Involvement of Oxidative Stress in Tumor Cytotoxic Activity of Hepatocyte Growth Factor/Scatter Factor

Abstract

In this study, we show that N-acetylcysteine (NAC), a precursor of glutathione and an intracellular free radical scavenger, almost completely prevented hepatocyte growth factor (HGF)-suppressed growth of Sarcoma 180 and Meth A cells, and HGF-induced apoptosis, assessed by DNA fragmentation, and increase in caspase-3 activity, in Sarcoma 180 cells. The reduced form of glutathione also prevented HGF-suppressed growth of the cells as effective as NAC. Ascorbic acid partially prevented the effect of HGF, but other antioxidants such as superoxide dismutase, catalase, and vitamin E, and the free radical spin traps N-t-butyl-␣-phenylnitrone and 3,3,5,5-tetramethyl-1-pyrroline-1-oxide did not have protective effects. HGF caused morphological changes of the cells, many cells showing condensation and rounding, and enhanced the generation of intracellular reactive oxygen species (ROS) as judged by flow cytometric analysis using 2,7-dichlorofluorescein diacetate. NAC completely prevented both HGF-induced morphological changes and the enhancement of ROS generation in the cells. However, NAC did not prevent the HGF-induced scattering of Madin-Darby canine kidney cells. To our knowledge, this is the first report that HGF stimulates the production of ROS, and our results suggest the involvement of oxidative stress in the mechanism by which HGF induces growth suppression of tumor cells.Hepatocyte growth factor (HGF), 1 which was initially isolated as a potent mitogen of hepatocytes in primary culture (1, 2), is now known to be a broad-spectrum mitogen of a variety of cell types including melanocytes and endothelial and epithelial cells (3). HGF has been found to be identical with the "scatter factor" (4), which dissociates and increases the motility of epithelial cells. In addition to these activities, HGF induces branching tubule formation of Madin-Darby canine kidney epithelial cells in a three-dimensional collagen gel matrix (5). HGF is a potent angiogenic factor in vivo (6, 7) and is involved in organ regeneration (8) and tumorigenesis (9). All these effects of HGF are mediated by the HGF receptor, identified as the product of the c-met proto-oncogene (10, 11). During embryogenesis, c-met is expressed in epithelial components of various organs, while hgf is expressed in the adjacent mesenchyme (12). Embryos of mutant mice homozygous for the hgf gene die due to abnormal development of the placenta or both the placenta and liver (13,14). Likewise, c-met-deficient embryos lack muscles of the limbs, diaphragm, and tip of the tongue, all derived from migratory precursors (15). Thus, HGF is now understood as a multifunctional cytokine that mediates epithelial-mesenchymal interactions.In addition to its mitogenic activity, HGF has tumor cytotoxic activity, which suppresses growth of several tumor cell lines including Sarcoma 180 cells, Meth A mouse sarcoma cells, and Hep G2 human hepatoblastoma cells (16 -18). Moreover, HGF has been shown to inhibit the growth of hepatocellular carcinoma cells in vivo and c-myc-induced hepatocarcin...

A positive feedback loop between hepatocyte growth factor receptor and β-catenin sustains colorectal cancer cell invasive growth

“…However, a proapoptotic role of HGF (Arakaki et al, 1998;Matteucci et al, 2003;Rasola et al, 2004) and b-catenin (Kim et al, 2000;van Gijn et al, 2001;Edlund et al, 2005) was also reported in several models. Here, we show that the HGFR transduction axis and the b-catenin/TCF pathway cooperate in underpinning survival signals, whose importance probably increases during carcinoma progression.…”

Protection by antioxidants against toxicity and apoptosis induced by the sulphur mustard analog 2‐chloroethylethyl sulphide (CEES) in Jurkat T cells and normal human lymphocytes

“…Studies with other toxic agents have implicated ROS generation and depletion of cellular antioxidants in toxicity and induced cell death (Arakaki et al, 1999;Wolfler et al, 2001;Lee et al, 2002). To provide greater insight into the mechanism of SM action in order to identify potential treatments for exposure to this agent, we have now investigated the possible contribution of ROS generation and depletion of cellular antioxidants to SM toxicity.…”

Epoxycyclohexenone Inhibits Fas-mediated Apoptosis by Blocking Activation of Pro-caspase-8 in the Death-inducing Signaling Complex

“…Caspase substrate-mimicking peptide inhibitors have been most frequently used to block apoptosis. Radical scavengers such as N-acetylcysteine and metalloporphyrin can protect against apoptosis induced by reactive oxygen species (15)(16)(17). Isatin sulfonamide derivatives directly inhibit effector caspases such as active caspase-3 and active caspase-7 and are able to block apoptosis induced by cycloheximide or camptothecin (18).…”

“…Functional and structural analyses of these regulators have contributed to our understanding of the molecular basis of Fas-mediated apoptosis. However, several non-peptide small molecules that modulate apoptosis also have been reported (15)(16)(17)(18)(19)(20)(21). These molecules are useful for dissecting the apoptosis signal transduction pathway and also may be potential candidates for therapeutic use.…”