Oncogenic MST1R activity in pancreatic and gastric cancer represents a valid target of HSP90 inhibitors.

AIM: To evaluate the effects of HSP90 blockade by EC154 on the oncogenic receptor tyrosine kinase macrophage-stimulating 1 receptor (MST1R) in gastric and pancreatic cancer. MATERIALS AND METHODS: Impact of EC154 on signaling pathways was investigated by western blotting. Cancer cell migration was evaluated in Boyden chambers. Transcriptional regulation of MST1R was examined by using promoter-luciferase reporter constructs. Effects on MST1R expression, and tumor growth were investigated in in vivo tumor models. RESULTS: MST1R was expressed by cancer cells without evidence of MST1R mutations. EC154 led to an effective inhibition of cancer cell growth, down-regulated MST1R, diminished its promoter activity, and disrupted oncogenic macrophage-stimulating protein 1 (MSP1) signaling. Moreover, pro-migratory activities of cancer cells were dramatically inhibited. In vivo, treatment with EC154 significantly reduced tumor growth, while MST1R expression was down-regulated. CONCLUSION: Wild-type MST1R is an HSP90 client protein that can be targeted in gastrointestinal cancer using HSP90 inhibitors.

Upregulation of recepteur d'origine nantais tyrosine kinase and cell invasiveness via early growth response-1 in gastric cancer cells.

Abnormal accumulation and activation of the recepteur d'origine nantais (RON) has been implicated in carcinogenesis of epithelial tumors. RON expression was induced by the tumor promoter, phorbol 12-myristate 13-acetate (PMA), in gastric adenocarcinoma AGS cells. Studies with deleted and site-directed mutagenesis of Egr-1 promoter and with expression vectors encoding Egr-1 confirmed that Egr-1 is essential for RON expression. In addition, AGS cells pretreated with PMA showed remarkably enhanced invasiveness, which was partially abrogated by siRNA-targeted RON and Egr-1. These results suggest that tumor promoter induces RON expression via Egr-1, which, in turn, stimulates cell invasiveness in AGS cells.

Ascorbic acid suppresses the 2,3,7,8-tetrachloridibenxo-p-dioxin (TCDD)-induced CYP1A1 expression in human HepG2 cells.

The mechanisms involving the inhibitory effects of ascorbic acid (AA) on carcinogenesis have not fully defined, except for its free-radical scavenging activity against oxidative DNA damage. In this study, we examined the effects of AA on the expression of the aryl hydrocarbon receptor (AhR)-regulated gene cytochrome P4501A1 (CYP1A1), which catalyzes the activation of genotoxic metabolites that can lead to mutagenesis. Cultured human HepG2 cells were incubated with AA with or without the potent AhR agonist/CYP1A1 inducer 2,3,7,8-tetrachloridibenxo-p-dioxin (TCDD). AA was highly effective at suppressing CYP1A1 induction following coincubation of the cells with 1nM TCDD. The preventive effects of AA were seen at the level of mRNA and protein expression as well as CYP1A1-specific 7-ethoxyresorufin O-deethylase (EROD) activity. A transient transfection assay using a dioxin response element (DRE)-linked luciferase reporter and an electrophoretic mobility shift assay revealed that AA reduced the amount of AhR that could form a complex with the DRE sequence in the promoter region of the CYP1A1 gene. In addition, AA inhibited the TCDD-induced Ecto-ATPase activity, which is known to be requiring for AhR translocation to the nucleus. These results suggest that AA may exert at least part of its anticarcinogenesis effect by controlling the expression of CYP1A1 at the transcription level.

EGF stimulates uPAR expression and cell invasiveness through ERK, AP-1, and NF-kappaB signaling in human gastric carcinoma cells.

