Mechanisms of invasion induced by hepatocyte growth factor in gallbladder carcinoma cell lines

人类胆囊癌是高度恶性的，其预后差取决于周围组织浸润的程度。我们在体外检测了四种胆囊癌细胞系（GB-d1、GB-h3、GB-d2和FU-GBC-1）在缺乏或存在肝细胞生长因子（HGF）的情况下的侵袭活性。在1型胶原凝胶培养中，HGF刺激细胞增殖，并诱导GB-d1、GB-h3和GB-d2中树突状或单个小管结构的侵袭性表型。在Matrigel侵袭实验中，HGF也诱导了这三种细胞系的侵袭，但FU-GBC-1没有。HGF对四种细胞系的细胞运动均有不同程度的刺激作用。HGF是由原代培养的人胆囊正常成纤维细胞产生的，而不是从胆囊癌细胞系中产生的。蛋白水解酶的酶谱图显示，即使在没有HGF的情况下，GB-d1、GB-h3和GB-d2中的IV型胶原酶和尿激酶型纤溶酶原激活物（u-PA）活性也很高。在HGF的作用下，GB-h3的72K型ⅳ型胶原酶（MMP-2）活性和GB-d1、GB-h3和GB-d2的u-PA活性均增强。相反，无论HGF是否加入，FU-GBC-1的MMPs和PAs活性都很微弱。Western blot分析显示，GB-d1、GB-h3和GB-d2中的190K c-met产物（HGF受体）水平高于FU-GBC-1。这些结果表明，除了蛋白水解能力的重要性外，通过HGF/HGF受体系统诱导的细胞运动对于胆囊癌细胞的侵袭性进展至关重要。

Human gallbladder cancers are highly malignant and its prognosis is poor depending on extend of surround tissue invasion. We examined in vitro the invasive activity of four gallbladder cancer cell lines (GB-d1, GB-h3, GB-d2 and FU-GBC-1) in the absence or presence of hepatocyte growth factor (HGF). In type 1 collagen gel culture, HGF stimulated cell proliferation and induced invasive phenotype of arborizing or individual tubule structures in GB-d1, GB-h3 and GB-d2. In Matrigel invasion assay, invasion was also induced in these three cell lines by HGF but not in FU-GBC-1. Cellular motility was however stimulated by HGF in all of the four cell lines at various extent. HGF was produced from primary cultured normal fibroblasts of human gallbladders but not from the gallgaldder cancer cell lines. Zymography for proteolytic enzymes demonstrated high levels of type IV collagenase and urokinase-type plasminogen activator (u-PA) activity in GB-d1, GB-h3 and GB-d2 even in the absence of HGF.In the presence of HGF,the 72K type IV collagenase (MMP-2) activity of GB-h3 and u-PA activities of GB-d1, GB-h3 and GB-d2 were enhanced. In contrast, MMPs and PAs activities of FU-GBC-1 were faint irrespective of HGF addition. Western blot analysis demonstrated higher levels of 190K c-met product (HGF receptor) in GB-d1, GB-h3 and GB-d2 than FU-GBC-1. These results suggest that in addition to the importance of proteolytic potent, the cellular motility induced via HGF/HGF-receptor system is essential for the invasive progression of the gallbladder carcinoma cells.