BACKGROUNDColon cancer is one of the major malignancies worldwide and it still remains resistant to much of the currently available chemotherapy. Downregulation of HGF/c-Met signaling pathway is an emerging therapy for cancer treatment.

METHODSIn this study, the inhibitory effects of c-Met phosphorylation were observed with SU11274 on different colon cancer cell lines in vitro.

RESULTSThe results revealed the significant inhibitory effects of SU11274 on cell proliferation and cell survival, in a time and dose-dependent manner. Furthermore, the inhibitory effects of SU11274 on different subgroups of colon cancer cells via the HGF/c-Met signaling pathway were implicated in this study.

CONCLUSIONThe results suggested the possible selective therapeutic effects of c-Met inhibitor on colon cancer.

Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Colonic Neoplasms ; drug therapy ; pathology ; Hepatocyte Growth Factor ; pharmacology ; Humans ; Indoles ; pharmacology ; Piperazines ; pharmacology ; Proto-Oncogene Proteins c-met ; antagonists & inhibitors ; physiology ; Signal Transduction ; Sulfonamides ; pharmacology

Aberrant activation of hepatocyte growth factor/scatter factor (HGF/SF) and its receptor, Met, is involved in the development and progression of many human cancers. In the cell-based screening assay, (-)epigallocatechin-3-gallate (EGCG) inhibited HGF/SF-Met signaling as indicated by its inhibitory activity on HGF/SF-induced cell scattering and uPA activation (IC50 = 15.8 microg/ml). Further analysis revealed that EGCG at low doses specifically inhibited HGF/SF-induced tyrosine phosphorylation of Met but not epidermal growth factor (EGF)-induced phosphorylation of EGF receptor (EGFR). On the other hand, high-dose EGCG decreased both Met and EGFR proteins. We also found that EGCG did not act on the intracellular portion of Met receptor tyrosine kinase, i.e., it inhibited InlB-dependent activation of Met but not NGF-induced activation of Trk-Met hybrid receptor. This inhibition decreased HGF-induced migration and invasion by parental or HGF/SF-transfected B16F10 melanoma cells in vitro in either a paracrine or autocrine manner. Furthermore, EGCG inhibited the invasion/metastasis of HGF/SF-transfected B16F10 melanoma cells in mice. Our data suggest the possible use of EGCG in human cancers associated with dysregulated paracrine or autocrine HGF/SF-Met signaling.
Animals ; Autocrine Communication/*drug effects ; Catechin/*analogs & derivatives/metabolism/pharmacology ; Cell Line, Tumor ; Cell Movement/drug effects ; Female ; *Hepatocyte Growth Factor ; Humans ; Mice ; Mice, Inbred BALB C ; Neoplasms, Experimental/*metabolism/pathology ; Paracrine Communication/*drug effects ; Phosphorylation/drug effects ; Proto-Oncogene Proteins c-met/antagonists & inhibitors/metabolism ; Receptors, Growth Factor/antagonists & inhibitors/metabolism ; Signal Transduction

Oxidative stress and apoptosis are the key factors that limit the hypothermic preservation time of donor hearts to within 4-6 h. The aim of this study was to investigate whether the histone deacetylase 3 (HDAC3) inhibitor RGFP966 could protect against cardiac injury induced by prolonged hypothermic preservation. Rat hearts were hypothermically preserved in Celsior solution with or without RGFP966 for 12 h followed by 60 min of reperfusion. Hemodynamic parameters during reperfusion were evaluated. The expression and phosphorylation levels of mammalian STE20-like kinase-1 (Mst1) and Yes-associated protein (YAP) were determined by western blotting. Cell apoptosis was measured by the terminal deoxynucleotidyl-transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) method. Addition of RGFP966 in Celsior solution significantly inhibited cardiac dysfunction induced by hypothermic preservation. RGFP966 inhibited the hypothermic preservation-induced increase of the phosphorylated (p)-Mst1/Mst1 and p-YAP/YAP ratios, prevented a reduction in total YAP protein expression, and increased the nuclear YAP protein level. Verteporfin (VP), a small molecular inhibitor of YAP-transcriptional enhanced associate domain (TEAD) interaction, partially abolished the protective effect of RGFP966 on cardiac function, and reduced lactate dehydrogenase activity and malondialdehyde content. RGFP966 increased superoxide dismutase, catalase, and glutathione peroxidase gene and protein expression, which was abolished by VP. RGFP966 inhibited hypothermic preservation-induced overexpression of B-cell lymphoma protein 2 (Bcl-2)-associated X (Bax) and cleaved caspase-3, increased Bcl-2 mRNA and protein expression, and reduced cardiomyocyte apoptosis. The antioxidant and anti-apoptotic effects of RGFP966 were cancelled by VP. The results suggest that supplementation of Celsior solution with RGFP966 attenuated prolonged hypothermic preservation-induced cardiac dysfunction. The mechanism may involve inhibition of oxidative stress and apoptosis via inactivation of the YAP pathway.
Acrylamides/pharmacology* ; Animals ; Apoptosis/drug effects* ; Cryopreservation ; Disaccharides/pharmacology* ; Electrolytes/pharmacology* ; Glutamates/pharmacology* ; Glutathione/pharmacology* ; Heart/physiology* ; Heart Transplantation/methods* ; Hepatocyte Growth Factor/antagonists & inhibitors* ; Histidine/pharmacology* ; Histone Deacetylase Inhibitors/pharmacology* ; Intracellular Signaling Peptides and Proteins/antagonists & inhibitors* ; Male ; Mannitol/pharmacology* ; Oxidative Stress/drug effects* ; Phenylenediamines/pharmacology* ; Proto-Oncogene Proteins/antagonists & inhibitors* ; Rats ; Rats, Sprague-Dawley ; Signal Transduction/drug effects* ; YAP-Signaling Proteins

