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说，我们在体外检测了人肝癌细胞株和细胞上ET-1受体的产生和分泌，并研究了ET-1和/或特异性ET受体拮抗剂对肝癌生长的影响。Northern印迹分析表明，ET-1前体基因在肝癌细胞系中有表达，特异性酶免疫分析法检测到细胞提取液和培养上清液中都有大量的ET-1表达。肝细胞癌免疫组织化学染色显示肝癌细胞ET-1阳性。在含有ET-1的半固体培养液中，ET-1可明显促进肝癌细胞系的集落生长，并呈剂量依赖关系。当ET受体拮抗剂106M BQ-123与之共同孵育时，108M ET-1对肝癌细胞生长的刺激作用被完全阻断。此外，马赫拉武和Alexan…的菌落形成单独与BQ-123或ET-1单抗孵育可显著抑制更多的DER细胞。Northern印迹分析表明，至少有两种人肝癌细胞株--Mahramu和Hep G2表达了人ET_B受体的mRNA。ET_A受体mRNA在所有肝癌细胞系中均不表达。用标记的内皮素作为配体进行的结合研究表明，HUH-7细胞上有高亲和力的结合位点(Kd=2.7×10^&lt；10&gt；M)。为了确定肝癌的生长是否可能依赖于ET，我们将两个肝癌细胞系Mahramu和Huh-7移植到裸鼠体内。在这两个实验中，给予波生坦(10 mg/kg/天)均可显著抑制肿瘤生长。这些结果表明，ET-1是由肝癌细胞产生和分泌的刺激其自身生长的物质，并且富含ET_B受体，这可能介导了ET-1对肝癌的促生长作用。结论：ET-1是肝细胞癌的生长因子，ET受体拮抗剂可能在肝细胞癌的药物治疗中发挥作用。较少

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