Expressions Of Protein Kinase C-δ In Human Tongue Squamous Cell Carcinomas And The Role Of PKC-δ In Heat-induced Apoptosis In Tca8113 Cells

Objective To study the expression character of protein kinase C-δ(PKC-δ) in human tongue squamous cell carcinoma and the role of PKC-δin heat-induced apoptosis in human tongue squamous cell carcinoma Tca8113 cells.Methods (1) Immunohistochemistry(ChemMate^{T M} En Vision) was used to examine expressions of PKC-δin 42 cases of human tongue squamous cell carcinoma tissue and 15 cases of human normal tongue tissue. Stained cells number and total epithelium number in every five high power field which was randomly observed were calculated. The ratio of stained cells number to total epithelium number >10% was considered positive stain, and <10% was considered negative stain. (2) Tca8113 cells were cultured conventionally. The cells were pretreated with rottlerin, a PKC-δspecific inhibitor and DMSO for 30min, and treated at temperature of 43 degree Celsius in a heating waterbath for Omin, 40min, 80min, 120min. Then the cells were incubated for 4 hours, 16 hours and 24 hours. The morphologic change of the cells were observed under inverted microscope, the cells were harvested to analysis apoptosis rate by FCM with propidium iodide (PI) stain , the cells were stained with Rhodamine123 to analysis mitochondrial transmembrane potential(â–³(?)m)intensity by FCM, the total proteins were extracted for immunoblotting assay of activation and cleavage of PKC-δand colorimetric assay of relative activity of caspase-3(A₄₀₅), and immunocytochemistry was used to locate PKC-δin Tca8113cells.Result (1) In the epithelium of normal tongue mucosa, expressions of PKC-δwere all positive, In the epithelium of tongue squamous cell carcinoma, expressions of PKC-δin 28 cases were negative and in 14 cases were positive. Positive expression rate of PKC-δin the epithelium of normal tongue mucosa compared with that in the epithelium of tongue squamous cell carcinoma, there was significantally statistical differences(P<0.01). There was not correlation between expressions of PKC-δand histopathological grade in tongue squamous cell carcinoma (P>0.05). (2) The expression of PKC-δwas found in cytoplasm and perinucleus in Tca8113 cells. Heat induced PKC-δactivation and formation of functional catalytic fragment, but rottlerin play an inhibitory role. PKC-δtranslocation to intra-nuclear was detected by immunocytochemistry after heating. The relative activity of caspase-3 and apoptotic ratio and percentage of hypofluorescence cells were increased, but the mitochondrial transmembrane potential (â–³(?)m) intensity decreased with prolongation of heating. The value of A₄₀₅ which present the relative activity of caspase-3 in the rottlerinpretreated cells treated for Omin, 40min, 80min, 120min was (0.08±0.01), (0.28±0.03), (0.52±0.07), (0.79±0.04) ((?)±s) respectively, in the DMSO- pretreated cells was (0.09±0.02), (0.39±0.04), (0.64±0.02), (0.99±0.10) ((?)±s) respectively. The hypofluorescence cells' percentage in the Rottlerin- pretreated cells treated for Omin, 40min, 80min, 120min was (4.43±2.22)%, (15.87±0.55)%, (31.3±1.83)%, (42.56±2.67) % ((?)±s) % respectively, in the DMSO-pretreated cells was (4.67±2.14)%,(23.43±2.85)%,(39.9±3.97)%,(53.6±2.62)% ((?)±s) % respectively. The apoptosis rate in rottlerin-pretreated cells treated for Omin, 40min, 80min, 120min was (4.76±0.88)%,(12.3±1.91)%,(31.5±2.61)%,(36.53±0.65)% ((?)±s) % respectively, in the DMSO-pretreated cells was (4.90±0.72%),(22.1±1.35)%,(43.2±1.57)%,(49.9±1.25)% ((?)±s) % respectively. There was no statistical difference in the relative activity of caspase-3, apoptosis rate and percentage of hypofluorescence between the rottlerin-pretreated group and the DMSO-pretreated group without heating (Omin) (P>0.05), but there were significantally statistical differences after the cells were heated for 40min, 80min, 120min (P<0.01).Conclusion PKC-δis lost or poor expression in human tongue squamous cell carcinoma tissues. Heat can induce apoptosis through activation and cleavage of PKC-δin Tca8113 cells. PKC-δtranslocates to intra-nucleus in heat-induced Tca8113 cells apoptosis.