The Effects Of Proliferation And Apoptosis In Pancreatic Cancer Cells Treated By COX-2 Inhibitor In Combination With Survivin Antisense Oligonucleotide And Their Possible Mechanism

Objective: 1. To investigate the expression of cyclooxygenase-2 ( COX-2 ) and survivin in human pancreatic cancer and its correlation with clinicobiological behavior, and to explore their correlation with VEGF, p53, ki67. 2. To explore the effects of proliferation and apoptosis in pancreatic cancer cells treated by COX-2 inhibitor in combination with cisplatin. 3. To investigate whether survivin antisense oligonucleotide (ASODN) down-regulate survivin mRNA, induce apoptosis , inhibit proliferation and enhance the sensitivity of pancreatic cancer cells to gemcitabine. 4. To investigate the effects of proliferation and apoptosis in pancreatic cancer cells treated by COX-2 inhibitor in combination with survivin ASODN and their possible mechanism.Methods: The expressions of COX-2, survivin , VEGF, p53, ki67 in 51 cases of human pancreatic ductal adenocarcinoma and 11 case of adjacent normal tissue were detected by immunohistochemistry of Envision. A 20-mer phosphorothioate antisense oligonucleotides targeting survivin mRNA and lipofectin was also used to encapsulate ASODN in transfection. After pancreatic cell line BxPC-3 cells were treated with ASODN, COX-2 inhibitor celecoxib, cisplatin and gemcitabine respectively., the cell relative viability was examined using 3(4,5-dimethylethiazoly 1-2-) 2,5-diphonyl tetrazolium bromide (MTT) assays, respectively. The expressions of COX-2 mRNA ,survivin mRNA, bcl-2 mRNA and mcl-1 mRNA were detected by RT-PCR. Flow cytometry and electron microscopy were used to demonstrate apoptotic morphological changes in treated cells .The apoptosis rates weredetected by flow cytometry , and caspase-3 activity was detected by caspases-3 colorimetric assay kito All the statistical analyses were performed using SPSS 10 software, a Spearman's non-parametrical correlation analysis and x2 were used ,The data are expressed as means.E.M. Statistical comparisons were made by using Student's t-test. P<0.05 was considered significant.Results: Expressions of COX-2, surviving, VEGF and p53 in pancreatic ductal adenocarcinoma were 74.5%, 80.4%, 68.6% and 60.8% respectively. ki67 LI (labeling index) was 26.52+ 12.22. No expressions of these genes in adjacent normal tissue were detected . The COX-2 expressions were significantly associated with clinical stages and lymph node metastasis status (P<0.05) but not with histological grade (P>.05).The survivin expression was significantly associated with histological grade (P<0.01) but not with clinical stages and lymph node metastasis status (P>0.05). The VEGF expression was significantly correlated with clinical stages and lymph node metastasis status (P<0.05) but not with histological grades (P>.05). The p53 expression were significantly correlated with lymph node metastasis status (P<0.05) but not with histological grades and clinical stages(P>.05). None of them had relation with age, sex, tumor size and location. The expression of COX-2 and survivin was closely correlate with VEGF, p53 and ki67 LI (P<0.01), respectively. Furthermore, we found that the expression of COX-2 had intimate correlation with survivin (P<0.01).After treated BxPC-3 cells with COX-2 inhibitor celecoxib, As measured by MTT, cell viability were inhibited in dose-dependent and time-dependent manner with an IC50 of 100uM at the time of 24h.The expressions of COX-2 mRNA could be significantly decreased by celecoxib, The mean RT-PCR product grey of COX-2/ B-actin for experiment guoup and control group was 0.31+0.03 and 0.86 + 0.06,respectively, there was significant difference between them, (P<0.01) . Cell viability for 80uM celecoxib at 24h was 70.68% + 3.37%. Cell viability for cisplatin at different concentration (1ug/ml 2.5ug/ml 5ug/ml 10ug/mll 15ng/ml )at24h was 88.41%+2.84%, 56.38% + 1.28%, 50.35% + 1.78%, 45.04% + 3.91%, 40.00% + 2.38%,respectively. After treated BxPC-3 cells with 80uM Celecoxib in combination with cisplatin at these different concentration for 24h, Cell viability was 53.54%+3.08% 37.7%+2.22%, 32.51%+1.24%, 29.55% +1.08% 25.53%+2.32% respectively; the experiment g...