The integrated effects of selected inducers of endoplasmic reticulum stress, the unfolded protein response and apoptosis on P-Glycoprotein mediated drug resistance in MCF-7 breast carcinoma cells
Purpose: One of the leading causes of death reported in women worldwide is breast cancer. Manytumours, including breast cancer, associated with poor prognosis, have received a renewed focus and increased perspective with regard to drug discovery and innovation towards developing rational combination regimens of first-line anticancer drugs with novel compounds that target diverse hallmarks of the cancer phenotype. Multidrug resistance (MDR), which has been found to significantly decrease the efficacy of anticancer drugs and causes tumor recurrence, has been a major challenge in clinical cancer treatment with chemotherapeutic drugs for decades. Several mechanisms of overcoming drug resistance have been postulated and the well known P-glycoprotein (P-gp) including other drug efflux transporters are considered to be critical in pumping anticancer drugs out of cells which in turn results in unsuccessful chemotherapy treatments. The endoplasmic reticulum (ER) is an interconnecting organelle which synthesizes proteins and its quality control processes ensures the proper protein folding, post-translational modifications and conformation of secretory and trans-membrane proteins. Previous studies demonstrated that geldanamycin (GA), a benzoquinone ansamycin antibiotic, the antibiotic, tunicamycin (TM) and the sesquiterpene lactone, thapsigargin (TG) have been found to cause ER stress and consequently, cellular arrest. GA is known to manifest anti-cancer activity through the inhibition of Hsp90-chaperone, TM interferes with N-glycosylation of newly synthesized proteins triggering the unfolded protein response, while TG inhibits intracellular Ca2+ ATPases resulting in increased cytosolic Ca2+. Cellular stress conditions, lead to accumulation of unfolded or misfolded proteins in the endoplasmic reticulum lumen which results in a unfolded protein response (UPR) to maintain cell survival in cancer cells. ERS has been previously reported to enhance MDR1 transcriptional induction and P-gp transport function in cancer cells, however, prolonged endoplasmic reticulum stress conditions and inadequate unfolded protein response force cells undergo apoptosis. In this study, we examined the effects of GA, TG and TM alone and in combination to determine the cellular response of the MCF-7 breast carcinoma cell line with regard to proliferation and P-gp-mediated drug efflux activity and apoptosis. Methods: Analyses of MCF-7 breast carcinoma cells exposed to Endoplasmic Reticulum Stress (ERS) inducers geldanamycin, thapsigargin and tunicamycin, alone and in combination, included growth curves alone and in the presence of 24 hour IC50 inhibitory concentrations of the 3 ERS inducers alone, dose-response curves (MTT cytotoxicity assays) of the ERS alone and in combination, analysis of P-glycoprotein-mediated efflux pump activity in the presence of the ERS inducers alone and in combination (Calcein-AM efflux assays), analysis of viability, cytotoxicity and early apoptosis via caspase-3/7 expression (Triplex assay) and morphological staining of apoptotic and/or necrotic cells in the presence of IC50 inhibitory concentrations of the ERS inducers alone with Annexin V-FITC. Results: This study investigated the effects of Endoplasmic Reticulum Stress (ERS) inducers on growth and proliferation of MCF-7 breast carcinoma cells in culture. The MCF-7 cell line was exposed to different concentrations of ERS inducers alone and in combination with each other. All responses occurred in a dose- and time- dependent manner. When combined at equimolar log dose concentrations, integrated effects yielded enhanced cytotoxic properties as IC50 values were drastically decreased in combination as opposed to single ERS inducer responses. Combined effect on P-glycoprotein-mediated drug efflux activity yielded minor but insignificant decreases in efflux pump activity at different time intervals as opposed to the increase in cellular efflux in the presence of the ERS inducers alone at different time intervals. Caspase-3/7 apoptotic protein expression was increased as log doses of ERS inducers alone were increased, leading to cell necrosis at higher cytotoxic concentrations. The determined IC50 growth inhibitory concentrations after 24 hours were confirmed by the Annexin V-FITC demonstrating early apoptotic, necrotic and viable cells in the presence of the ERS inducers alone. Conclusion: This study demonstrated a significant growth inhibition of MCF-7 breast carcinoma cells upon exposure to ERS inducers alone. Results suggested that when ERS inducers are used in combination, their efficacy is enhanced as 50 percent inhibitory concentrations were considerably lower in combination as opposed to when used alone. The present study is consistent with previous studies with geldanamycin, and was the 1st to investigate the effects of geldanamycin, thapsigargin and tunicamycin in combination and with reference to P-gp efflux activity. Results suggested that in combination, efflux activity may be reduced, and efficacy may be enhanced. To enhance efficacy would be a major breakthrough in cancer drug discovery and development-targeting specific populations of cancer cells and reducing ERS-induced toxicity to normal cells and vital organs.