The Mechanisms Of Id1 Invovled In The Angiogenesis Of Gastric Cancer

Angiogenesis is necessary for tumor formation and growth and studies on antiangiogenesis have become one of the most promising and active fields in anticancer research. Inhibitor of DNA binding and differentiation-1(Id1) is one of members of HLH transcription factors subfamily. It has been shown that Id1 protein plays a critical role in angiogenesis during tumor formation and progression, so it is identified as a quasi-protooncogene and a proangiogenesis factor. Mounting evidence have confirmed the key roles of Id1 in tumorigenesis by controlling the transcriptional levels of its'downstream moleculars, however, its regulation and mechanisms are still unclear.Matrix metalloproteinases (MMPs) are a family of enzymes that proteolytically degrade various components of the extracellular matrix (ECM). MMPs participate in remodeling of basement membranes and ECM. However it has become clear that MMPs contribute more to angiogenesis than just degrading ECM components. MMPs are generally abundantly expressed in virtually all human cancers. High expression levels of certain MMPs are correlated with tumor invasive and/or metastatic potential and poor prognosis. It has been reported that the activation of MMP-2 in the supernatant of fibroblast in Id1-/- mice was reducted, so we detected the potential effect of Id1 on the expressions of MMP-2 and MMP-9 in angiogenesis in gastric cancer.Cyclooxygenase-2 (COX-2) is generally thought to play important roles in the tumorigenesis and progression of many cancer. One of the important mechanisms by which COX-2 acts as a tumor promoter is through the stimulation of angiogenesis. However, the mechanism of COX-2 contribution to tumor angiogenesis and progression is still not fully understood. Our previous study has revealed that the expression of Id1 was found significantly downregulated by NSAIDs and in COX-2-deprived 7901 cells through gene chip method. In the present study, we aimed to investigate the effect of Id1 by COX-2 in angiogenesis in gastric cancer.[Objective](1)To investigate the possible relationship between Id1 and MMPs in angiogenesis of gastric cancer; (2) To explore the potential role of COX-2 in Id1-mediated angiogenesis in stomach neoplasms. [Methods](1)Expressions of Id1 and MMPs in gastric cancer tissues were examined by immunohistochemistry; (2) Establish the human gastric cancer SGC7901 sub-cell lines of Id1-siRNA expression; (3) Expressions of MMP-2 and MMP-9 in Id1-siRNA transfectants were examined by semi-quantitative RT-PCR and Western blot; (4) Enzyme activity of MMP-2 and MMP-9 in Id1-siRNA cells were detected by zymography method; (5) Expressions of MMP-2 and MMP-9 in Id1-siRNA cell-transplanted tumor tissues in nude mice were identified by immunohistochemical method; (6) The abilities of migration and tube formation of HUVECs(Humane Umbilical Vein Endothelial Cells) in the CM(conditioned medium) of Id1-overexpression gastric cancer cells were observed, and the effect of the neutralizing antibody of MMP-2 and MMP-9 into the CM; (7) MMP-2 and MMP-9 promoter activities were evaluated by dual luciferase reporter assay when Id1 was blocked; (8) Establish the human gastric cancer SGC7901 sub-cell line of forced expression of COX-2 and COX-2–siRNA; (9) Western blot and RT-PCR were performed to detect the protein and mRNA levels of Id1 in these transfectants; (10) The content of secreted VEGF protein in the supernatant of COX-2 over expression and COX-2 -siRNA cells were determined by ELISA; (11) MTT assay was used to examine the proliferation ability of HUVECs in the CM of COX-2 over expression and COX-2-siRNA cells; (12) Tumorigenesis and MVD in SGC7901/COX-2/Id1-siRNA cell-transplanted tumor tissues in nude mice were observed, and expression of Id1 were identified by immunohistochemical method.[Results](1) Id1 and MMP-2,-9 co-expressions were identified in the gastric cancer tissues; (2) Significantly decreased expressions of both Id1 and MMP-2,-9 on both protein and mRNA levels in SGC7901 Id1-siRNA transfectants were found; (3) Data from zymography method showed that the activations of MMPs in Id1-siRNA cell lines were reduced compared to the control groups;(4) Decreased expression of MMP-2 and MMP-9 in the Id1-siRNA transplanted tumor tissues in nude mice were found compared to those from the control cells; (5) The effect of inhibition of the migration and tube formation of HUVECs in Id1 overexpressed CM were reversed by adding the neutralizing antibody of MMP-2 and MMP-9; (6) By bioinformatics, the promoter sequences of MMP-2 and MMP-9 were found(-2000~+200).The dual luciferase reporter assay demonstrated that Id1 block in the gastric cancer cells led to a 2- to 2.5-fold decrease of MMP-2 and MMP-9 transcriptional activities compared to control cell, indicating that Id1 caused transactivation of MMP-2 and MMP-9 promoter and then the upregulation of MMP-2 and MMP-9 expression and activities; (7) The subclones were picked out and confirmed that COX-2 was highly expressed in SGC7901/COX-2 cells and was expressed at lower level in SGC7901/COX-2-siRNA cells compared to the controls; (8) Id1 mRNA and protein were elevated in SGC7901/COX-2 cell and suppressed in SGC7901/COX-2-siRNA cells; (9) VEGF content was comparatively higher in the SGC7901/COX-2 cells and was decreased according to the level of Id1 in SGC7901/COX-2-siRNA cells; (10) HUVECs exhibited higher proliferation rate in SGC7901/COX-2 CM while lower proliferation rate in SGC7901/COX-2-siRNA CM; (11) The increase of VEGF in SGC7901/COX-2 cells and the effect of contribution to the proliferation of HUVECs were both abrogated when Id1 was knocked out in SGC7901/COX-2 cells; (12) Tumorigenesis inhibition and declined MVD in the transplanted tumor tissues in nude mice were found when Id1 was knocked down in SGC7901/COX-2 cells, compared to the control cells.[Conclusions](1) Id1 and MMP-2,-9 were co-overexpressed in gastric cancer tissues; (2) Id1 can upregulate the expression of MMP-2 and MMP-9 in gastric cancer, and thus contribute to the migration and the tube formation of HUVECs. Id1 block in the gastric cancer cell inhibited the transctriptional activities of MMP-2 and MMP-9. So we cound presume that Id1 might promote angiogenesis by transactivating MMP-2 and MMP-9; (3) Id1 was upregulated by COX-2 in the gastric cancer and stimulate the downtream VEGF and enhance the proliferation of HUVECs. Blocking this pathway might be great helpful to the tumor therapeutics, so Id1 can be exploited as antiangiogenesis therapeutic target of gastric cancer.