Role Of JNK Signal Transduction Pathway In The Apoptosis Of Hepatocellular Carcinoma Cell Induced By Norcantharidin

Hepatocellular carcinoma (HCC) is one of the malignant tumor in theworld. In the treatment of liver cancer it shows the circumstances like diffi-culty early diagnosis, smaller radical resection of opportunity, the high rate ofrecurrence and metastasis, nonsensitive to the chemotherapy, the overall effi-cacypoor. Norcantharidin (NCTD) is derivatives of removal of the cantharidinchemical structure synthetic1,2-methyltransferase. Mainly used in the treat-ment of esophageal cancer, bile duct cancer, and breast cancer, especially inliver cancer. he experimental study that the NCTD can induce apoptosis inliver cancer cells to anti-tumor effect, but the specific mechanism causedapoptosis especially which signal transduction pathway involved remains to befurther elucidated. The c-Jun N-terminal kinase (JNK) signal transductionpathway and nuclear factor-kappa B (NF-κB) signal transduction pathway inthe mechanism research in the regulation of apoptosis. We have studied the invitro experiments, to explore the effect of NCTD on proliferation and apop-tosis of hepatoma cells and JNK and NF-κB signal transduction system inwhich the role.Objective: The experiment mainly through different concentration ofnorcantharidin intervention in cultured human liver cancer cell SMMC–7721，to investigate its effect on proliferation, apoptosis and the possible mechanismof signal transduction pathways JNK and NF-κB.Methods: To detect the Proliferation effects of NCTD on activatedhepatoma carcinoma cell by MTT test; morphologica changes which stainingwith Hoechst33342were observed under fluorescence microscope; Cell apop-tosis rate were analyzed by flow cytometry. The expression of JNK, p-JNK,NF-κBp65and IκBα were detected by Western blot.Results: 1The effect of norcantharidin on SMMC-7721proliferationThe results of MTT assay cell proliferation showed that, no significantdifference was found between the the group without DMSO and the controlgroup on A492value(P>0.05).0.05%DMSO has no significant effect on pro-liferation of SMMC-7721. Different concentrations norcantharidin (5μg/ml,10μg/ml,20μg/ml,40μg/ml,80μg/ml)on SMMC-7721proliferation at differ-ent time(12h、24h、36h、48h):The results of MTT assay showed that A492value of norcantharidin groups decreased compared with control group whichhad statistically significant differences(P<0.05). And the result showed thatthe inhibion rate on SMMC-7721proliferation had a time-and-dose depedentmanner. After different concentrations norcantharidin interval in HCC cell atdifferent time. the inhibition rate was reached to the peak at48h, and the IC50is40μg/ml.2The effect of norcantharidin on SMMC-7721apoptosis2.1The effect of norcantharidin on SMMC-7721apoptosis at differenttimes.We find that the nuclear enrichment and the formation of apoptotic bodyin SMMC-7721after40μg/ml norcantharidin intervention for12h,24h,36h,48h,staining SMMC-7721cell with Hoechest33342and imaging by fluores-cence microscopy. no significant difference was found between the controlgroup and no DMSO group (P>0.05). norcantharidin groups apoptosis rateincreased compared control group,which had obviously statistically differ-ences(P<0.05).The results suggested that0.05%DMSO has not significanteffect on SMMC-7721apoptosis,and norcantharidin had effect on the rate ofHCC cell apoptosis. After40μg/ml norcantharidin interval12h(11.2%±0.20%),SMMC-7721apoptosis rate was began to increase; At48h(34.13%±1.00%)the apoptosis rate reached the peak.2.2The effect of different concentration noncantharidin on SMMC-7721apoptosis rate by flow cytometry detecting: With the different concentrationtreatment at the same time, the increase in the rate of apoptosis is adose-dependent maner. The overall mean of the apoptotic rates treatment withdifferent concentration norcantharidin have significantly increased, Which have obviously statistically differences compared with the controlgroup(P<0.05), However, compared with40μg/ml NCTD group(43.2%±0.95%), the apoptosis rate decreased in the SP600125+NCTDgroup(28.67%±0.93%),(P<0.05). The resuls suggested that SP600125couldinhibit the apoptosis effection induced by norcantharidin.3Effects of norcantharidin and SP600125on JNK and NF-κB signal transdu-tion pathway of SMMC-7721cells3.1Effects of DMSO on the each pathway of cell:The results of Western blotshowed that, the protein expression of gray value was no significant differencebetween without DMSO group and the control group (P>0.05), no significanteffect of DMSO on JNK and NF-κB two signal transduction pathway inSMMC-7721cells.3.2Effects of norcantharidin and SP600125on JNK signal transdution path-way: The protein expression of gray value of the JNK signaling pathwayshowed that, except DMSO group, the other group and the control group havea significant difference (P <0.05) in the expression of p-JNK. And the expres-sion of p-JNK increased in a dose-dependent maner. However, compared with40μg/ml NCTD group the expression of p-JNK decreased in theSP600125+NCTD group (P<0.05).The content of total JNK had no significantchange. The resuls suggested NCTD increased activation of JNK, andSP600125effectively inhibit the activation.3.3Effects of norcantharidin and SP600125on NF-κB signal transdutionpathway: The protein expression of gray value of the NF-κB signaling path-way showed that, except DMSO group, the other group and the control grouphave a significant difference (P<0.05) in the expression of NF-κBp65andIκBα. And with the concentration of the drug increased the expression ofNF-κBp65and IκBα gradually reduced. However, compared with40μg/mlNCTD group the expression of NF-κBp65and IκBα increased in theSP600125+NCTD group (P<0.05). The resuls suggested NCTD inhibit activa-tion of NF-κB. SP600125, by blocking the phosphorylation of c-Jun, inhibitedJNK phosphorylation, and attenuated apoptosis significantly. JNK inhibition also induced IκBα degradation and NF-κB p65translocation.Conclusions:1The results of vitro cell culture studies show that, norcantharidin caninhibite SMMC-7721cell proliferation.2Norcantharidin could induced the SMMC-7721cell apoptosis. Theproapo-ptotic effect on HCC cell was significantly in a time-anddose-depedent manner.3Norcantharidin could Promote apoptosis of SMMC-7721cells bypromoting the activation of the JNK signaling pathway and inhibition ofNF-κB activity. And JNK pathways have some inhibitory effect on NF-κB,both interactions jointly regulate cellapoptosis.