Understanding the pharmacogenetics of drug resistance in the treatment of prostate cancer

Abstract

INTRODUCTION Drug resistance has been identified as one of the major causes of chemotherapeutic treatment failure in prostate cancer patients. One of the Multidrug resistance mechanisms for cancer resistance, explored in this study, is the overexpression of transport proteins involved in transporting chemo drugs in and out of the cells. The study analyses the expression of the ABC (ATP-binding cassette) transporter genes, ABCC1 and ABCB1 genes and their relative protein products (mrp-1 and p-gp respectively) involved in MDR in prostate cancer, and explores them as targets for combating chemo resistance. ABC transporters are trans-membrane proteins that facilitate the translocation of different substrates across membranes as well as non-transport-related biological functions including RNA translation and DNA repair by using the energy of adenosine triphosphate (ATP) breakdown. Medicinal plants are important sources of therapeutic agents and are good sources of anticancer agents; they can be also used for the restoration of sensitivity in resistant cancer cells and reverse drug resistance in vitro. Ruta graveolens and Malva parviflora are medicinal plants reported to exhibit antioxidant, antibacterial and anti-inflammatory effects but their anticancer activity has not been fully elucidated. The study aimed to assess the anticancer and potential drug resistance reversal potential of these plant species against prostate cancer cell lines, and whether their mechanism of action involves upregulation of ABCC1 and ABCB1 genes and their relative protein products. This is the first study to report the anticancer effects of the DCM extract of R. graveolens and that of DCM+MeOH and MeOH extracts of M. parviflora. METHODOLOGY The DU-145 prostate cancer cell line was cultured according to established procedures. Resistant sublines were established by continuously exposing the DU-145 cell line to increasing concentrations of Doxorubicin and Cisplatin for 6 months. Cell viability and chemosensitivity was assessed by MTT assay. RT-qPCR and western blot were used to determine the mRNA and protein expression levels of the ABCC1 and ABCB1 gene and their relative protein products, mrp-1 and p-gp. Calcein AM assay was used to determine the function of the ABC transporters in the drug resistant cells, DU-145/DOX and DU-145/CDDP cells. R. graveolens and M. parviflora were authenticated and subjected to extraction by different solvents. RESULTS AND DISCUSSION Acquisition of drug resistance by DU145 prostate cancer cell line was confirmed. DU-145/CDDP and DU-145/DOX were 2,156 and 54,23-fold resistant to Cisplatin and Doxorubicin respectively, when compared to the DU1-45/WT cells. mRNA expression levels of ABCC1 and ABCB1 gene were successfully quantified by RT-qPCR, protein products of ABCC1 and ABCB1, mrp-1 and p-gp were below detectable levels when determined by western blot. Function of the ABC transporters in the drug resistant prostate cancer DU-145 cells was successfully determined by the Calcein AM assay, the results obtained were comparable to that of positive controls verapamil and cyclosporine A. R. graveolens DCM extract exhibited >50% growth inhibitory effect at 5μg/ml on the DU-145 wild type cell line. In addition to the anti-proliferative activity against the DU-145 wild type, M. parviflora DCM+MeOH and MeOH extracts exhibited activity against DU-145/DOX at a concentration of 5μg/ml and 10μg/ml respectfully, and the M. parviflora DCM+MeOH exhibited growth inhibitory effect at 10μg/ml for DU-145/CDDP. CONCLUSION This study unequivocally confirms that Ruta graveolens and Malva parviflora have the potential to provide anticancer lead compounds for further development. This is proven by the biological activity observed. Further studies to identify the bioactive compounds in the extracts that exhibited anti-proliferative activity and the mechanism of action are recommended. Moreover, combination studies are necessary to validate the potential drug resistance reversal activity of M. parviflora.