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

Abstract

The purpose of the present study was to investigate the differential proteins that interact with protein kinase C‑δ (PKC‑δ) 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‑δ 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˚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‑δ were analyzed by western blot analysis. The proteins that interacted with PKC‑δ 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‑δ. 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‑δ cleavage fragmentation was also identified. By contrast, the apoptotic rate of the TT‑Tca8113 cells was not significantly increased following hyperthermia and no PKC‑δ cleavage fragmentation was observed. Among the proteins interacting with PKC‑δ, 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‑δ 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.