| Primary liver cancer, predominantly consisting of hepatocellularcarcinoma(HCC), is one of the most common and aggressive humanmalignancies worldwide. Prognosis of HCC is very poor, the number ofHCC-related deaths is more than600,000, and the5-year survival rate isbelow9%. Nuclear factor-κappa B(NF-κB) is a class of nucleoprotein withmultidirectional transcriptional regulation factor that exists in multiple cells ofa variety of tissues.It was involved in a variety of biological processes, suchas immune response, inflammation, apoptosis, tumor and so on.Recent studieshave shown that NF-κB and the occurrence, development, resistance, invasionand metastasis of primary liver cancer are related very closely.Norcantharidin(NCTD) is the demethylated analog of cantharidin isolated from natural blisterbeetles.It has the anti-tumor, elevated white blood cell, antiviral and regulatingimmune function, less side effects than cantharidin. NCTD has been used forthe treatment of primary hepatoma, esophageal carcinoma, etc. But the changeof the NF-κB pathway in the process of NCTD induced SMMC-7721cellsapoptosis, and the influence on JNK seldom reported at home and abroad. Inthis study We observed the influence of NCTD on morphology, proliferationand apoptosis of SMMC-7721cells, in addition, the change of the NF-κBpathway in the process of NCTD induced SMMC-7721cells apoptosis and theinfluence on JNK were observed before and after add PDTC. To furtherclarify mechanism of NCTD treatment for liver cancer, and providetheoretical basis for its further development and utilization.Objective: We aim to detect the influence of different concentrationsNCTD on morphology, proliferation and apoptosis of SMMC-7721cells,after PDTC used to restrain the NF-κB pathway, and to detect the change ofthe NF-κB pathway in the process of NCTD induced apoptosis of SMMC-7721cells and the influence on JNK.Methods:1The influence of different concentrations NCTD on proliferation ofSMMC-7721cells was detected by MTT assay.2To study the influence of NCTD on apoptosis of SMMC-7721cellsthrough the Hoechst33342staining.3The influence of NCTD on apoptosis of SMMC-7721cells wasdetected by AnnexinⅤ-FITC/PI method of flow cytometry.4To detect the expression of nuclear NF-κBp65and cytoplastic IκBα,JNK and pJNK protein through Western blotting.Results:1The influence of different concentrations NCTD on proliferation ofSMMC-7721cells was detected by MTT assayAccording to the results determined by MTT method, The A492value ofSMMC-7721cells with different concentrations (5ug/ml,10ug/ml,20ug/ml,40ug/ml,80ug/ml) norcantharidin after12hours,24hours,36hours and48hours decreased, which compared with the control group was statisticallysignificant (P<0.05),12hours (0.599±0.02,0.585±0.01,0.540±0.01,0.505±0.01,0.428±0.01) and control group (0.635±0.01);24hours(1.068±0.08,1.053±0.09,0.961±0.01,0.878±0.11,0.699±0.12) and controlgroup (1.218±0.07);36hours (0.582±0.01,0.533±0.01,0.506±0.02,0.400±0.03,0.283±0.02) and control group (0.720±0.02);48hours(0.699±0.06,0.595±0.02,0.510±0.06,0.435±0.04,0.252±0.03) and controlgroup (0.885±0.07), The results show that norcantharidin has inhibitoryinfluence on proliferation of SMMC-7721and characteristic of the dosedependent.The inhibition rate of SMMC-7721cells with different concentrations(5ug/ml,10ug/ml,20ug/ml,40ug/ml,80ug/ml) norcantharidin after12hours,24hours,36hours and48hours increased, which compared with the controlgroup was statistically significant (P<0.05). In addition significant differencewas found among the different concentrations and time groups (P<0.05),12h (5.63±0.10,7.97±0.14,15.22±0.66,20.56±0.89,32.66±1.51) and controlgroup (0);24h (11.47±0.72,13.86±0.83,21.78±1.26,27.09±0.94,43.30±0.09)and control group (0);36h (19.32±0.94,25.51±1.06,29.58±1.48,44.30±1.35,59.30±1.37) and control group (0);48h (21.28±1.15,33.20±1.30,42.40±1.31,50.91±1.32,70.74±0.83) and control group (0). The results show thatnorcantharidin has inhibitory influence on proliferation of SMMC-7721andcharacteristics of the dose and time dependent (Fig.1,Table2).2The influence of NCTD on apoptosis of SMMC-7721cells through theHoechst33342stainingHoechest33342dyeing results show that the cell size of control group isuniform.The condensed nuclei and apoptotic body was found in othergroups.The