The Antiproliferative Effects Of Somatostatin Receptor Subtype 2 On Breast Cancer Cells

In the present study, the mRNA expression level of hSSTR2 was compared among different human cancer cell lines including neuroendocrine tumor cells and nonendocrine tumor cells by quantitative real-time PCR. The lower expression of hSSTR2 was found in human breast cancer cell line MCF-7. Thus, a plasmid pIRES2-EGFP-SSTR2 (pSIG) bearing hSSTR2 was constructed and transfected into MCF-7 cells. The apoptotic and cytostatic effects were more significant in hSSTR2 overexpressing MCF-7 cells than control groups. Furthermore, the expression of epidermal growth factor receptor (EGFR) was decreased in MCF-7 cells transfected with pSIG (MCF-7/pSIG) and the high expression of hSSTR2 may counteract the effect of EGF. Conclusion was made that the enhanced hSSTR2 expression played an antiproliferative role in MCF-7 cells through inducing apoptosis, G1/S cell cycle arrest and decreasing the EGFR expression, therefore reversing the effects of EGFR. In order to study the antiproliferative effct of SSTR2 in vivo, we continued to prepare a recombinant adenovirus Ad-SIG. Our data seem to indicate that developing a new therapeutic agent that could both activate hSSTRs and inhibit EGFR overexpression could potentially be a way to block tumor progression.1. Construction and expression of pSIG plasmid bearing human somatostatin receptor subtype 2 (hSSTR2)hSSTR2 gene was amplified from plasmid pGEM-T/hSSTR2 by PCR and was inserted into plasmid of pIRES2-EGFP to yield the recombinant plasmid of pIRES2-EGFP/hSSTR2 (pSIG). The recombinant plasmid of pSIG was constructed correctly confirmed by restricted endonuclease enzymes analysis, PCR analysis and DNA sequence analysis.Fluorescence microscope was applied to observe the expression of EGFP in MCF-7/pSIG. In addition, Real-time PCR technique, FCM, and immunofluorescence assay were performed to detect expression of hSSTR2 on MCF-7 cells. On mRNA level, Real-time PCR revealed that the expression level of hSSTR2 mRNA in MCF-7/pSIG cells was about 2730 folds to those of MCF-7 and MCF-7/pIRES2-EGFP groups. On protein level, FCM analysis confirmed that the percent of red fluorescence emitting cells rose from 1.63±0.15% (MCF-7 group) and 2.33±0.75% (MCF-7/pIRES2-EGFP group) to 49.67±1.35% (MCF-7/pSIG group). There were significant differences in hSSTR2 mRNA and protein expression level between MCF-7/pSIG group and negative controls (P<0.01). Immunofluorescence staining showed that MCF-7/pSIG cells could emit both green and red fluorescence. Most of the red fluorescence was found at the plasma membrane and green fluorescence mainly in cytoplasm.2. The antiproliferative effects of hSSTR2 on breast cancer cellsAfter interacting with its ligand SS analogue Octreotide, hSSTR2 play a role of antiproliferation via:1) Causing apoptosis of MCF-7 cells and G1/S cell cycle arrest. FCM analysis showed that on MCF-7/pSIG group, 40.40±14.30% cells underwent apoptosis. On the contrary, MCF-7 cells or MCF-7/pIRES2-EGFP cells underwent little apoptosis (1.31±0.38%, 10.09±6.18%, P<0.01). Cell cycle analysis showed that 5.61% MCF-7/pSIG cells were in S phase. But that figures rose evidently to 22.36% and 32.52% in untransfected and the vector controls, respectively.2) Decreasing the expression of EGFR on MCF-7 cells. Results showed that the percentage of EGFR expressing cells in MCF-7/pSIG group was lowered to 64.53±10.05% (P<0.01 vs controls). However, the expression level of EGFR on both the vector control and the untransfected MCF-7 cells remained high, to 93.01±1.92% and 92.14±1.49%, respectively.3) Counteracting the proliferative effect of EGF. Our data showed that the Inhibition Ratio of cell growth (IR%) in MCF-7/pSIG group was significantly higher (70.5±11.1%, 70.7±7.0%, 81.1±3.6%, 88.5±2.7%, 86.5±2.5% and 79.8±4.9% with different dose of EGF) than MCF-7/pIRES2-EGFP group (28.6±7.4%, 40.4±6.5%, 49.5±11.4%, 60.6±14.3%, 54.6±10.3% and 42.1±9.7%) and MCF-7 cells (0±0%) (P<0.01).3. Preparation and identification of recombinant adenovirus Ad-SIG.hSSTR2-IRES-EGFP gene was digested from pSIG plasmid and then subcoloned into plasmid pShuttle-CMV to yield the recombinant plasmid pShuttle-CMV/SIG. After linearized by PmeⅠ, pShuttle-CMV/SIG was transformed into competent BJ5183 containing pAdEasy-1 vector. Positive clones of homologous recombination (Ad-SIG) were selected and identificated. Ad-SIG was then transfected into packaging 293 cell to generate recombinant adenovirus Ad-SIG. The adenoviruses were typically generated within 7 to 10 days. The formation of recombinant adenovirus was confirmed by monitoring EGFP expression with fluorescence microscope.After packing, amplification, purificationand tittering, the recombinant adenovirus was infected into MCF-7 cells at different MOI. Expression of hSSTR2 was detected by real-time PCR technique, FCM and Laser Scanning Confocal Microscope.Conclusions:1. It was sucessfully constructed the reombinant plasmid pSIG which encoded the human somatostatin receptor subtype 2. In pSIG transfected human breast adenocarcinoma MCF-7 cells, the high expression of hSSTR2 exerted antiproliferative effects through causing the apoptosis of MCF-7 cells, the cell cycle arrest, lowering the expression of EGFR on MCF-7 membrane and counteracting the effect of proliferative effect of EGF.2. Based on the above recombinant plasmid pSIG, recombinant adenovirus Ad-SIG was prepared, which provides a basis for further study of hSSTR2 in vitro and in vivo.