The aim of this study was to investigate the molecular effects of paclitaxel and IFN-γ on cultured human keratinocyte cells (HaCaT) assessing the induction of both the apoptotic pathway and cell survival signals. Cellular cytotoxicity assays were performed by MTT dye assay. Caspases 8, 3 and AKT (Ser473 and Thr308 residues) were assessed by Western blot analysis. Morphological characteristics were examined by Wright stain analysis. Paclitaxel reduced keratinocyte growth in a 3-day bioassay with an effective ED50 of 6-600 ng/ml. A large variation in ED50 can be attributed to the asynchronous population of cells. Paclitaxel treatment induced activation of the AKT survival pathway in a time-dependent manner. The down-regulation of AKT signal was preceded by the subsequent activation of caspases 8 and 3 leading to apoptosis. These results indicate that paclitaxel activates both the PI3-K/AKT cell survival pathway followed by induction of apoptotic signals in cultured human keratinocytes. The induction of apoptosis in paclitaxel-treated cells is enhanced by coadministration of IFN-γ. The synergistic effect of these two agents on HaCaT cells relies on a pathway involving caspases 8 and 3, with activity increasing by 48 h. Collectively, our data indicate that i) paclitaxel-induced apoptosis is enhanced by IFN-γ; ii) the down-regulation of PI3-K/AKT survival pathway may help potentiate the apoptotic effects of paclitaxel and iii) the apoptotic signaling pathways are initiated with the activation of caspases 8 and 3 activities.

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