apoptosis rate of SMMC-7721cells with40ug/ml norcantharidinafter12hours,24hours,36hours and48hours icreased in turn, whichcompared with the control group was statistically significant (P<0.05). Inaddition significant difference was found among the different time groups(P<0.05),(11.21±0.20%,19.83±0.76%,28.00±1.00%,34.13±1.00%) andcontrol group (4.33±0.15%).The results show that norcantharidin can inducethe apoptosis of SMMC-7721cells and has the characteristic of timedependent (Fig.2, Table3).3The influence of NCTD on apoptosis of SMMC-7721cells wasdetected by AnnexinⅤ-FITC/PI method of flow cytometryThe apoptosis rate of SMMC-7721cells with different groups (5ug/mlNCTD,20ug/ml NCTD,40ug/ml NCTD, PDTC, PDTC+40ug/ml NCTD)after48hours compared with the control group was statistically significant(13.27±0.56%,36.80±0.36%,43.17±0.55%,17.53±0.52%,50.23±1.35%）and control group (5.70±0.20%)(P<0.05). In addition significant differencewas found among the different concentrations groups (P<0.05), The resultshow that apoptosis of SMMC-7721induced by NCTD has the characteristicof the concentration dependent.The apoptosis rate of PDTC group(17.53±0.52%) has significantly difference compared with thecontrol group (5.70±0.20%)(P<0.05), which suggest that the the apoptosis of SMMC-7721cells was induced by the inhibition of NF-κB pathway. Inaddition, the apoptosis rate of PDTC+40ug/ml NCTD group (50.23±1.35%)have significantly difference compared with the40ug/ml NCTDgroup (43.17±0.55%)(P<0.05). The rate of apoptosis increased furthersuggest that NCTD induced apoptosis by inhibiting the activity of NF-κBpathway (Fig3，Table4).4The expression of nuclear NF-κBp65and cytoplastic IκBα, JNK andpJNK protein was detected through Western blottingDifferent groups (5ug/ml、20ug/ml、40ug/ml、PDTC、PDTC+40ug/mlNCTD) acting on SMMC-7721cells after48hours, the expression of nuclearNF-κBp65(1.85±0.01,1.74±0.01,1.42±0.01) and cytoplasmic IκBα(1.34±0.01,1.20±0.02,0.85±0.01) protein with the different concentrationsnorcantharidin (5ug/ml,20ug/ml,40ug/ml) was decreased, while pJNKprotein (1.54±0.01,1.72±0.01,1.82±0.01) was elevated. The significantdifference was found among groups (P<0.05); The overall mean ofnuclear p65and cytoplasmic IκBα protein with the different concentrationsnorcantharidin have significantly difference compared with the controlgroup (2.02±0.01,1.55±0.01,1.36±0.01, P<0.05). But the expression of JNKprotein (1.98±0.01,1.98±0.01,1.98±0.01) with the different concentrationsnorcantharidin has no significant change. No significant difference was foundamong groups (P>0.05); The overall mean of JNK protein with thedifferent concentrations norcantharidin have no significantly differencecompared with the control group (1.98±0.01, P>0.05). The results show thatNCTD has the influence on expression of nuclear p65,cytoplasmic IκBα andpJNK protein of SMMC-7721cells and characteristic of the dose dependent.The inhibition of NF-κB and activation of JNK pathway was enhanced withthe increasing concentration.The expression of nuclear p65(2.02±0.01,1.63±0.01) and cytoplasmicIκBα (1.63±0.01,1.55±0.01) protein with the control group and the PDTCgroup was decreased.The significant difference was found between the twogroups (P <0.05). The results indicate that PDTC can obviously inhibit the NF-κB pathway. The expression of nuclear p65(1.42±0.01,1.06±0.01) andcytoplasmic IκBα (0.85±0.01,0.62±0.01) protein with the PDTC+40ug/mlNCTD group and40ug/ml NCTD group was further decreased, while pJNKprotein (1.82±0.01,2.12±0.01) was elevated. The significant difference wasfound between the two groups (P<0.05); But the expression of JNK protein(1.97±0.01,1.98±0.01) with the PDTC+40ug/ml NCTD group and40ug/mlNCTD group is no significant change. No significant difference was foundbetween the two groups (P>0.05); The results show that the suppression ofNF-κB pathway may promote the activation of JNK pathway.Conclusions:1Cell cultured in vitro studies show that norcantharidin can inhibiteSMMC-7721cells proliferation.2Norcantharidin can induce the apoptosis of SMMC-7721cells.3NCTD can inhibit the NF-κB pathway of SMMC-7721cells andaccelerate the apoptosis of SMMC-7721cells.4The suppression of NF-κB pathway may promote the activation of JNKpathway. |
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