The Study Of Effects And Mechanisms Of TNF-α Pathway And Beyond During Apoptosis Of H22 Cells

Purpose: To investigate the effects and mechanism of tumor necrosis factor a' (TNF- α ) signaling and beyond (protein kinase C- α , PKC-α ) during the apoptosis of mouse liver cancer (H22) cells induced by TNF-α.Method: Apoptotic rates and the expression of PKC- α, phosphorylation-PKC- α (p- PKC- α ), c-Jun N-terminal kinase (JNK), p-JNK, p-P38, caspase-8 of H22 cells were separately detected by flow cytometer (FCM) and westernbot in different time after H22 cells were treated with different concentration of TNF- α . When inhibiting activity of JNK with sp600125, P38 with SB203580, PKC-α with Go6976 or activating PKC- α with 12-Otetradecanoylphorbol-13-acetate (TPA) during the H22 cells were treated with TNF-a, the apoptotic rates and the related protein of H22 cells were detected; the expression of p-JNK was detected with immunofluorescence microscope; the expression and translocation of p-PKC- α were detected by confocal microscope; the activity of nuclear factor κB (NF-κB) combined with the DNA probe was analyzed by electrophoretic mobility shift assay (EMSA). The interactions of PKC- α , JNK, p-P38, caspase-8 and the relationships between these protein and apoptosis of H22 cells induced by TNF- α were analyzed. Necrotic cells of ascite liver cancer cells and life span were observed when mice with ascite liver cancer cells were treated with TNF-α and Go6976 or TNF-α only.Result: The apoptotic rates and the expression of PKC- α , p-PKC- α , JNK, p-JNK, p-P38, caspase-8 of H22 cells augmented with increasing concentration of TNF-a and prolongation of time. The expression of PKC- α , p-PKC-α up-regulated; JNK, p-JNK, p-P38, caspase-8 down-regulated; activity of NF-κB combined with DNA probe increased and the apoptotic rates of H22 cells dropped when activity of JNK was inhibited with sp600125 or P38 was inhibited with SB203580. When activity of PKC- α was inhibited with Go6976, the expression of p-JNK, caspase-8 up-regulated; activity of NF-kB combined with DNA probe attenuated and H22 cellswere sensitized to TNF- α . On the contrary, PKC- α was activated with TPA, the expression of p-JNK, caspase-8 down-regulated; the expression of p-PKC-α enhanced and distributed in cytoplasma and nuclear; the activity of NF-kB combined with DNA probe increased significantly. Compared with being treated with TNF- α only, the necrotic tumor cells in mice ascites increased and life span was prolonged significantly in mice with ascites liver cancer cells treated with TNF-α and Go6976. Conclusion: The expression of JNK, p-JNK, PKC-α, p-PKC-α, p-P38, caspase-8 and the activity of NF-kB combined with DNA probe increase when the H22 cells were treated with TNF-α . Activation of JNK and P38 are pro-apoptotic but the activation of PKC- α and NF-kB are anti-apoptotic when signal transduction mediated by TNF-α. Anti-apoptotic effect of PKC-α is carried out through increasing the activity of NF-kB. Inhibition of the activation of PKC-a with Go6976 can significantly enhance the toxicity of TNF- α to H22 cells. The life span of Mice with ascite liver cancer cell can be prolonged significantly when treated with TNF-α combined with Go6976.