ENDOTHELIN-1 IS AN AUTOCRINE GROWTH FACTOR FOR HEPATOCELLULAR CARCINO

为了验证内皮素-1 （ET-1）可能是肝细胞癌（HCC）的自分泌或旁分泌生长因子的假设，我们检测了人肝癌细胞系和ET-1受体在体外产生和分泌ET-1，并研究了ET-1和/或特异性ET受体拮抗剂对HCC生长的影响。Northern blot分析表明，在HCC细胞系中有ET-1前体mRNA的表达，并且在细胞提取物和培养基中都发现了大量的ET-1，这是通过特定的酶免疫测定法确定的。肝癌细胞免疫组化显示ET-1阳性。当HCC细胞在含有ET-1的半固体培养基中生长时，ET-1显著增加这些HCC细胞系的集落生长，并呈剂量依赖性。当特异性ET受体拮抗剂10 ^6M BQ-123共孵育时，10 ^8M ET-1对HCC细胞生长的刺激作用被完全阻断。此外，BQ-123或ET-1单克隆抗体孵育可显著抑制Mahlavu和Alexan…More der细胞的集落形成。Northern blot分析显示，人类ET_B受体mRNA在至少两种HCC细胞系Mahlavu和Hep G2中表达。ET_A受体mRNA在所有HCC细胞系中均未表达。使用标记内皮素作为配体进行的结合研究显示，Huh-7细胞具有高亲和力的结合位点（Kd=2.7 x 10 ^<10>M）。为了确定肝细胞癌的生长是否依赖et，我们将两种肝细胞癌细胞系Mahlavu和Huh-7植入胸腺裸鼠体内。在两个实验中，波生坦（10mg /kg/天）对肿瘤生长均有显著抑制作用。这些结果表明，ET-1是由HCC产生和分泌的，能够刺激其自身的生长，而HCC细胞中富含ET_B受体，可能介导ET-1对HCC的促生长作用。我们得出结论，ET-1是HCC的生长因子，ET受体拮抗剂可能在HCC的医学治疗中有用。少

To test the hypothesis that endothelin-1 (ET-1) may be an autocrine or paracrine growth factor for hepatocellular carcinoma (HCC), we examined the in vitro production and secretion of ET-1 by human HCC cell lines and ET-1 receptors on those cells, and studied the effect of ET-1 and/or specific ET receptor antagonists on HCC growth. Northern blot analysis demonstrated the expression of mRNA for ET-1 precursor in HCC cell lines and a substantial amount of ET-1 as determined by a specific enzyme immunoassay was found both in cell extracts and in culture media. Immunohistochemistry of HCC cells showed HCC cells positive for ET-1. When HCC cells were grown in semi-solid medium containing ET-1, ET-1 markedly increased colony growth of these HCC cell lines in a dose-dependent manner. When 10 ^6M BQ-123, a specific ET receptor antagonist, was coincubated, the stimulatory effect of 10 ^8M ET-1 on the growth of HCC cells was completely blocked. Furthermore, colony formation of Mahlavu and Alexan … More der cells was significantly inhibited by incubation with BQ-123 or the monoclonal antibody for ET-1 alone. Northern blot analysis demonstrated the expression of mRNA for human ET_B receptor in at least two HCC cell lines, Mahlavu and Hep G2. The ET_A receptor mRNA was not expressed in any of the HCC cell lines examined. A binding study conducted using a labeled endothelin as a ligand showed high affinity binding sites on Huh-7 cells (Kd=2.7 x 10 ^<10>M). To determine whether HCC growth may be ET-dependent then, two HCC cell lines, Mahlavu and Huh-7, were implanted into athymic nude mice. Administration of bosentan (10 mg/kg/day) caused a significant inhibition of tumor growth in both experiments. These results indicate that ET-1 is produced and secreted by HCC,which stimulates its own growth, and that HCC cells are enriched with ET_B receptors, which may mediate the growth-stimulatory effect of ET-1 on HCC.We conclude that ET-1 is a growth factor for HCC,and that ET receptor antagonists may be useful in the medical treatment of HCC. Less