Analysis Of Proteins Interacting With Protein Kinase C-δ In The Apoptosis Induced By Hyperthermia Of Oral Tongue Squamous Cell Carcinoma Tca8113Cells

[Objective]To analysis of proteins interacting with protein kinase C-δ in the apoptosis induced by hyperthermia of oral tongue squamous cell carcinoma Tca8113cells.[Methods](1) Tca8113cells were cultured and induced apoptosis with water bath heating at43℃80min.(2) Thermotolerant Tca8113cells were got with gradient heating technology.(3) Tca8113cells were stained with propidium iodide and followed analysis through flow cytometry. Sub-G0/G1phase was considered as apoptosis peak and the apoptotic rate was measured.(4) The expression and translocation of PKC-8was observed with En Vision immunocytochemistry staining.(5) Activation of PKC-δ was analyzed with Western blot.(6) Those proteins interacting with the protein kinase C-δ were analyzed with co-immunoprecipitation coupled mass spectrometry. The proteins promoting or inhibiting apoptosis in interaction with PKC-δ were screened and put into STRINGD Database(http://string.embl.de/) to retrieve their relationship with the PKC-δ.(7) Statistical analysis of differences between the groups were tested with one-way ANOVA (SPSS11.0). The values were two-sided and statistical significant difference was defined as P<0.05.[Results](1)Thermotolerant Tca8113cells (TT-Tca8113) were got with gradient heating technology. Flow cytometry measurement showed that the apoptosis rate of Tca8113cells was (40.10±6.86)%and TT-Tca8113was (18.03±3.23)%(P<0.05). PKC-δ was found in Tca8113cells and it is mainly distributed in the cytoplasm and perinuclear. The PKC-δ was translocated to nuclear in Tca8113cells but not in TT-Tca8113cells after hyperthermia induction. The Western blot analysis showed that the PKC-δ in Tca8113cells was fragmented but not in TT-Tca8113cells after hyperthermia induction.(2) With co-immunoprecipitation coupled mass spectrometry,39following proteins were screened which may promote apoptosis in interaction with the PKC-δ:TNRC6B、 MINA、HLCS、ZNF12、LGALS8、POLR3E、S100A9、FAM115A、TNR、ACTB、 TTN、ITSN1、TCEB3、KIAA0776、LYZ、THAP2、CTNNA1、102kDa、RECQL5、 PTCHD2、P2RX5、HSD17B10、PMS1、ZNF746、PRRT2、TRAPPC3、CEP290、 ARFGAP1、FLNC、IPPK、TUBB2C、DNAH2、LMNB2、MGC3032、FLJ22374、 C3orf63、WDR42A、KIAA0467、cDNA FLJ59573.32C21orf88. These proteins include cell proliferation-related proteins, apoptosis-related proteins, signal transduction related proteins, cytoskeleton proteins, transcription factors, protease, intracellular transport proteins and some others.16following proteins were screened which may inhibit apoptosis in interaction with the PKC-δ:HSPA9、CDK2、PCMT1、Poly (ADP-ribose)、 Polymerase PARP1、Cytoskeletal calmodulin and Titin OBSCN Ribosomal protein L11、L22and some others. Some of the proteins have been confirmed to be associated with PKC-δ directly or indirectlyby STRING database retrieval analysis. Such as ACTB、LMNB2、or PARP1has direct interaction with PKC-δ. P2RX5、CTNNA1、 HSPA9、ZNF12、BANF1、CDK2、PSMD1、TTN、OBSCN、HBB or ALDOA has indirect interaction with PKC-δ through ACTB、LMNB2or PARP1.[Conclusion]PKC-δ plays an important role in the hyperthermia-induced Tca8113cells apoptosis and its function and regulation involves a variety of other proteins.