The Experimental Research Of Recombinant Human TRAIL In Treating Gallbladder Carcinoma

Part OneResearch of the proapoptotic effect of recombinant human TRAIL on humangallbladder carcinoma cell line SGC-996 and GBC-SD【Abstract】Objective To detect the proapoptotic effect of recombinant humanTRAIL(rhTRAIL) on human gallbladder carcinoma cell line SGC-996 and GBC-SD.Methods The half maximal inhibitory concentration (IC50) was calculated throughgrowth curve. Apoptosis of gallbladder carcinoma cells were detected in three ways:(1) Phase microscopy of the cells. (2) Detection of activity of effector caspase-3 andcaspase-7.(3) Proportion of apoptotic cells was labeled by Annexin V-PI,flowcytometric analysis was performed using CELLQUEST software. Results InrhTRAIL treated group,cell growth of SGC-996 and GBC-SD was inhibited.Shrinkage and significantly elevated pyknosis in a subset of cells under phasemicroscopy in treated group was observed.Caspase-3/7 activation and theproportion of apoptotic cells in samples treated by rhTRAIL were also markedlyincreased. IC50 of rhTRAIL on gallbladder cell lines SGC-996 and GBC-SD was245.8 ng/ml and 270.2 ng/ml,which was higher than that in treating other tumor celllines. Conclusions rhTRAIL could inhibit the growth of gallbladder carcinoma celllines in mode of dose-dependent. The growth inhibition was caused by rhTRAILinduced-apoptosis.Part TwoThe expression of c-FLIP in gallbladder carcinoma tissues and the effect ofc-FLIP on rhTRAIL induced apoptosis in gallbladder carcinoma.【Abstract】Objective To detect the expression of c-FLIP either in gallbladdercarcinoma tissues or in the cell lines,then to explore the effect of c-FLIP on rhTRAILinduced apoptosis in gallbladder carcinoma. Methods To determine the expressionlevel of c-FLIP, 35 carcinoma,10 normal and 10 adenomatous gallbladder tissueswere enrolled. Immunohistochemistry was performed using the standard streptavidin-biotin-peroxidase complex technique. Semi-quantitation was performed bydetermining the percentage of positive cells.Small interference RNA(SiRNA) of c-FLIP was synthesized to down-regulate the expression of c-FLIP. Apoptosis of thecombined treatment with c-FLIP siRNA and rhTRAIL were detected. Results Outof the 35 gallbladder carcinoma tissues, positive c-FLIP expression was found in 26samples (6/positive+++, 13/++,7/+), whereas negative or weak c-FLIP staining wasexamined in normal (1/+, 9/-) and adenomatous (2/+, 8/-) gallbladder tissues. SiRNAof c-FLIP can substantially down-regulate the expression levels of c-FLIP. Thecombined treatment with c-FLIP siRNA and rhTRAIL significantly inducedapoptosis in gallbladder carcinoma cells compared to other groups. ConclusionsExpression of c-FLIP in gallbladder carcinoma tissues was significantly higherthan that in normal gallbladder and gallbladder adenoma tissues. SiRNA of c-FLIPcould effectively down-regulate the expression of c-FLIP in gallbladder carcinomacell lines. Down-regulation of c-FLIP could sensitize rhTRAIL-induced apoptosis ingallbladder carcinoma cell lines.Part ThreeThe effect of combined treatment with rhTRAIL and Celecoxib on gallbladdercarcinoma and the possible causes of the effect【Abstract】Objective Observe the effect of combined treatment with rhTRAILand selective Cox-2 inhibitor Celecoxib on gallbladder carcinoma in vitro and invivo and also to analyze the possible causes of the effect. Methods Western-blotanalysis was used to detect the expression of c-FLIP and death receptors after beingtreated by Celecoxib. Apoptosis of the combined treatment with Celecoxib andrhTRAIL in gallbladder cell line SGC-996 was detected. A nude mice model beatinggallbladder carcinoma xenografts was established to assess the suppressive effect ofcombination with rhTRAIL and Celecoxib in vivo. After being treated for 2 weeks,the L(length) and W(width) of xenografts was recorded, the weight of xenograftswas measured and inhibition rate was calculated. Results Celecoxib could down-regulatethe expression of c-FLIPs and up-regulate the expression of DR5 in a doseand time dependent mode on cell line SGC-996. Apoptotic level in the combinedtreatment group on on cell line SGC-996 were significantly higher that in single drugtreatment group and control group. The volume of xenografis in the combinedtreatment group were markedly prohibited. Inhibition rate of xenografts in combinedtreatment group was 23.3%,which is higher than that in rhTRAIL treated group(14.3%) or in Celecoxib treated group(15.5%). However, the difference of the weight of xenografts among different groups is not of statistical significance.Conclusion Celecoxib could markedly sensitize rhTRAIL-induced apoptosisthrough down-regulation of c-FLIPs and up-regulation of DR5 in gallbladdercarcinoma cell line SGC-996. In vivo, as compared to other group, the combinedtreatment group with rhTRAIL and Celecoxib could inhibit more of growth ofgallbladder carcinoma xenografts in nude mice.The results deserved to be furtherstudied.