Overexpression of epidermal growth factor (EGF) and urokinase plasminogen activator receptor (uPAR) have been observed in human gastric cancers. However, the interaction between EGF and uPAR in gastric cancer has not been well elucidated. In this study, we investigated the effect of EGF on uPAR expression and the underlying signal pathways in human gastric cancer AGS cells. EGF induced uPAR mRNA expression in a time- and concentration-dependent manner. EGF also induced uPAR promoter activity. In addition, EGF induced the activation of extracellular signal regulated kinase-1/2 (ERK-1/2) and P38 mitogen-activated protein kinase (MAPK) but not the activation of c-Jun amino terminal kinase. A specific inhibitor of MEK-1 (an upstream effector of ERK-1/2) and a dominant negative MEK-1 were able to suppress the EGF-induced uPAR promoter activity. Site-directed mutagenesis and electrophoretic mobility shift assays demonstrated that the binding sites of transcription factors, activator protein-1 (AP-1) and nuclear factor (NF)-kappaB, are involved in the EGF-induced uPAR transcription. Suppression of the EGF-induced uPAR promoter activity by the AP-1 decoy oligonuclotide, as well as expression vectors encoding mutated-type NF-kappaB-inducting kinase and I-kappaB, confirmed that the activation of AP-1 and NF-kappaB are essential for the EGF-induced uPAR upregulation. The AGS cells pretreated with EGF showed a remarkably enhanced invasiveness and this effect was partially abrogated by uPAR neutralizing antibodies and by the inhibitors of ERK-1/2, AP-1, and NF-kappaB. The above results suggest that EGF induces uPAR expression via ERK-1/2, AP-1, and NF-kappaB signaling pathways and, in turn, stimulates cell invasiveness in human gastric cancer AGS cells.

Triptolide inhibits tumor promoter-induced uPAR expression via blocking NF-kappaB signaling in human gastric AGS cells.

The overexpression of urokinase-type plasminogen activator receptor (uPAR) is closely related to tumor cell invasion. Therefore, strategies for down-regulating uPAR expression may be of clinical utility. This study examined the effects of triptolide, which is a diterpenoid triepoxide extracted from the Chinese herb Tripterygium wilfordii Hook F., on the induction of uPAR in human gastric cancer AGS cells. Triptolide inhibited the phorbol 12-myristate 13-acetate (PMA)-induced uPAR mRNA and protein expression in a dose-dependent manner, and reduced the uPAR transcriptional activity. The stability of the uPAR transcripts was not altered by the triptolide treatment. The signals involved in uPAR induction by PMA were investigated to determine the mechanisms for the triptolide-mediated regulation of uPAR. The inhibitors of extracellular signal-regulated kinases 1 and 2 (Erk-1/2, PD98059), c-Jun N-terminal kinases (JNK, SP600125) and nuclear factor-kappa B (NF-kappaB, Bay11-7082) inhibited the PMA-induced expression of uPAR, which suggests that PMA induces uPAR through multiple signals. Triptolide suppressed the PMA-induced activation of NF-kappaB but not Erk-1/2 and JNKI The inhibitory effect of triptolide on the activation of NF-kappaB was confirmed by an electrophoretic mobility shift assay and NF-kappaB dependent transcription studies. AGS cells treated with PMA showed a remarkably enhanced invasiveness, which was partially abrogated by triptolide and uPAR-neutralizing antibodies. This suggests that triptolide may exert at least part of its anti-invasive effect in gastric cancer by controlling the expression of uPAR through the suppression of NF-kappaB activation.

Helicobacter pylori stimulates urokinase plasminogen activator receptor expression and cell invasiveness through reactive oxygen species and NF-kappaB signaling in human gastric carcinoma cells.

