Department of Receptor Biology & Tumour Metastasis, Chittaranjan National Cancer Institute, Kolkata, India.
Oncol Res. 2011;19(7):335-48. doi: 10.3727/096504011x13079697132925.
Cell adhesion to extracellular matrix (ECM) initiates signaling cascade regulated by cell surface integrin receptors, which affects the proliferation and invasion of cells. Cells cultured in the presence of ECM ligand fibronectin (FN) stimulate secretion of matrix metalloproteinases (MMPs), facilitating cancer cell invasion and metastasis. Among all the members of the MMP family, MMP-9 is of crucial importance in tumor invasion and metastasis. The present study aims at studying the effects of integrin receptor alpha5beta1 and its ligand FN on expression of MMP-9 in murine melanoma cell line B16F10 and understanding the molecular mechanism(s) involved. The main experimental methods performed in the study were gelatin zymography, immunoblot, real-time RT-PCR, immunocytochemistry, enzyme linked immunosorbent assay (ELISA), transwell chamber assay, and in vivo metastasis assay in syngenic (C57BL6J) mice. The study reports that FN induces the activity, mRNA, and protein expression of MMP-9 and initiates its proteolytic activation in B16F10 cells. Blockage of the alpha5 receptor abrogated the FN-mediated stimulatory response on MMP-9 in B16F10 cells. Inhibitor studies and immunoblot analysis strongly suggest the involvement of focal adhesion kinase (FAK), extracellular regulated kinase (ERK), and phosphatidylinositol-3-kinase (PI-3K) in the FN-mediated responses. Immunocytochemical analysis showed the nuclear localization of nuclear factor-kappaB (NF-kappaB) might lead to activation of MMP-9 gene upon FN treatment. This study demonstrates that integrin receptor alpha5beta1 and FN interaction induces the invasive potential of B16F10 cells and MMP-9 induction is the downstream effectors in the process. This system serves as a novel model system to understand the molecular mechanism of melanoma growth and invasion.
细胞黏附到细胞外基质（ECM）启动由细胞表面整合素受体调节的信号级联反应，从而影响细胞的增殖和侵袭。在 ECM 配体纤连蛋白（FN）存在的情况下培养的细胞会刺激基质金属蛋白酶（MMPs）的分泌，从而促进癌细胞的侵袭和转移。在 MMP 家族的所有成员中，MMP-9 对肿瘤的侵袭和转移至关重要。本研究旨在研究整合素受体α5β1及其配体 FN 对小鼠黑色素瘤细胞系 B16F10 中 MMP-9 表达的影响，并了解所涉及的分子机制。该研究主要采用明胶酶谱法、免疫印迹、实时 RT-PCR、免疫细胞化学、酶联免疫吸附测定（ELISA）、Transwell 室测定以及在同基因（C57BL6J）小鼠体内转移测定等实验方法。该研究报告称，FN 诱导 B16F10 细胞中 MMP-9 的活性、mRNA 和蛋白表达，并启动其蛋白水解激活。阻断α5 受体可消除 FN 介导的对 B16F10 细胞中 MMP-9 的刺激反应。抑制剂研究和免疫印迹分析强烈表明，黏着斑激酶（FAK）、细胞外调节激酶（ERK）和磷脂酰肌醇-3-激酶（PI-3K）参与了 FN 介导的反应。免疫细胞化学分析显示，核因子-κB（NF-κB）的核定位可能导致 FN 处理后 MMP-9 基因的激活。本研究表明，整合素受体α5β1 和 FN 相互作用诱导了 B16F10 细胞的侵袭潜力，而 MMP-9 的诱导是该过程中的下游效应物。该系统为理解黑色素瘤生长和侵袭的分子机制提供了一个新的模型系统。
