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Tumor cells are known to produce larger amounts of reactive oxygen species (ROS) than normal cells. Although numerous reports have indicated the importance of ROS in urokinase plasminogen activator (uPA) production, the precise mechanisms remain controversial. In our study, we investigated the effect of ROS on uPA generation in human hepatoma cells, HepG2 and Hep 3B. We determined the effects of hepatocyte growth factor (HGF) on the regulation of ROS, which resulted in suppression of ROS production, as measured with the fluorescent probe, 2'-7'-dichlorofluorescein diacetate. The role of HGF in modulating ROS production, particularly that regulated by Rac-1, was determined. HGF suppressed the increment in Rac-1-regulated ROS in both cell lines. Treatment with 200 micrometer of H(2)O(2) showed a 1.6-2.1 fold increment in HGF, but a little increment occurred at 500 micrometer of H(2)O(2). It looks no dose dependent manner. Combined treatment with H(2)O(2) and HGF, resulted in a slightly increased production of HGF compared to no treatment (control). Also, H(2)O(2) upregulated uPA expression in both hepatoma cell lines. To identify the downstream pathways regulated by ROS, we treated cells with PD 98059, an MEK inhibitor, and SB 203580, a p38 inhibitor, after treatment with H(2)O(2), and showed negative control between ERK and p38 kinase activities for uPA regulation. We found that HGF modulate Rac-1-regulated ROS production through activation of Akt and ROS regulates uPA production via MAP kinase, which provides a novel clue to clarify the mechanism underlying hepatoma progression.

experimental and molecular medicine/experimental and molecular medicine

Reactive oxygen species regulate the generation of urokinase plasminogen activator in human hepatoma cells via MAPK pathways after treatment with hepatocyte growth factor