Direct evidence that hepatocyte growth factor-induced invasion of hepatocellular carcinoma cells is mediated by urokinase

Background/Aims: We have shown that hepatocyte growth factor secreted by human hepatic myofibroblastsincreased the in vitro invasion of the hepatocarcinoma cell line HepG2 through Matrigel. Our aim in this study was to evaluate the role of urokinase in this process. Methods: Expression of urokinase in HepG2 cells was measured by Northern blot and zymography, and plasminogen activation was shown by a chromogenic substrate assay. Cell invasion was assayed on Matrigelcoated filters. Urokinase and urokinase receptor transcripts in hepatocarcinoma were detected by reverse transcription-polymerase chain reaction. Activated hepatocyte growth factor was detected by Western blot with a hepatocyte growth factor-β chain-specific antibody. Results: HepG2 cells expressed urokinase mRNA and secreted active urokinase. Urokinase expression was enhanced by hepatocyte growth factor at the protein and mRNA level. Notably, cell-surface-associated urokinase was increased 22-fold by hepatocyte growth factor. Hepatocyte growth factor also increased urokinase receptor mRNA expression. B428, a urokinase inhibitor, decreased by up to 70% HepG2 invasion induced by myofibroblasts and by 90% that induced by recombinant hepatocyte growth factor. This was not due to a decrease in the generation of activated hepatocyte growth factor by myofibroblasts. Finally, all 17 hepatocarcinoma samples tested expressed urokinase and urokinase receptor transcripts. Conclusion: Hepatocyte growth factor-dependent, myofibroblasts-induced invasion of HepG2 cells is secondary to the induction of urokinase expression on tumor cells.