| Malignant melanoma (MM) is a tumor that responds poorly to chemotherapy. Chemoresistance is a major cause of treatment failure in patients with metastatic disease. The refractory nature of MM suggests the presence of inherent cellular resistance mechanisms. To elucidate a possible mechanism of chemoresistance, the present study was undertaken to establish the protective role of three primary intracellular enzymatic antioxidant defenses (superoxide dismutases [SOD], catalase, and glutathione peroxidase) against chemotherapeutic agents that cause apoptosis by inducing oxidative stress. We generated chemoresistant (CML-10R) MM cell lines by exposing a parental canine melanoma cell line (CML-10P) to sublethal concentrations of carboplatin, cisplatin, doxorubicin, lomustine or gemcitabine over time. Using the MTT assay, a 2 to 13 fold increase in IC50 values to the test anticancer agents was observed in the chemoresistant variants compared to that of the CML-10P cell line. The increase in IC50's values for cisplatin, doxorubicin and gemcitabine chemoresistant variants was statistically significant (P ≤ 0.05) when compared to that of the parental line. Using the Annexin-V binding flow cytometry assay, CML-10R cell lines showed a 1.5 to 3.5 fold increase in apoptotic indices when compared to the parental line after 48 hr and 72 hr exposures to the test chemotherapeutic agents. The cisplatin resistant variant was found to be 3.5 fold less sensitive to cisplatin compared to CML-10P exposed to cisplatin. Enzyme activity assays were employed to determine whether this decreased chemosensitivity was mediated by intracellular antioxidants. Enzyme assays showed that mitochondrial SOD (Mn-SOD), cytosolic SOD (Cu/Zn-SOD), catalase and glutathione peroxidase activities varied among CML-10P, cisplatin treated CML-10 P and cisplatin treated CML-10RCis. Both Mn-SOD and Cu/Zn-SOD levels were higher in CML-10RCis cells than in the CML-10P and cisplatin treated CML-10P. Catalase activity was significantly lower in CML10RCis compared to CML-10 P and cisplatin treated CML-10P. The change in glutathione peroxidase activity was of smaller magnitude when CML-10P, cisplatin treated CML-10P and CML-10RCis were compared.; In summary, MM cells that are exposed to sublethal doses of ROS generating chemotherapeutic agents are able to modulate antioxidant defenses as an adaptive response. By upregulating Mn-SOD without commensurate increase in catalase activities, an imbalance in oxidant processing occurs. This imbalance in the antioxidant defense system results in accumulation of peroxides, which can act as secondary messengers to enhance cell survival pathways. |
