Cancer cells acquire drug resistance via various mechanisms including enhanced cellular cytoprotective and antioxidant activities. Heme oxygenase-1 (HO-1) is a key enzyme exerting potent cytoprotection, cell proliferation and drug resistance. We aimed to investigate roles of HO-1 in human cholangiocarcinoma (CCA) cells for cytoprotection against chemotherapeutic agents. KKU-100 and KKU-M214 CCA cell lines with high and low HO-1 expression levels, respectively, were used to evaluate the sensitivity to chemotherapeutic agents, gemcitabine (Gem) and doxorubicin. Inhibition of HO-1 by zinc protoporphyrin IX (ZnPP) sensitized both cell types to the cytotoxicity of chemotherapeutic agents. HO-1 gene silencing by siRNA validated the cytoprotective effect of HO-1 on CCA cells against Gem. Induction of HO-1 protein expression by stannous chloride enhanced the cytoprotection and suppression of apoptosis caused by anticancer agents. The sensitizing effect of ZnPP was associated with increased ROS formation and loss of mitochondrial transmembrane potential, while Gem alone did not show any effects. A ROS scavenger, Tempol, abolished the sensitizing effect of ZnPP on Gem. Combination of ZnPP and Gem enhanced the release of cytochrome c and increased p21 levels. The results show that HO-1 played a critical role in cytoprotection in CCA cells against chemotherapeutic agents. Targeted inhibition of HO-1 may be a strategy to overcome drug resistance in chemotherapy of bile duct cancer.
Abstract. Simvastatin is a low density lipoprotein-lowering drug that is widely used to prevent and treat cardiovascular disease by inhibiting the mevalonate pathway. Simvastatin also exhibits inhibitory effects on a number of types of cancer. In the present study, the effects of simvastatin on the activity of doxorubicin in the breast cancer MCF-7 cell line, and the mechanisms by which this interaction occurs were investigated. The effect of simvastatin and doxorubicin treatment, alone and in combination, on the growth of MCF-7 cells was evaluated by a sulforhodamine B and colony formation assay. To delineate the mechanisms of cell death, the following parameters were measured: Reactive oxygen species (ROS) production using the fluorescence probe dihydroethidium; caspase 3 activity by the fluorometry method; gene expression by quantitative polymerase chain reaction; and apoptotic-and proliferative-related protein levels by western blotting. MCF-7 cell proliferation was significantly suppressed by 24-48 h treatment with simvastatin alone. Doses of 10-50 µM simvastatin also enhanced the cytotoxicity of doxorubicin against MCF-7 cells in a dose-dependent manner, and decreased the colony-forming ability of MCF-7 cells. Simvastatin alone or in combination with doxorubicin significantly increased ROS levels. Combination treatment significantly decreased expression of the cell cycle regulatory protein Ras-related C3 botulinum toxin substrate 1 and numerous downstream proteins including cyclin-dependent kinase (Cdk) 2, Cdk4 and Cdk6. Additionally, simvastatin in combination with doxorubicin significantly induced expression of the cyclin-dependent kinase inhibitor p21, increased cytochrome c and caspase 3 expression and reduced cyclin D1 expression. In conclusion, simvastatin acts synergistically with the anticancer drug doxorubicin against MCF-7 cells, possibly through a downregulation of the cell cycle or induction of apoptosis. Although additional studies are required, simvastatin and doxorubicin combination may be a reasonable regimen for the treatment of breast cancer.
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