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）自分泌或旁分泌生长因子的假设，我们检测了人HCC细胞系体外产生和分泌ET-1及其受体，并研究了ET-1和/或特异性ET受体拮抗剂对HCC生长的影响。北方印迹分析证实了ET-1前体mRNA在HCC细胞系中的表达，并且通过特异性酶免疫测定法测定，在细胞提取物和培养基中均发现了大量的ET-1。免疫组化显示肝癌细胞ET-1阳性。当肝癌细胞在含有ET-1的半固体培养基中生长时，ET-1以剂量依赖的方式显著增加这些肝癌细胞系的集落生长。当与ET受体特异性拮抗剂10^6MBQ-123共孵育时，10^8MBET-1对肝癌细胞生长的刺激作用被完全阻断。此外，Mahlavu和Alexan的殖民地形成 关于我们 BQ-123或ET-1单克隆抗体单独孵育对der细胞有明显的抑制作用。北方印迹分析证实至少在Mahlavu和Hep G2两个肝癌细胞系中有ET_（B）受体mRNA的表达。ET_A受体mRNA在所有肝癌细胞系中均不表达。使用标记的内皮素作为配体进行的结合研究显示了Huh-7细胞上的高亲和力结合位点（Kd=2.7 × 104 μ <10>M）。为了确定肝癌生长是否可能依赖于ET，将两种肝癌细胞系Mahlavu和Huh-7植入无胸腺裸小鼠体内。在两项实验中，波生坦（10 mg/kg/天）给药均显著抑制肿瘤生长。这些结果表明，ET-1由HCC产生和分泌，刺激HCC的生长，HCC细胞富含ET_B受体，可能介导ET-1对HCC的生长刺激作用，提示ET-1是HCC的生长因子，ET受体拮抗剂可能在HCC的药物治疗中有应用价值。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