BACKGROUND: Up-regulation of the hepatocyte growth factor (HGF), its transmembrane tyrosine kinase receptor (c-Met), and urokinase type plasminogen activator (uPA), is associated with the development and metastasis of various types of cancers. However, the mechanisms by which HGF/c-Met signaling mediates cancer progression and metastasis are unclear. METHODS: We investigated the roles of HGF/c-Met in tumor progression and metastasis in NUGC-3 and MKN-28 stomach cancer cell lines. RESULTS: Treatment with HGF increased c-Met phosphorylation in a dose-dependent manner, as well as increasing cell proliferation. HGF treatment also increased the protein level and the activity of uPA in NUGC-3 and MKN-28 cells. A monoclonal antibody against human uPA receptor (uPAR), mAb 3936, inhibited HGF-mediated tumor cell invasion in a dose-dependent manner. Down-regulation of uPA using uPA-shRNA induced a decrease in in vitro cell invasion in NUGC-3 cells. CONCLUSIONS: These results suggest that NUGC-3 and MKN-28 cells express functional c-Met, which may provide a therapeutic target for interfering with metastases of cancer cells by inhibiting uPA and uPAR-mediated proteolysis.
Urinary Plasminogen Activator/antagonists & inhibitors/*metabolism ; Stomach Neoplasms/drug therapy/*enzymology ; Signal Transduction/*drug effects ; Receptors, Growth Factor/*drug effects ; Receptor Protein-Tyrosine Kinases/*drug effects ; Proto-Oncogene Proteins c-met/*drug effects ; Neoplasm Metastasis ; Humans ; Hepatocyte Growth Factor/*metabolism ; Disease Progression ; Adenocarcinoma/drug therapy/enzymology

Paraquat (1,1'-dimethyl-4,4'-bipyridinium dichloride; PQ), an effective and widely used herbicide, was commercially introduced in 1962. It is reduced by the electron donor NADPH, and then reduced PQ transfers the electrons to molecular oxygen, resulting in the production of reactive oxygen species (ROS), which are related to cellular toxicity. However, the influence of continuous hypoxia on PQ-induced ROS production has not fully been investigated. We evaluated in vitro the protective effect of continuous hypoxia on PQ-induced cytotoxicity in the human carcinogenic alveolar basal epithelial cell line (A549 cells) by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and live and dead assay, and by measuring lactate dehydrogenase (LDH) release. To elucidate the mechanism underlying this effect, we monitored the immunofluorescence of intracellular ROS and measured malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GPx) activities. Continuous hypoxia protected the A549 cells from PQ-induced cytotoxicity. Continuous hypoxia for a period of 24 h significantly reduced intracellular ROS, decreased MDA concentration in the supernatant, and normalized SOD and GPx activities. Continuous hypoxia attenuated PQ-induced cell toxicity in A549 cells. This protective effect might be attributable to the suppression of PQ-induced ROS generation.
Apoptosis/drug effects ; Cell Line, Tumor ; Cell Proliferation/drug effects ; Flavonoids/pharmacology ; *Gene Expression Regulation, Neoplastic ; Hepatocyte Growth Factor/*pharmacology ; Humans ; Inhibitor of Apoptosis Proteins/antagonists & inhibitors/*genetics ; MAP Kinase Kinase Kinases/antagonists & inhibitors ; Neoplasm Invasiveness ; Promoter Regions, Genetic ; Protein Binding ; Proto-Oncogene Proteins c-jun/genetics/*metabolism ; Stomach Neoplasms/genetics/*metabolism/pathology ; Urokinase-Type Plasminogen Activator/*genetics

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 microM of H2O2 showed a 1.6-2.1 fold increment in HGF, but a little increment occurred at 500 microM of H2O2. It looks no dose dependent manner. Combined treatment with H2O2 and HGF, resulted in a slightly increased production of HGF compared to no treatment (control). Also, H2O2 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 H2O2, 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.
Cell Line, Tumor ; Fluorescent Dyes/chemistry ; Hepatocyte Growth Factor/pharmacology/*physiology ; Humans ; Hydrogen Peroxide/pharmacology ; Imidazoles/pharmacology ; Liver Neoplasms/drug therapy ; Mitogen-Activated Protein Kinases/antagonists & inhibitors/*metabolism ; Pyridines/pharmacology ; Reactive Oxygen Species/*metabolism ; Recombinant Proteins/pharmacology ; Urokinase-Type Plasminogen Activator/*biosynthesis ; rac1 GTP-Binding Protein/metabolism

