The Research Of The Combination Treatment Of Pancreatic Carcinoma Cells With TRAIL And Camptothecin

Pancreatic carcinoma is one of the most malignant tumors with a lowsurvival rate. The majority of patients with diagnosed pancreatic cancer presentwith an already unresectable tumor stage. chemotherapy, radiotherapy, andimmunotherapy are not sensitive to the desease. So, the therapeutic options arevery limited. Tumor necrosis factor-Related Apoptosis-Inducing Ligand(TRAIL) is a new member of the tumor necrosis factor family, and hasrecently emerged as a cancer therapeutic agent, because it is capable ofpreferentially induced apoptosis in human cancer over normal cells, so it hasbecome a hot topic. Recently, a large number of study indicate that some tumorcells are resistant to TRAIL, however, fortunately these resistant tumors will besensitive to TRAIL by combination treatment with chemotherapy. socombinated treatment with chemotherapy and TRAIL can overcome TRAILresistance and enhance TRAIL therapeutic efficacy in treating pancreaticcarcinoma.Our experiments take the pancreatic carcinoma cell line PANC-1 as theresearch object, and compare the difference of inhibition rate of TRAIL groupand CPT compared with TRAIL group, and further discuss the mechanismswhich may exist.Firstly, our results indicate that pancreatic cancer cell line PANC-1 wasnot sensitive to TRAIL alone, we divided TRAIL group into five different concentration ( 10ng/ml, 30ng/ml, 100ng/ml, 1000ng/ml ) , the growthinhibition rate after treated for 24 hours were 0.62±0.41, 1.03±0.66, 7.2±0.79,8.02±1.33 and 10.60±2.36 respectively, so we can conclud that PANC-1 is notsensitive to TRAIL alone. The growth inhibition rate of cell combined TRAIL(100ng/ml) with different concentration camptochecin ( 10ng/ml, 30ng/ml,100ng/ml, 300ng/ml, 1000ng/ml ) for 24 hours were respectively 3.3±0.66%,8.2±1.35%, 0.5±1.97%, 33.6±4.52% and 41.5±3.95%,It shows that theco-treated group of TRAIL and CPT significantly kill tumor cells,however,thegroup of CPT is not. For making out the mechanism of killing tumor cells,wedetected that many cells combined TRAIL with CPT appeared nucleus together,margina, nuclear fragmentation and other topical apoptotic morphology byfluorescence staining of Hoechst 33258,However, The group of TRAIL alonehad no obvious signs of apoptosis. It shows that CPT can enhance thesensitivity of TRAIL-induced apoptosis in PANC-1 cells. In addition,wedetected the apoptosis from the level of protein by the western-blot, wedetected the expression levels of Caspase-8,Caspase-3 and their cleavages inevery group, we only saw the pro-caspase-8 and pro-caspase-3 from theTRAIL group and CPT group. In the joint group, we not only saw thepro-caspase-8 and pro-caspase-3 , and saw their cleavages, this study suggestedthat the combination of CPT and TRAIL have synergistic apoptosis effects andprovide effective treatment for pancreatic cancer cells.To further investigate theeffect of that, we use Western-blot to detect the expression of Caspase-8 andCaspase-3 protein in TRAIL group and CPT in combination with TRAILgroup,the result shows that the TRAIL alone can not activate pro-caspase-8, soit can not induce cells apoptosis, however, the two application, Caspas-8 andCaspas-3 obviously appeared activation with cleavage. It indicated thatchemotherapy drugs combined with TRAIL release the suppression ofcaspase-8 and restore the apoptosis signaling transduction by some mechanism.In order to futher explore the mechanism that CPT combined withTRAIL activated caspase-8, we detected DR4 and DR5 on tumor cellular faceand upstream regulated protein c-Flip of caspase-8 which were influenced byCPT combined with TRAIL, the result showed that the expression of DR4 andDR5 had no significant difference between Medium group and CPT group.However, we significantly saw down-regulation of the expression of c-Flipprotein in CPT group and joint group, so we concluded that CPT enhance thesensitivity of PANC-1 to TRAIL just because CPT inhibit c-Flip proteinexpression ,Moreover, there is no things with DR4 and DR5. At the same time,it showed that after pre-CPT treatment, it can release the inhibition of caspase-8by down-regulation of the expression of c-Flip and then resume apoptosissignaling transduction .Based on the above experimental results, first, CPT can enhance thersensitivity to TRAIL in PANC-1 cells. next, TRAIL combined with CPTinduce PANC-1 death by apoptosis pathway. finally, TRAIL and CPT ininducing apoptosis of PANC-1 have significant synergies. The mechanism mayrelease the inhibition of caspase-8 by down-regulation of the expression ofc-Flip and then resume apoptosis signaling transduction.