Schedule-Dependent Cytotoxic Interaction between Epidoxorubicin and Gemcitabine in Human Bladder Cancer Cells in Vitro

Purpose: The aim of the study was to evaluate the activity of epidoxorubicin (EPI) and gemcitabine (GEM) and to define the most effective schedule in human bladder cancer cells. Experimental Design: The study was performed on HT1376 and MCR cell lines. Cells were exposed for 1 and 24 h to drugs used in different schemes. Cytotoxic activity was evaluated by the sulforhodamine B assay, potential clinical activity was estimated by relative antitumor activity, and the type of drug interaction was assessed using the method of Chou and Talalay. Cell cycle perturbations and apoptosis were assessed by flow cytometry; BAX, BCL-2, and P53 expression was evaluated by Western blot; and DNA damage was assessed using the alkaline Comet assay. Results: EPI and GEM produced a cytotoxic effect in both cell lines, with 50% inhibitory concentration and relative antitumor activity values suggestive of a high clinical activity. Simultaneous treatment with EPI and GEM and the sequence GEM→EPI caused an antagonistic interaction (combination index > 1) after both 1- and 24-h treatments. Conversely, the inverse sequence, EPI→GEM, produced a synergistic interaction that was more pronounced in MCR cells than in HT1376 cells. The increase in DNA-damaged cells from 10% to 20% after single-drug exposure to 40–60% at the end of EPI→GEM treatment may explain the synergistic interaction produced by the anthracycline-antimetabolite sequence. Conclusions: Our findings show that the efficacy of the EPI and GEM combination is highly schedule dependent and indicate that the most active scheme is EPI followed by GEM, which is currently being validated in an ongoing intravesical Phase I-II clinical protocol.