HGF upregulates and modifies subcellular distribution of proteins in colon cancer cell enterocytic differentiation.

Hepatocyte growth factor (HGF) and its receptor, c-Met, are involved in cell transformation. To study their role in intestinal cell differentiation, we used Caco-2 colon cancer cells, which differentiate spontaneously into enterocytes during culture. Cells grown continuously in the presence of HGF reached confluence more quickly than control cells. Markers of enterocytic differentiation, such as alkaline phosphatase and sucrase-isomaltase activities, adhesion molecules, and structural proteins such as E-cadherin, villin, and F-actin were upregulated by HGF throughout the 35 days of culture, and actin fibers were reorganized. HGF also stimulated expression and tyrosine phosphorylation of c-Met and Gab-1 as well as protein kinase C (PKC)-alpha expression. PKC-alpha has been shown to be involved in intestinal differentiation. We therefore investigated the possibility that increases in PKC-alpha protein levels were responsible for the HGF-promoted events. We did this by incubating cells with Gö-6976, an inhibitor of PKC-alpha and -beta1, concomitantly with HGF. This inhibitor abolished the HGF-induced increase in villin levels before, but not after, confluence. Thus HGF accelerates Caco-2 cell differentiation and stimulates the metabolic and structural events accompanying this process. These HGF-promoted events may be mediated partly by Gab-1, and the effects of HGF on villin before confluence seem to involve PKC.

Hepatocyte growth factor induces colonic cancer cell invasiveness via enhanced motility and protease overproduction. Evidence for PI3 kinase and PKC involvement.

Tumour progression to the metastatic phenotype is mainly dependent on tumour cell invasiveness. Cell migration is a crucial step in this process. Here we investigate the effect of hepatocyte growth factor (HGF) on the induction of in vitro invasiveness of poorly aggressive Caco-2 colonic cancer epithelial cells. Invasion assays through a Matrigel barrier were performed. Proteases were assessed by zymography, reverse transcription-polymerase chain reaction and immunoblotting. Caco-2 cells were found to express HGF receptor but not HGF and to secrete several proteases, namely matrix metalloproteinase-1 (MMP-1), MMP-2, possibly MMP-9 and urokinase plasminogen activator (uPA). Exogenous HGF promoted invasiveness of Caco-2 cells through an artificial basement membrane matrix and enhanced their production of proteases. In addition, analyses of media at the end of invasion assays indicated that anti-HGF antibody inhibited protease production in parallel with cell invasion. The involvement of proteases in the HGF-induced invasion process was further investigated using either a synthetic general MMP inhibitor or neutralizing antibodies against MMPs or uPA. All components significantly inhibited HGF-promoted cell invasion. Moreover, specific inhibitors of PKCalpha/beta1 and PI3 kinase also decreased both HGF-promoted cell invasion and protease expression in invasion assay media. Thus, our findings provide evidence that the process of HGF-activated invasiveness of Caco-2 cells involves PI3 kinase and PKC and results from close association of two events, stimulation of cell motile activity and concomitant overproduction of proteases, which permits cell migration through a degraded extracellular matrix.

Transforming growth factor-alpha and epidermal growth factor receptor in colonic mucosa in active and inactive inflammatory bowel disease.

Transforming growth factor-alpha (TGF-alpha) is overexpressed in colonic carcinomas and promotes mucosal wound healing. It may be implicated in chronic inflammatory bowel disease (IBD). We analyzed the expression of TGF-alpha and its receptor, epidermal growth factor receptor (EGF-r), in the colonic mucosa of patients with Crohn's disease (CD) or ulcerative colitis (UC), in active or inactive stages, as compared with controls. Proteins and mRNA were detected in biopsies from the right and left colon and in surgical colonic specimens. Immunoblot analysis revealed TGF-alpha protein as a 29 kDa band. This band was normally expressed in uninvolved colonic mucosa of patients with CD or UC whether in active or inactive stages, but decreased or absent in involved mucosa of active IBD, even when TGF-alpha mRNA and EGF-r protein were detected. In the unaffected mucosa of CD, the intensity of TGF-alpha immunoreactivity was similar to that of controls in the right colon but stronger (P = 0.05) in the left colon. There was no TGF-alpha overexpression in dysplastic regions. In conclusion, in active IBD disease, the decreased TGF-alpha protein amount seems not only related to epithelial cell loss but reflects a down-regulation at least at the protein level. We speculate that TGF-alpha does not play a role within the active stage but may be implicated later in the repair process.

