MRP2 as a Targetable Oxaliplatin Resistance Factor in Gastrointestinal Cancer
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Despite the severe adverse effects, toxicity and limited efficacy due to the development of multidrug resistance (MDR), chemotherapy is still the treatment of choice for most of the cancer patients. One of the formidable MDR mechanisms is the up-regulation of various efflux pumps, known as the ATP binding cassette (ABC) transporters, which can efficiently remove drugs from the cell, thus causing the decreased efficacy of chemotherapeutic drugs. The platinum-based regimens are important in the clinical treatment of gastrointestinal cancer. While many membrane transporters are reported to play important roles in platinum-based chemoresistance, a recent study showed that multidrug resistance-associated protein 2 (MRP2) mediated the ATP-dependent active transport of oxaliplatin-derived platinum in membrane vesicle models. This study suggested that oxaliplatin is a substrate for MRP2. However, the mechanistic mechanisms of MRP2-oxaliplatin interactions and the contribution of MRP2 to oxaliplatin resistance remain unclear. The purpose of this thesis is to investigate oxaliplatin interaction with MRP2 transporter using a colourimetric ATPase assay and assess whether MRP2 confers oxaliplatin resistance in MRP2 overexpressing gastrointestinal cancer cells, particularly colorectal cancer cells (Caco-2), hepatocellular cancer cells (HepG2) and pancreatic cancer cells (PANC-1). Human MRP2-expressing membrane vesicles prepared from Sf9 insect cells were used for ATPase studies (Chapter 3). The amount of inorganic phosphate released from substrate-stimulated ATP hydrolysis was measured by a colourimetric assay. Oxaliplatin stimulated vanadate-sensitive Sf9/MRP2 ATPase activity appeared linear within 30 min. Concentration-dependent effects of oxaliplatin on Sf9/MRP2 ATPase activity were determined at 20 min and the data was best fit with a sigmoidal dose-response model to generate an EC50 value of 8.3 ± 0.7 µM and a Hill coefficient of 2. Oxaliplatin-stimulated Sf9/MRP2 ATPase activity was significantly inhibited by well-defined MRP2 inhibitors benzbromarone and myricetin. Oxaliplatin does not interact with wild-type ABC transporters in Sf9 cells. Taken together, our results suggest oxaliplatin is a human MRP2 substrate possibly with two binding sites on MRP2. In above mentioned human gastrointestinal cell lines, silencing the ABCC2 gene led to increased cellular accumulation of oxaliplatin-derived platinum and enhanced anticancer activity of oxaliplatin (Chapter 4 and 5). In HepG2, Caco-2 and PANC-1 cells, after siRNA transient knockdown of ABCC2 gene, the ABCC2 mRNA level and cell surface MRP2 expression were significantly decreased. The sensitivity to oxaliplatin-induced growth inhibition were enhanced in ABCC2-siRNAs transfected HepG2, Caco-2 and PANC-1 cells by up to two-fold compared with control-siRNA transfected cells. To explore whether the marked suppression of tumour proliferation was attributed to the inhibition of MRP2-mediated platinum efflux, the mechanisms underlying the enhanced efficacy were explored. Silencing ABCC2 gene resulted in about 2-fold increase in the oxaliplatin-derived platinum accumulation in HepG2 and Caco-2 cells. The apoptosis assays revealed that modulating MRP2 transporter either by siRNA knockdown of ABCC2 gene or by an MRP2 inhibitor myricetin, significantly enhanced the oxaliplatin-induced apoptosis rate in gastrointestinal cancer cells (Chapter 6). In conclusion, oxaliplatin was confirmed as a substrate of MRP2 transporter. Furthermore, modulation of MRP2 in human gastrointestinal cancer cells using either siRNA-mediated transient gene knockdown or an MRP2 inhibitor myricetin increased the sensitivity of oxaliplatin and cellular accumulation of oxaliplatin-derived platinum. Screening tumour MRP2 expression levels to select patients for treatment with oxaliplatin alone or in combination with MRP2 modulation, could improve outcomes of gastrointestinal cancer treatment.