Half of the test wells were then incubated with 25 M P-gp inhibitor 29 diluted into phenol red free RPMI-1640 and the other half of the test wells were supplemented with phenol red free RPMI-1640 containing 0

Half of the test wells were then incubated with 25 M P-gp inhibitor 29 diluted into phenol red free RPMI-1640 and the other half of the test wells were supplemented with phenol red free RPMI-1640 containing 0.5% DMSO (vehicle). and inhibitor resulted in trapping of the chemotherapeutics within the cancerous cells. This trapping led to decreased cell viability, survival, and motility, and increased indicators of apoptosis in the cancerous cells. In contrast, extended exposure of non-Pgp-overexpressing cells to the inhibitor during and after similar chemotherapy treatments did not lead to decreased cell viability and survival, indicating that toxicity of the chemotherapeutic was not increased by the inhibitor. Increases in efficacy in treating MDR cancer cells without increasing toxicity to normal cells by such extended inhibitor treatment might translate to increased clinical efficacy of chemotherapies if suitable inhibitors can be developed. Introduction Chemotherapy treatments are often part of cancer therapies, either before surgery to decrease the size of existing tumors, or after surgery to target metastatic cells that may have migrated out of the primary site of the disease. For cancers that are not surgically accessible, chemotherapy is often the only treatment option. Some of these therapies can be remarkably effective, but unfortunately many cancers recur after initial, Rabbit polyclonal to LRRC48 seemingly successful treatments and still others simply do not respond well to chemotherapies [1]. One common reason for the failure of chemotherapies is the expression of biochemical defense mechanisms in the cancer cells that have evolved to keep normal cells and tissues healthy. The phenomenon of multidrug resistances (MDR) in cancer chemotherapies is one such example, where certain members of the ABC transporter superfamily of membrane proteins [2], when expressed in cancerous cells, actively keep the cells free of the cytotoxic chemotherapeutics [3C8]. When expressed at high levels, proteins like P-glycoprotein (ABCB1, P-gp) [9], the breast cancer resistance protein (ABCG2, BCRP) [10], and/or the multidrug resistance associated protein 1 (ABCC1, MRP-1) [11], have the ability to remove most of the approved cancer chemotherapeutics from the cells, making chemotherapies ineffective. In previous work from our group, we used computational methods to develop structural models of one of these pumps, P-gp,[12, 13] which were used in ultrahigh throughput screening UPGL00004 approaches to identify[14] and characterize [15, 16] drug-like compounds that inhibited P-gp and reversed multidrug resistance in several cancer cells in culture. The compounds were selected to inhibit P-glycoprotein by interfering with the transporters ability to utilize ATP to power drug efflux and to not be transport substrates of the pump. These inhibitors have been shown to resensitize MDR cancer cells in culture and to enhance the killing of MDR UPGL00004 cancer cells in 3-dimensional microtumor spheroids[15, 16]. Most of the inhibitors of P-gp that were assessed previously were transport substrates of the pump [6, 17C19]. The P-gp inhibitors identified in [14] were found to not be transported out of cells by the transporter[16] as was the original premise of the computational search employed[14]. This characteristic is viewed as an important improvement over previous generations of P-gp inhibitors. Active removal of P-gp inhibitors from the cells likely requires overall higher extracellular concentrations for efficacy, causing off-target toxicities once UPGL00004 the compounds are geared towards clinical applications as co-therapeutics to treat chemotherapy insensitive cancers. We show here in a multidrug resistant cancer cell line that over-expresses P-gp, that the continued presence of an inhibitor of P-glycoprotein after a short exposure of the cells to chemotherapeutic in the presence of the inhibitor, and the subsequent removal of the chemotherapeutic from the medium in the presence of the inhibitor, significantly increases the effectiveness of the therapy. We have shown here that this extended P-gp inhibitor treatment correlated with significantly increased cellular retention of chemotherapeutic, reduced cancer cell viabilities, reduced cancer cell migration, and increased morphological indicators of apoptosis and cancer cell mortality, thereby demonstrating the increased efficacy of the treatment. In isogenic cancer cells with low expression of P-gp, no increases in toxicity and associated effects from this “extended P-gp inhibition” were observed, so the observed effects are target (P-glycoprotein) specific. We have explored these effects with one of the P-glycoprotein inhibitors previously identified by us [14C16], but it is likely that these effects will be generalizable and work with other P-gp inhibitors as well. Our results suggest that the UPGL00004 efficacy of chemotherapeutics in killing cancerous cells can be extended beyond the actual treatment with the chemotherapeutic drug.