Matrix metalloproteinase inhibition prevents colon cancer peritoneal carcinomatosis development and prolongs survival in rats.

Matrix metalloproteinases (MMP) are enzymes responsible for extracellular matrix degradation which play a role in cancer progression and metastatic spreading. We investigated the effects of the MMP inhibitor, batimastat, in vitro on the proliferation and invasiveness of the rat colon cancer cell line DHD/K12, and in vivo on the growth of an aggressive model of peritoneal carcinomatosis producing haemorrhagic ascites and metastases, obtained in the rat by i.p. injection of DHD/K12 cells. MMP production was studied in conditioned culture media, solid tumors and ascitic fluid. In vivo, after injection of tumor cells on day 0, rats received i.p. daily either batimastat (30 mg/kg) or equal volume of vehicle from day 2 until killing on day 43 (series I) or from day 13 until death (series II). The grade of peritoneal carcinomatosis, ascite volume, number and size of liver metastases were evaluated in both series, and survival in series II. MMPs-1, -2 and -9 were identified in culture media, tumors and ascites. In vitro, batimastat did not modify DHD/K12 cell proliferation and slightly reduced cell invasion. In vivo, in series I, batimastat treatment totally prevented peritoneal carcinomatosis and liver metastasis development. In series II, it significantly prolonged survival (P < 0.0002) and reduced peritoneal carcinomatosis (P < 0.001) and hepatic metastases number as compared with controls. However, batimastat-treated rats of the two series had peritoneal inflammation with marked ascites. Nevertheless, inhibition of MMP is a new therapeutic approach which may be promising in treatment of microtumors as in more advanced cancer stages.

Exposure to ionizing radiation modifies circulating gastrin levels and gastrointestinal endocrine cell densities in the rat.

PURPOSE: Gastrointestinal functions, controlled partly by gut peptides, are disturbed by ionizing radiation exposure. The effect of whole-body irradiation on circulating gastrin levels, densities of gastrointestinal endocrine cells and gastric acid secretion was investigated. MATERIALS AND METHODS: Rats were exposed to 2 or 6 Gy gamma-radiation. They were killed 3 or 7 days later and compared with shams. Plasma gastrin and basal acid output were measured. Endocrine cells were identified by argyrophilia or immunohistochemistry and their densities estimated. RESULTS: Radiation exposure significantly increased gastrinaemia and gastric acid output at the times studied (p<0.05-p<0.001). Endocrine cells displayed different sensitivities to irradiation. In the gastric mucosa, a 6 Gy dose induced a decrease in fundic argyrophil cell, antral gastrin and somatostatin cell densities, always accentuated 7 days after irradiation, while in the intestinal mucosa it induced an increase, with highest values often at 7 days post-irradiation (p<0.01-p<0.001). This was true for neurotensin cells in the jejunum and ileum, substance P cells in ileum and enteroglucagon cells in the descending colon. CONCLUSIONS: Whole-body irradiation in rats significantly alters plasma gastrin levels, and several gut endocrine cell densities. This has repercussions on hormonal function, such as that exerted on acid secretion, and may explain gastrointestinal dysfunction observed following radiation exposure.

Developmental expression and functionality of hepatocyte growth factor and c-Met in human fetal digestive tissues.

BACKGROUND & AIMS: Hepatocyte growth factor (HGF) and its receptor c-Met are presumed to play a morphogenic role during embryogenesis. The possible implication of HGF and c-Met during the digestive system development was approached by investigating their ontogeny, distribution, and functionality in human fetal tissues. METHODS: Thirty fetuses, 7-24 weeks old, were obtained. HGF and c-Met messenger RNAs and proteins were detected in liver, pancreas, esophagus, stomach, and small and large intestine. Tyrosine phosphorylation assays were realized on homogenates and membrane preparations from fetal tissues. RESULTS: The temporal appearance of HGF and c-Met was established between 7 and 8 weeks of gestation in digestive tissues. Immunoblot analysis showed the presence of the c-Met beta-subunit 145-kilodalton band and of the HGF alpha-subunit 70-kilodalton band. c-Met was localized in epithelia, especially in fundic parietal cells, pancreatic and gut endocrine cells, and in muscular layers. HGF immunoreactivity was first detected in epithelia and then in mesenchyme and muscular layers. In young fetal stages, the c-Met immunoprecipitated 145-kilodalton band showed tyrosine phosphorylation after HGF stimulation. CONCLUSIONS: This study provides evidence for HGF and c-Met expression early in all human fetal digestive tissues and implicates HGF-c-Met in the digestive system morphogenesis.