The semaphorin and plexin family of ligands and receptor proteins provides important axon growth and guidance cues required for development. the MGC803 gastric cancer cell line, it was observed Rabbit Polyclonal to MMP-19 that knocking down plexin-A1 signaling led to a decreased expression of VEGFR2 at the messenger RNA and protein levels. Sema6D recognized and activated plexin-A1, which subsequently activated its downstream target, VEGFR2. The activation of VEGFR2 functioned as a positive regulator of tumor angiogenesis. Our data provided an understanding of the complex signaling cascades involved in the angiogenesis-related pathway in tumor cells. In light of our observations, pharmacological interventions targeting Sema6D/plexin-A1/VEGFR2 signaling may potentially be used as a target for the development of novel anti-angiogenic drugs in gastric cancer. strong class=”kwd-title” Keywords: semaphorin 6D, plexin-A1, VEGFR2, gastric cancer, angiogenesis Introduction The semaphorin family is a large super-protein family containing secreted, transmembrane and glycosylphosphatidylinositol-linked proteins, and it has been divided into eight hypotypes based on their structures and amino acid sequence similarity: Invertebrate semaphorins consist of classes 1 and 2, while classes 3C7 are vertebrate semaphorins (with the exception of class 5C semaphorins, which are encoded by viral genomes) (1). The semaphorin family was initially identified to be involved in mediating axonal guidance in the developing nervous system (2). Certain members of the semaphorin family have been also shown to exert diverse and important functions in other physiological processes, including heart morphogenesis (3,4), vascular growth (5), immune cell regulation (6) and tumor progression (7,8). Important regulatory functions of certain members of the semaphorin family within tumor angiogenesis have been reported. For example, semaphorin 3A (Sema3A) inhibits angiogenesis through competition for vascular endothelial growth factor (VEGF) (9), and Sema3B has also been indicated as a putative tumor suppressor that inhibits tumor growth and angiogenesis (10). By contrast, Sema3C presumably may promote angiogenesis by stimulating integrin phosphorylation and VEGF120 secretion (11), and Sema4D has been demonstrated to serve a role in tumor-induced angiogenesis (12). Sema6D, mapped on chromosome 2, is the best characterized factor of the class 6 semaphorins, which are single-pass membrane-bound semaphorins (13). It was reported that Sema6D regulates the late-phase activity of T cells during PA-824 manufacturer PA-824 manufacturer the primary immune response (14), and it functions as a promoter of tissue remodeling (15) and tumorigenesis (16). Additionally, Sema6D also functions as a ligand for plexin-A1 during T cell-dendritic cell interactions (6). Plexin-A1 serves as a main receptor for Sema6D and contributes to cardiac morphogenesis (17). Notably, plexin-A1 forms complexes with VEGF receptor-2 (VEGFR-2), which undergoes phosphorylation upon stimulation by Sema6D (15), and VEGFR is responsible for the transduction of pro-angiogenic signals (18). The association between plexin-A1 expression and gastric carcinoma angiogenesis has been previously explored by the present authors (19). In the current study, Sema6D was observed to be highly expressed in tumor tissue compared with normal gastric mucosa and was determined to be responsible for tumor promotion. Its putative receptors were detected, and it was speculated that plexin-A1 may be the main receptor for Sema6D. Next, the levels of Sema6D and plexin-A1 were detected, and it was noticed that they were highly expressed in tumor vascular endothelial cells. Furthermore, both were positively correlated with VEGFR2. These observations PA-824 manufacturer indicate that they may function as a modifier in the formation of tumor angiogenesis. This is consistent with a previous report showing that Sema6D activates VEGFR2 through plexin-A1-mediated signal transduction and controls cardiac morphogenesis (20). Materials and methods Human tissue specimens Gastric tissues were obtained from The 266th General Hospital of People’s Liberation Amy (Chengde, China) with the institutional approval and informed consent of the patients. The procedures to obtain human gastric tissues were in accordance with the Ethical Principles for Medical Research Involving Human Subjects, as formulated in the World Medical Association Declaration of Helsinki (revised in 2008). During surgical resection, gastric tumor tissues and normal gastric tissues (located ~5 cm away from the macroscopic margin of the resected tumors) were obtained from 10 patients who were diagnosed as gastric carcinomas by pathologists. The patient ages ranged between 37 and 82 years, with a median age of 58 years. There were six male and four female patients. None of the patients had received PA-824 manufacturer any chemotherapy or radiotherapy prior to biopsy or surgery. Reagents RPMI-1640 medium, fetal bovine serum (FBS), 0.25% trypsin and 0.02% ethylenediaminetetraacetic acid (EDTA) were purchased from Gibco (Thermo Fisher Scientific, Inc., PA-824 manufacturer Waltham, MA, USA). SuperScript III First-Strand Synthesis System kit and GoTaq? qPCR Master Mix were purchased from Promega Corporation (Madison, WI, USA). Anti-VEGFR2 mouse monoclonal antibody (ab9530), anti-plexin-A1 rabbit monoclonal antibody (ab32960) and anti–actin mouse monoclonal antibody (ab8226) were purchased from Abcam (Cambridge, MA, USA), while anti-Sema6D goat monoclonal antibody (sc-67965) was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Peroxidase-conjugated AffiniPure goat anti-mouse immunoglobulin G (IgG), peroxidase-conjugated AffiniPure goat anti-rabbit IgG, peroxidase-conjugated AffiniPure donkey anti-goat IgG,.