Role And Mechanism Of Somatostatin Receptor 2 In Pancreatic Cancer

Part 1: EphA2 and E-cadherin expression and significance in pancreatic cancerObjective: To detect EphA2 and E-cadherin expression in pancreatic cancer, the relationship with clinicopathologic characteristic, and determine the role of EphA2 and E-cadherin in pancreatic cancer.Methods: The expression of EphA2 and E-cadherin in 56 cases of pancreatic cancer and 23 cases of adjacent pancreas tissue were detected by immunohistochemistry, and their relationships with clinicopathologic characteristics were analyzed. EphA2 mRNA expression in pancreatic cancer cell line(BxPc-3, Panc-1 and PC-3) was detected by RT-PCR.Results: EphA2 was expressed in 37 of 56 cases (66.07%), which was significantly higher than in adjacent pancreas tissue(P<0.05). E-cadherin was expressed in 24 of 56 cases (42.86%), which was significantly lower than in adjacent non-cancerous pancreas tissue(P<0.05). EphA2 expression is related to differentiation level, TNM stages and lymphatic metastasis but E-cadherin expression is only related to TNM stages and lymphatic metastasis. Negative correlation between EphA2 and E-cadherin expression in pancreatic cancer is found. High level of EphA2 is detected in BxPc-3 and Panc-1, low level of EphA2 is detected in PC-3.Conclusions: EphA2 and E-cadherin expression in pancreatic cancer suggest they may play important roles in the development and metastasis in pancreatic cancer. These two proteins may be useful to judge the metastasis potential of pancreatic cancer. Part 2: Reconstruction of AdvCMV.SSTR2 and its re-expression in human pancreatic cancer cell lineObjective: To explore the mechanisms further, AdvCMV.SSTR2 is constructed and pancreatic cancer cell lines re-expressing human SSTR2 are established.Methods:1. SSTR2-cDNA was inserted into adenovirus shuttle plasmid-pAdtrackCMV to generate a recombinant plasmid pAdtrackCMV-SSTR2, then the Pme I linearized plasmid pAdtrackCMV-SSTR2 was transfected into E. coli BJ5183 cells that had been transfected with adenovirus backbone plasmid pAdEasy-1. The DNA contained the identified recombinant plasmid was digested with Pac I and transfected into 293 cells to package the adenovirus. After amplified in 293 cells, the Adenovirus was transfected into 293 cells. The green fluorescence protein expression was detected by fluorescent microscopy.2. AdvCMV.SSTR2 was transfected into human pancreatic cancer cell line BxPc-3, PC-3 and Panc-1. At 48h and 96h after transfection, the expression of SSTR2 through green fluorescence protein expression was observed under fluorescent microscopy. Furthermore, the mRNA and protein expression of SSTR2 was detected by RT-PCR and Western blot.Results:1. AdvCMV.SSTR2 was constructed successfully, which was identified by restriction enzyme digestion and GFP expression.2. After transfected with AdvCMV.SSTR2 in BxPc-3, PC-3 and Panc-1, the green fluorescence protein expression was observed under fluorescent microscopy. And the green fluorescence protein expression in PC-3 was more powerful than in BxPc-3 and Panc-1 at 96h after transfection. 3. SSTR2 was re-expressed in BxPc-3, PC-3 and Panc-1 after transfection of AdvCMV/SSTR2.Conclusion : The successful reconstruction of AdvCMV.SSTR2 and establishment of pancreatic cancer cell line re-expressing human SSTR2 provide the basis of gene therapy for pancreatic cancer. Part 3: The effect of somatostatin receptor type 2(SSTR2) on the proliferation and invasion of human pancreaticcancer cells in vitroObjective: To investigate effect and mechanism of somatostatin receptor type 2 (SSTR2) gene transfection mediated by adenovirus on proliferation and invasion of human pancreatic carcinoma cell line.Methods: The full length human SSTR2 cDNA was introduced into pancreatic cancer cell line BxPc-3 by adenovirus-mediated transfection. Expression of SSTR2 mRNA and protein was detected by RT-PCR and Westen-blot. The apoptotic index (AI) of experimental group, vector control and mock control was determined by FCM. Proliferation of these cells was analyzed by MTT assays. The migratory and invasive ability of these cells was deteced by Transwell. Furthermore, the expression of matrix metalloproteinase-2 (MMP-2), tissue inhibitor of metalloproteinase-2(TIMP-2) and EphA2 were detected by RT-PCR.Results: The stable expression of SSTR2 was detected in BxPc-3 transfected by AdCMV.SSTR2. The AI was significantly higher in the experimental group compared with the vector control and mock control (P<0.05). The proliferation of experimental group was dramatically decreased(P<0.05). The number of migrated BxPc-3 cells in experimental group was far less compared with vector control and mock control (P<0.01). Moreover, MMP-2 and EphA2 mRNA were significantly reduced in the experimental group. Conversely, TIMP-2 mRNA were significantly increased in the experimental group compared with vector control and mock control (P<0.01). The expressions of EphA2 protein in the experimental group were decreased.Conclusion: The growth and metastasis of pancreatic cancer cells can be inhibited by SSTR2 obviously. SSTR2 may play a role by down-regulating the expressions of MMP-2 and EphA2 and up-regulating the expressions of TIMP-2. Part 4: The effect of SSTR2 on the proliferation and invasion of human pancreatic cancer cells in vivoObjective: To investigate mechanism of growth and metastatic progression inhibition of pancreatic carcinoma in xenografts implantation model after adenovirus-mediated gene transfer of SSTR2 and administration of somatostatin analog Octrotide.Methods: The pancreatic cancer cell BxPc-3 was transfected with AdvCMV/SSTR2 and cell line stablely expressing SSTR2 was established. Twenty-two nude mice were randomly divided into 3 groups. 8 mice were xenografted with SSTR2 expressing cells (experimental group), empty vector control group, and mock control group cells respectively. Then the experimental group and control group nude mice received an intratumoral injection of AdCMV.SSTR2, AdvCMV.GFP, PBS respectively 3 times (once / two days). Octreotide(250μg.kg^-1 q.o.d) was given intraperitoneally in each groups for 7 weeks. Eight weeks after implantation, tumor weight, apoptotic index (AI) and the presence of metastasis were evaluated respectively after the mice were killed and the tumors were taken. The expressions of SSTR2 were detected by RT-PCR and Westen-blot. Furthermore, the expressions of mRNA of MMP-2, TIMP-2 and EphA2 were detected by RT-PCR.Results:1. The stable expression of SSTR2 was detected in experimental group.2. Compared with vector control(2.74±0.16) and mock control (2.70±0.13), the tumor weight (1.89±0.07) of experimental group were significantly reduced(P<0.05). The AI was significantly higher in experimental group (5.18±1.13%) compared with vector control(2.42±0.40%) and mock control (2.34%±0.53%).3. The significant decrease of MMP-2, EphA2 and increase of TIMP-2 mRNA expressions were detected in the experimental group compared with control groups (P<0.05).4. Furthermore, the incidences of lymphatic metastasis, liver metastasis decreased significantly in the experimental group compared with control groups (P<0.05).Conclusion: The administration of somatostatin analog Octreotide can inhibit the growth and metastasis of pancreatic carcinoma after adenovirus-mediated gene transfer of SSTR2. The mechanisms may involved in inducing apoptosis, down-regulating the expressions of MMP-2, EphA2 and up-regulating the expressions ofTIMP-2.