| Research Abstract |
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
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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
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