The Sensitization Effects And Mechanisms Of Selective Cyclooxygenase 2 Inhibitor Celecoxib With Chemotherapy Or Radiotherapy Of Human Pancreatic Carcinoma

Objective: To investigate the sensitization effects and the mechanisms of selective cyclooxygenase-2(COX-2) inhibitor celecoxib with chemotherapy of pancreatic cancer.Methods: 1. Effects of gemcitabine combined with celecoxib on the proliferation in pancreatic cancer cell SW1990 were investigated by MTT assay and soft agar assay. Pancreatic cancer xenograft tumor model was eastablished in nude mice by inoculating SW1990 cells subcutaneously, and gemcitabine was administered intraperitoneally twice a week, and celecoxib was given via water daily. Expressions of proliferating cell nuclear antigen (PCNA) and cyclin D1 were detected by immunohistochemical staining. 2. Effects on pancteatic cancer cell apoptosis were studied by flow cytometry, DNA ladder electrophoresis, and TUNEL. Apoptosis was detected in tumor tissue using TUNEL. The expressions of bcl-2 and bax, members of bcl-2 family of apoptosis-associated genes were assayed by RT-PCR. 3. Pancreatic cancer cell cycle distribution was assayed by flow cytometry, and effects on the protein expression of cyclin A, B1 and D1 were assessed by Western Blot. 4. In vitro angiogenesis assay and cell invasion assay were used in pancreaticcancer cells. Chorioallantoic membrane (CAM) grafted model were also used to evaluate the angiogenesis and cell invasion of pancreatic cancer cell. Neovasculature was detected by immunohistochemistry staining labeled with anti-IV collagen antibody and was indicated by microvascular density (MVD). Expressions of MMP-2, MMP-9, TIMP-1 and TIMP-2 were assessed by RT-PCR. Medium form cells was examined for the presence and activity of MMP-2, MMP-9.TIMP-1 and TIMP-2 by zymography and reverse zymography. Results: 1. Celecoxib and gemcitabine inhibited pancreatic carcinoma cell growth dose and time dependently, and the mean volume of xenograft tumor was significantly reduced in gemcitabine group, celecoxib group and combination group. Combination of celecoxib with gemcitabine inhibited cell growth and tumor volume to a greater degree than either compound alone. The expression of PCNA and cyclin Di was significantly reduced in gemcitabine group and can hardly be detected in combination group, but without significant change in celecoxib group. 2. Flow cytometry, DNA ladder electrophoresis, and TUNEL all demonstrated a significant increase of apoptotic cells after treatment with celecoxib alone or combined with gemcitabine. The expression of bax, the pro-apoptotic member of bcl-2 family was increased, and the anti-apoptotic bcl-2 was decreased. 3. Treatment with celecoxib only or combined with gemcitabine altered the cell cycle phase distribution in SW1990, cells were mainly arrested in G0/G1 phase, S phase and G2/M phase development were greatly inhibited. Expression of Cyclin A, B-i and D1 were closely related with the cell cycle distribution induced by different drug. 4. In vitro and in vivo angiogenesis and cell invasion potential of SW1990 were inhibited by celecoxib, but without significant change in gemcitabine compared with the control group. Combination of celecoxib and gemcitabine compressed the angiogenesis and cell invasion potential of SW1990 as well, but with no significant difference from celecoxib group. Expressions and secretion of MMP-2 and MMP-9 closely related to the changes in angiogenesis and cell invasion potential induced by different drug, while the expressions of TIMP-1 and TIMP-2 did not altered significantly in all the groups.Conclusions: The selective cyclooxygenase 2 inhibitor celecoxib potently enhances the effect of gemcitabine in the treatment of pancreatic cancer.Induction of apoptosis and cell cycle arrest are involved in the progress of celecoxib. Inhibition of angiogenesis and cell potential in invasion enhances the sensitization effect of celecoxib on gemcitabine indirectly.Part 2 Radiosensitization effect of celecoxib in humanpancreatic carcinomaObjective: To investigate the sensitization effects and the mechanisms of selective cyclooxygenase-2(COX-2) inhibitor celecoxib with radiotherapy of pancreatic cancer.Methods: 1. Radiosensitization effects of celecoxib on the proliferation in pancreatic cancer cell SW1990 were investigated by colony forming assay. Pancreatic cancer xenograft tumor model was used in the assessment of the tumor growth delay effects of celcoxib in radiation-treated nude mice. Then mice bearing tumor divided into four groups randomly. Celecoxib was given via water daily for consecutive 10 days when the mean diameter of tumor was about 6mm, 10 Gy tumor irradiation was given when tumor diameter was 8mm. Regression and regrowth of tumors were observed until the tumor diameter reached 16mm. Expression of PCNA was detected by immunohistochemical staining and Western Blot. Expression of Cyclin Di was assessed by Western Blot. 2. Effects on apoptosis in SW1990 and tumor tissue were studied by TUNEL. The expressions of bcl-2 and bax were assayed by RT-PCR. 3. In vitro angiogenesis assay and cell invasion assay were used in pancreatic cancer cells. Expressions of MMP-2, MMP-9, TIMP-1 and TIMP-2 were assessed by RT-PCR. Medium form cells was examined for the presence of MMP-2, MMP-9JIMP-1 and TIMP-2 by zymography and reverse zymography.Results: 1. Celecoxib incubated with SW1990 cells for 24h, 48h, and 72h all showed radio potentiating effct. The sensitization enhancement ratios (SERDq) were 1.10, 1.25 and 1.37 respectively. Combination of celecoxib with radiation in xenograft tumor resulted in significant growth delay, and the tumor growth...