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BACKGROUND:
Epidemiologic studies suggested potential of Metformin, a widely used hypoglycemiant drug for type 2 diabetes mellitus, to reduce cancer-related mortality in several types of cancer including prostate cancer. Although activity of Metformin against various cancer cells in vitro was shown, the drug concentrations in most previous studies substantially exceeded clinically useful. We evaluated activity of Metformin in a therapeutically relevant micromolar range of up to 50uM against androgen independent prostate cancer cell lines in vitro. The goal of the study was to determine if Metformin alone or in combination with docetaxel, a first-line chemotherapeutic for prostate cancer, might be beneficial to treat advanced prostate cancers.
METHODS:
Prostate cancer cell lines PC3 and DU145 were used in this study. We also studied docetaxel-resistant derivatives of these cell lines PC3-8B2 and DU145-T2. Previous studies identified substantial levels of docetaxel-resistance in these sublines as well as genes mediating resistance to docetaxel. All cell lines were maintained in standard RPMI1640 media (10mM Glucose) supplemented with 5% fetal bovine serum, sodium pyruvate and penicillin/streptomycin. Sextuplicate cultures per each drug concentration were tested in colorimetric cell viability and chemosensitivity assays. MTT assay is based on tetrazolium conversion to formazan by mitochondria and Sulphorhodamine B stain (SRB) assay measures total cellular protein and directly corresponds to cell numbers. The results were evaluated after 96-hour drug treatment. Data curve fitting and statistical analyses were accomplished using XLfit software. Significance of findings was restricted by 95% confidence interval. To determine synergistic and/or antagonistic activity of Metformin with docetaxel, Metformin was co-administered concurrently with docetaxel. To improve the assay read-out variance, Metformin was maintained at either 25 or 50uM steady concentration, whereas docetaxel was serially diluted in an appropriate concentration range. All experiments were repeated at least 3 times.
RESULTS:
Both SRB and MTT assays detected concentration dependent reduction in cellular viability and metabolic activity in Metformin treated cultures at tested drug concentrations (p-value=<0.05). At maximum 50uM concentration, Metformin reduced the total protein and mitochondrial activity in both PC3 and PC3-8B2 cells by 8%, whereas DU145 cells showed 10% decline in viability and 29% in mitochondrial activity. No changes were identified in viability of DU145-T2 cells by SRB test, but mitochondrial activity was reduced by 35%. Co-administration of 25 uM Metformin with docetaxel resulted in reduction of IC50 values for docetaxel by 21% and 35% in PC3 and PC3-8B2 cells respectively. Increasing Metformin concentration to 50uM provided no additional benefit. Apparent Metformin effect on both DU145 and DU145-T2 cells did not translate into appreciable changes in IC50 values for docetaxel in either cell line.
CONCLUSIONS:
Biologic effect of Metformin on prostate cancer cells was concentration-dependent and produced detectable toxicity at above 10uM levels in all cell lines tested. Co-administration of Metformin with docetaxel revealed their synergistic activity and improved sensitivity to docetaxel in PC3 cells. Metformin completely overturned resistance to docetaxel in PC3-8B2 cells. Our findings provide further support to clinical evaluation of Metformin as potentially useful adjuvant in docetaxel chemotherapy of androgen independent prostate cancers.