of paclitaxel resistance in human ovarian cancer cells. Treatment of ovarian cancer cell lines with either chemically synthesized siRNAs or transfection with specific vectors that express targeted siRNAs resulted in decreased mRNA and protein levels. In this study, MTT assays of siRNA-treated cells demonstrated 7 to 12.4-fold reduction of paclitaxel resistance in the lines treated with the synthesized siRNA of MDR1 and 4.7 to 7.3-fold reduction of paclitaxel resistance in the cell lines transfected with siRNA of MDR1 expressing vectors.24 A more recent study surprisingly showed that the MDR1 phenotype in human hepatoma cells was completely reversed by using two transfected clones.25 Aside from the more frequently studied MDR1 phenotype, reversal of the drug-resistant MXR and MRP2 phenotype using both siRNA and shRNA-mediated approaches was also demonstrated in human carcinoma cells.26, 27

In a pre-clinical study the ablation of MDR1 in cells stably transduced with shRNA was functionally confirmed by increased sensitivity of MDR1-transfected cells toward the cytotoxic drugs vincristine, paclitaxel, and doxorubicin as well as by transport of 99mTc-sestamibi. In the same study, shRNA-mediated down-regulation of MDR activity in tumor implants in living animals was followed by direct noninvasive bioluminescence imaging using the fluorophore coelenterazine, a known MDR1 transport substrate. Additionally, a MDR1-firefly luciferase (MDR1-FLuc) fusion construct was used to document the effect of shRNA delivered in vivo on MDR1-FLuc protein levels with D-luciferin bioluminescence imaging.28 A similar study validated selective MRP2 gene function inhibition: after the intravenous delivery of siRNA effectors into mice, researchers observed a significantly reduced calcein excretion rate and resultant siRNA accumulation in the kidney.29

RNAi is proving to be a powerful laboratory tool for better understanding the multi-drug resistance genotype and phenotype. Its future therapeutic utility in suppressing gene expression in cancer patients will likely be dependent on the availability of effective RNAi delivery systems. Lessons can be learned from the history of gene therapy and antisense technologies. These technologies ultimately failed to produce successful clinical outcomes due to potentially harmful and inefficient delivery systems. The use of RNAi in complex genetic diseases, such as cancer, will not see a quick and straightforward transition from research to clinical success, but with time the promise of viable RNAi therapies may be realized.30 Additionally, innovative technologies combined with new directions in the study of ABC transporters will lead to an understanding of whether or not ABC transporters are important molecular targets for anticancer drug development.

*Technical content provided by: Balázs Sarkadi31, Gergely Szakács31,32 and András Váradi33

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References

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