Activation of JNK by TPA promotes apoptosis via PKC pathway in gastric cancer cells.
OBJECTIVE
JNK cascade plays an important role in cell proliferation, differentiation and apoptosis. However, the exact function of JNK cascade for apoptosis induction remains largely unknown. In this study, the role of JNK activation stimulated by TPA in the process of apoptosis induction and its signaling transduction pathway in gastric cancer cells were investigated and determined.


METHODS
Expressions of mRNA and protein were detected by Northern blot and Western blot. Transcription activity was measured by transient transfection and CAT assay. Apoptotic cells were displayed through staining the nucleus with DAPI and were observed under fluorescence microscope. The apoptotic index was determined by counting 1000 cells randomly.


RESULTS
JNK protein was stimulated rapidly by TPA, and reached its highest peak within 3 hr, then decreased in a time-dependent manner, but the expression level of JNK protein induced by TPA was always keeping higher than that in untreated cells. Similar pattern was seen in c-jun mRNA level induced by TPA. TPA significantly activated the transcriptional activity of activator protein-1 with a TPA-dose-dependent manner. Furthermore, activation of JNK was mediated through PKC pathway. Treatment of cells with PKC specific inhibitor, Wortmannin, led to repression of JNK even in the presence of TPA. More importantly, all these effects were associated with induction of apoptosis in gastric cancer cells. TPA inducted apoptosis obviously in gastric cancer cells. The apoptotic cells became smaller and rounded, and their nuclei became condensation and fragmentation with brightly stained chromatin. However, suppression of JNK by PKC specific inhibitor, Wortmannin, resulted in the decrease of apoptosis induced by TPA in a time-dependent manner, apoptotic index dramatically decreased from 32.56 % to 8.71 %.


CONCLUSIONS
TPA stimulates JNK cascade, including up-regulation of JNK protein expression level and c-jun mRNA expression level, and activation of activator protein-1 transcriptional activity. Activation of JNK is mediated through PKC pathway, which has an association with induction of apoptosis by TPA. Thus, activation of JNK via PKC pathway may represent one of important mechanisms for TPA to induce apoptosis in gastric cancer cells.
Chen
Yan
Y
Key Laboratory of the Ministry of Education for Cell Biology and Tumor Cell Engineering, The School of Life Sciences, Xiamen University, Fujian Province, 361005, China.
Wu
Qiao
Q
Song
Si-Yang
SY
Su
Wen-Jin
WJ
D017209:Apoptosis / Q000187:drug effects*; D004789:Enzyme Activation / Q000187:drug effects; D016758:Genes, jun / Q000187:drug effects; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012334:RNA, Neoplasm / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D013274:Stomach Neoplasms / Q000201:enzymology* / Q000235:genetics / Q000473:pathology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014407:Tumor Cells, Cultured
D012333:RNA, Messenger; D012334:RNA, Neoplasm; D018808:Transcription Factor AP-1; D011493:Protein Kinase C; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D013755:Tetradecanoylphorbol Acetate