Identification of proteins interacting with protein kinase C-δ in hyperthermia-induced apoptosis and thermotolerance of Tca8113 cells

The purpose of the present study was to investigate the differential proteins that interact with protein kinase C-delta (PKC-delta) in hyperthermia-induced apoptosis as well as thermotolerance in Tca8113 cells, and furthermore, to investigate the mechanisms of these processes in tumor cells. Activation of PKC-d was previously indicated to be involved in the heat sensitivity and thermal resistance of tongue squamous carcinoma cells. Tca8113 cell apoptosis was induced by incubation at 43 degrees C for 80 min and the thermotolerant Tca8113 cells (TT-Tca8113) were generated through a gradient temperature-elevating method. The apoptotic rate of the cells was determined by flow cytometry, while cleavage and activation of PKC-delta were analyzed by western blot analysis. The proteins that interacted with PKC-delta in the Tca8113 and TT-Tca8113 cells were identified by co-immunoprecipitation coupled with mass spectrometry. Co-immunoprecipitation analysis followed by electrospray ionization mass spectrometric analysis were utilized to identify the pro-and anti-apoptotic proteins that interacted with PKC-delta. Significant cell apoptosis was observed in Tca8113 cells following hyperthermia, and the apoptotic rate was significantly higher than that in the control group (P<0.05). Marked PKC-d cleavage fragmentation was also identified. By contrast, the apoptotic rate of the TT-Tca8113 cells was not significantly increased following hyperthermia and no PKC-d cleavage fragmentation was observed. Among the proteins interacting with PKC-delta, 39 were found to be involved in the promotion of apoptosis and 16 in the inhibition of apoptosis of Tca8113 cells; these proteins were known to be involved in the regulation of cell proliferation, apoptosis, transcription and intracellular protein transport. The results of the present study provided evidence that PKC-delta is a crucial factor in the heat sensitivity and thermal resistance of tongue squamous carcinoma cells and elucidated the underlying molecular basis, which may aid in the improvement of hyperthermic cancer treatments.