10568 Background: Adrenomedullin (AM) is a 52 amino acid peptide that has an important role on tumor cell proliferation and neoangiogenesis through its receptors CRLR/RAMP2 and CRLR/RAMP3. AM gene expression is stimulated by Hypoxia Inductible Factor-1 (HIF- 1) and 60–80% of the human conventional renal carcinomas (cRCC) display mutations in the tumor suppressor protein von Hippel-Lindau, which leads to constitutively elevated HIF-1. Methods: Tumors and non-malignant kidney tissues were obtained from patients who underwent unilateral nephrectomy. We studied VEGFA, AM and its receptors expression by quantitative RT-PCR in 62 frozen renal tumors including: 40 cRCC; 5 cRCC metastasis; 5 chromophobe carcinomas; 5 Papillary carcinomas; 2 oncocytomas; 2 collecting duct carcinomas; 2 normal adjacent renal tissue; 1 unclassified renal tumor. AM and AM-receptors expression was studied in 42 paraffin-embedded renal tumors by immunohistochemistry using rabbit anti-AM, anti-CRLR, anti-RAMP2 and anti-RAMP3 polyclonal antibodies. Effects of these anti AM or anti AM- receptors antibodies on cell proliferation were examined in vitro on BIZ cell line (cRCC cells). Results: VEGFA, AM and AM-receptors genes were overexpressed in cRCC compared to other renal tumors (Except AM gene in chromophobe carcinoma). Their expressions were independent of classical prognostic factors (such as Fuhrman Grade and pT status). In cRCC, there was a strong positive correlation between VEGF-A gene expression and AM (r=0.7; p=0.01) and AM-receptors genes expressions (RAMP2 r=0.61; p= 0.01 and RAMP3 r=0.58; p= 0.01). Immunohistochemistry confirmed a tumor expression of CRLR, RAMP2 and AM in more than 80% cRCC. RAMP3 was expressed by inflammatory cells but not by tumoral cells. The cell proliferation assay showed a significant inhibition of BIZ cell proliferation by AM antibody or AM-receptors antibodies. Conclusion: AM and its receptor CRLR/RAMP2 are overexpressed in cRCC. In vitro cell proliferation assay results support that AM inhibition may suppress cRCC growth, independently of potential antiangiogenic effects. Further studies on animal models are needed but AM pathway may be a potential therapeutic target in cRCC. No significant financial relationships to disclose.
In Vitro Cell Proliferation Assay Method Using Rat Gastric Cultured Cells and Effect of Anti-ulcer Drugs on the Proliferation of Cultured Cells