PURPOSE: Rearrangement of the proto-oncogene rearranged during transfection (RET) has been newly identified potential driver mutation in lung adenocarcinoma. Clinically available tyrosine kinase inhibitors (TKIs) target RET kinase activity, which suggests that patients with RET fusion genes may be treatable with a kinase inhibitor. Nevertheless, the mechanisms of resistance to these agents remain largely unknown. Thus, the present study aimed to determine whether epidermal growth factor (EGF) and hepatocyte growth factor (HGF) trigger RET inhibitor resistance in LC-2/ad cells with CCDC6-RET fusion genes. MATERIALS AND METHODS: The effects of EGF and HGF on the susceptibility of a CCDC6-RET lung cancer cell line to RET inhibitors (sunitinib, E7080, vandetanib, and sorafenib) were examined. RESULTS: CCDC6-RET lung cancer cells were highly sensitive to RET inhibitors. EGF activated epidermal growth factor receptor (EGFR) and triggered resistance to sunitinib, E7080, vandetanib, and sorafenib by transducing bypass survival signaling through ERK and AKT. Reversible EGFR-TKI (gefitinib) resensitized cancer cells to RET inhibitors, even in the presence of EGF. Endothelial cells, which are known to produce EGF, decreased the sensitivity of CCDC6-RET lung cancer cells to RET inhibitors, an effect that was inhibited by EGFR small interfering RNA (siRNA), anti-EGFR antibody (cetuximab), and EGFR-TKI (Iressa). HGF had relatively little effect on the sensitivity to RET inhibitors. CONCLUSION: EGF could trigger resistance to RET inhibition in CCDC6-RET lung cancer cells, and endothelial cells may confer resistance to RET inhibitors by EGF. E7080 and other RET inhibitors may provide therapeutic benefits in the treatment of RET-positive lung cancer patients.
Adenocarcinoma/drug therapy/*genetics ; Cell Line, Tumor ; Cetuximab/pharmacology ; Drug Resistance, Neoplasm/drug effects/*genetics ; Epidermal Growth Factor/metabolism/*pharmacology ; *Gene Rearrangement ; Hepatocyte Growth Factor/*pharmacology ; Humans ; Indoles/pharmacology ; Lung Neoplasms/drug therapy/*genetics ; MAP Kinase Signaling System ; *Mutation ; Niacinamide/analogs & derivatives/pharmacology ; Phenylurea Compounds/pharmacology ; Piperidines/pharmacology ; Protein Kinase Inhibitors/therapeutic use ; Proto-Oncogene Proteins c-ret/*antagonists & inhibitors/genetics ; Pyrroles/pharmacology ; Quinazolines/pharmacology ; RNA, Small Interfering/pharmacology ; Receptor, Epidermal Growth Factor/genetics/metabolism ; Signal Transduction/drug effects ; fms-Like Tyrosine Kinase 3/metabolism

The regulatory mechanisms for the proliferation and the particular invasive phenotypes of stomach cancers are not still fully understood. Up-regulations of hepatocytes growth factor (HGF), its receptor (c-Met), and urokinase-type plasminogen activator (uPA) are correlated with the development and metastasis of cancers. In order to investigate roles of HGF/c-Met signaling in tumor progression and metastasis in stomach cancers, we determined effects of a specific MEK1 inhibitor (PD098059) and a p38 kinase inhibitor (SB203580) on HGF-mediated cell proliferation and uPA expression in stomach cancer cell lines (NUGC-3 and MKN-28). HGF treatment induced the phosphorylations of ERK and p38 kinase in time- and dose- dependent manners. Pre-treatment with PD098059 reduced HGF-mediated cell proliferation and uPA secretion. In contrast, SB203580 pre-treatment enhanced cell proliferation and uPA secretion due to induction of ERK phosphorylation. Stable expression of dominant negative-MEK1 in NUGC-3 cells showed a decrease in HGF-mediated uPA secretion. These results suggest that interaction of a MEK/ERK and a p38 kinase might play an important role in proliferation and invasiveness of stomach cancer cells.
Cell Line, Tumor ; Cell Proliferation/drug effects ; Culture Media, Serum-Free ; Dose-Response Relationship, Drug ; Enzyme Activation/drug effects ; Enzyme Inhibitors/pharmacology ; Extracellular Signal-Regulated MAP Kinases/metabolism ; Flavonoids/pharmacology ; Hepatocyte Growth Factor/*pharmacology ; Humans ; Imidazoles/pharmacology ; Kinetics ; MAP Kinase Kinase 1/metabolism ; Mitogen-Activated Protein Kinases/antagonists & inhibitors/*metabolism ; Neoplasm Metastasis ; Phosphorylation/drug effects ; Pyridines/pharmacology ; Research Support, Non-U.S. Gov't ; Stomach Neoplasms/*enzymology/*pathology ; Urinary Plasminogen Activator/*secretion ; p38 Mitogen-Activated Protein Kinases/metabolism