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AIM:JNK cascade plays an important role in cell proliferation,differentiation and apoptosis.However,the exact functionof JNK cascade for apoptosis induction remains largelyunknown.In this study,the role of JNK activation stimulatedby TPA in the process of apoptosis induction and its signalingtransduction pathway in gastric cancer cells were investigatedand determined.METHODS:Expressions of mRNA and protein weredetected by Northern blot and Western blot.Transcriptionactivity was measured by transient transfection and CATassay.Apoptotic cells were displayed through staining thenucleus with DAPI and were observed under fluorescencemicroscope.The apoptotic index was determined bycounting 1000 cells randomly.RESULTS:JNK protein was stimulated rapidly by TPA,andreached its highest peak within 3 hr,then decreased in atime-dependent manner,but the expression level of JNKprotein induced by TPA was always keeping higher thanthat in untreated cells.Similar pattern was seen in c-junmRNA level induced by TPA.TPA significantly activated thetranscriptional activity of activator protein-1 with a TPA-dose-dependent manner.Furthermore,activation of JNK wasmediated through PKC pathway.Treatment of cells with PKCspecific inhibitor,Wortmannin,led to repression of JNK evenin the presence of TPA.More importantly,all these effectswere associated with induction of apoptosis in gastric cancercells.TPA inducted apoptosis obviously in gastric cancer cells.The apoptotic cells became smaller and rounded,and theirnuclei became condensation and fragmentation with brightlystained chromatin.However,suppression of JNK by PKC specificinhibitor,Wortmannin,resulted in the decrease of apoptosisinduced by TPA in a time-dependent manner,apoptotic indexdramatically decreased from 32.56 % to 8.71%.CONCLUSION:TPA stimulates JNK cascade,including up-regulation of JNK protein expression level and c-jun mRNAexpression level,and activation of activator protein-1 transcriptional activity.Activation of JNK is mediated throughPKC pathway,which has an association with induction ofapoptosis by TPA.Thus,activation of JNK via PKC pathwaymay represent one of important mechanisms for TPA toinduce apoptosis in gastric cancer cells.
AIM: JNK cascade plays an important role in cell proliferation, differentiation and apoptosis. However, the exact function of JNK cascade for apoptosis induction remains largelyunknown. In this study, the role of JNK activation stimulated by TPA in the process of apoptosis induction and its signalingtransduction pathway in gastric cancer cells were investigated and determined. METHODS: Expressions of mRNA and protein were detected by Northern blot and Western blot. Transcription was was used by transient transfection and CATassay. Poptotic cells were displayed by staining thenucleus with DAPI and were observed under fluorescence microscope. The apoptotic index was determined bycounting 1000 cells randomly.RESULTS: JNK protein was stimulated rapidly by TPA, andreached its highest peak within 3 hr, then decreased in a time-dependent manner, but the expression level of JNKprotein induced by TPA was always keeping higher thanthat in untreated cells .Similar pattern was seen in c-jun mRNA level indu ced by TPA.TPA significantly activated the transcription activity of activator protein-1 with a TPA-dose-dependent manner. Durthermore, activation of JNK wasmediated through PKC pathway. Treatment of cells with PKCspecific inhibitor, Wortmannin, led to repression of JNK even in the presence of TPA. Importantly, all these effects associated with induction of apoptosis in gastric cancer cells. TPA inducted apoptosis obviously in gastric cancer cells. apoptotic cells became smaller and rounded, and their nuclei have been condensed and fragmentation with brightly stained chromatin. Nearly, suppression of JNK by PKC specific inhibitor, Wortmannin, resulted in the decrease of apoptosis induced by TPA in a time-dependent manner, apoptotic index decreased from 32.56% to 8.71% .CONCLUSION: TPA stimulates JNK cascade, including up-regulation of JNK protein expression level and c- jun mRNAexpression level, and activation of activator protein-1 transcriptional activity. Activation of JNK is mediated through PKC pathway, which has an association with induction ofapoptosis by TPA.Thus, activation of PKN pathway in JNK one of the important mechanisms for TPA to apoptosis in gastric cancer cells.