The gastric pathogen, helicobacter pylori (H. pylori), has been associated with the progression of gastric cancer. It was previously reported that H. pylori induced urokinase plasminogen activator receptor (uPAR) expression and stimulated cell invasiveness in human gastric cancer AGS cells. However, the precise mechanisms for how H. pylori upregulates uPAR are unclear. This study investigated the underlying signal pathways in H. pylori-induced uPAR in human gastric cancer AGS cells. The intracellular H2O2 content, as determined using H2O2-sensitive probe 2',7'-dichlorodihydrofluorescein, increased after the H. pylori treatment. N-acetyl cysteine (NAC), an antioxidant, prevented the H. pylori-induced production of H2O2 and uPAR expression. In addition, exogenous H2O2 was found to increase uPAR mRNA expression and its promoter activity. Site-directed mutagenesis of the potential NF-kappaB element in the uPAR promoter showed that the redox-sensitive transcription factor NF-kappaB was essential for H. pylori-induced uPAR expression. The expression of vectors encoding a mutated-type NF-kappaB-inducing kinase and I-kappaB, and a specific inhibitor of NF-kappaB (BAY11-7082) decreased the H. pylori-induced uPAR promoter activity. Chromatin immunoprecipitation and the electrophoretic mobility shift assay confirmed that H. pylori increased the DNA binding activity of NF-kappaB. With the aid of NAC and H2O2, it was determined that reactive oxygen species (ROS) is an upstream signaling molecule for activating the NF-kappaB induced by H. pylori. The enhanced AGS cell invasiveness by H. pylori was partially abrogated by an NAC and BAY11-7082 treatment. These results suggest that the ROS and NF-kappaB signaling pathway is important in H. pylori-induced uPAR expression and the increased cell invasiveness of human gastric cancer AGS cells.

(-)-Epigallocatechin-3-gallate inhibits monocyte chemotactic protein-1 expression in endothelial cells via blocking NF-kappaB signaling.

Monocyte chemotactic protein-1 (MCP-1) is a potent chemoattractant for monocytes and plays a key role in various inflammatory responses, including atherosclerosis. In this study, we examined the effect of (-)-epigallocatechin-3-gallate (EGCG), a major green tea catechin, on the expression of MCP-1 in human endothelial ECV304 cells. EGCG markedly inhibited the phorbol 12-myristate 13-acetate (PMA)-induced MCP-1 mRNA and protein levels in a dose-dependent manner. EGCG was also found to reduce the MCP-1 transcriptional activity. The upregulation of MCP-1 by PMA was significantly inhibited by blockade of P38 mitogen-activated protein kinase (MAPK) and NF-kappaB, but not by blockade of extracellular-signal-regulated kinase and c-Jun N-terminal kinase pathway. Furthermore, The PMA-induced p38 MAPK and NF-kappaB activation were obviously attenuated after pretreating ECV304 cells with EGCG. The conditioned media from the endothelial ECV304 cells treated with PMA could remarkably stimulate the migration of THP-1 monocytes and this effect was partially abrogated by MCP-1 neutralizing antibodies. Moreover, the media from the EGCG-pretreated ECV304 cells lost the stimulatory activity for THP-1 migration. These results suggest that EGCG may exert an anti-inflammatory effect in endothelial cells by controlling MCP-1 expression, at least in part, mediated through the suppression of p38 MAPK and NF-kappaB activation.

Epigallocatechin-3-gallate inhibits the PDGF-induced VEGF expression in human vascular smooth muscle cells via blocking PDGF receptor and Erk-1/2.

Platelet-derived growth factor (PDGF) has been known to induce vascular endothelial growth factor (VEGF) expression in human vascular smooth muscle cells (hVSMCs). We previously reported that Erk-1/2 and AP-1 pathways are crucial in the PDGF-induced VEGF expression in hVSMCs . In this study, we investigated the effect of epigallocatechin-3-gallate (EGCG), the major green tea catechin, on the PDGF-induced VEGF expression in hVSMCs and the underlying mechanisms. EGCG were found to inhibit dose-dependently the VEGF expression and activation of PDGF receptor, Erk-1/2 and AP-1 induced by PDGF. In addition, cell free studies demonstrated that EGCG could directly inhibit the Erk-1/2 activity. Conditioned media from the hVSMCs treated with PDGF could remarkably stimulate the in vitro growth of human umbilical vein endothelial cells (HUVECs) but the media from the EGCG-pretreated hVSMCs lost its stimulatory activity for HUVEC proliferation. These results suggest that EGCG may exert the anti-angiogenic effect by inhibiting the PDGF-induced VEGF expression at multiple signaling levels.

Extracellular signal-regulated kinases and AP-1 mediate the up-regulation of vascular endothelial growth factor by PDGF in human vascular smooth muscle cells.

Endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) have been shown to communicate with each other via cytokine signaling during neovascularization. In this study, we investigated the effect of platelet-derived growth factor (PDGF), a cytokine released from tumors and ECs, on vascular endothelial growth factor (VEGF) expression in human VSMCs and underlying signal transduction pathways. PDGF induced VEGF expression in a time- and concentration-dependent manner. PDGF induced the activation of extra-cellular signal-regulated kinase-1/2 (ERK-1/2), but not the activation of c-jun amino terminal kinase (JNK) and P38 mitogen-activated protein kinase (MAPK). Specific inhibitor of mitogen-activated protein kinase kinase (MEK)-1 was found to suppress VEGF expression and promoter activity. The expression of vectors encoding a mutated-type MEK-1 decreased the VEGF promoter activity. Electrophoretic mobility shift assay revealed that PDGF dose-dependently increased the DNA binding activity of AP-1. Transient transfection studies using an AP-1 decoy oligonucleotide confirmed that the activation of AP-1 is involved in PDGF-induced VEGF upregulation. Conditioned media from the human VSMCs pretreated with PDGF could remarkably stimulate the in vitro growth of human umbilical vein endothelial cells and this effect was partially abrogated by VEGF neutralizing antibodies. The above results suggest that ERK-1/2 and AP-1 signaling pathways are involved in the PDGF-induced VEGF expression in human VSMCs and that these paracrine signaling pathways induce endothelial cell proliferation.

Urokinase plasminogen activator receptor is upregulated by Helicobacter pylori in human gastric cancer AGS cells via ERK, JNK, and AP-1.

The gastric pathogen Helicobacter pylori (H. pylori) is suggested to be associated with gastric cancer progression. In this study, we investigated the effect of H. pylori on urokinase plasminogen activator receptor (uPAR) expression which has been known to correlate closely with gastric cancer invasion. H. pylori induced the uPAR expression in a time- and concentration-dependent manner. Specific inhibitors and inactive mutants of MEK-1 and JNK were found to suppress the H. pylori-induced uPAR expression and the uPAR promoter activity. Electrophoretic mobility shift assay and transient transfection study using an AP-1 decoy oligonucleotide confirmed that the activation of AP-1 is involved in the H. pylori-induced uPAR upregulation. The AGS cells treated with H. pylori showed a remarkably enhanced invasiveness, and this effect was partially abrogated by uPAR-neutralizing antibodies. These results suggest that H. pylori induces uPAR expression via Erk-1/2, JNK, and AP-1 signaling pathways and, in turn, stimulates the cell invasiveness in human gastric cancer AGS cells.

Extracellular signal-regulated kinase and AP-1 pathways are involved in reactive oxygen species-induced urokinase plasminogen activator receptor expression in human gastric cancer cells.

Overexpression of urokinase plasminogen activator receptor (uPAR) is known to correlate closely with tumor cell invasion and metastasis. In gastric cancer, however, the mechanism for induction of uPAR remains to be elucidated. In this study, to investigate the effect of reactive oxygen species (ROS) on uPAR expression and the underlying signal pathways in human gastric cancer AGS cells, phenazine methosulfate (PMS) and H2O2 were used as ROS generator. PMS and H2O2 induced the uPAR expression in time- and concentration-dependent manner. PMS and H2O2 also induced the activation of extracellular signal-regulated kinases (Erk)-1/2. A specific inhibitor of MEK-1 (PD980590) was found to suppress the PMS-induced uPAR expression and the uPAR promoter activity. Expression of vectors encoding a mutated-type MEK-1 resulted in decreases in the uPAR promoter activity. Electrophoretic mobility shift assay revealed that PMS increased time-dependently the DNA binding activity of AP-1. Transient transfection studies using AP-1 decoy confirmed that the activation of AP-1 is involved in PMS-induced uPAR upregulation. The AGS cells pretreated with PMS showed a remarkably enhanced invasiveness and this effect was partially abrogated by uPAR neutralizing antibodies. The above results suggest that ROS induces uPAR expression via Erk-1/2 and AP-1 signaling pathways and, in turn, stimulates the cell invasiveness in human gastric cancer AGS cells.

Interleukin-1beta stimulates IL-8 expression through MAP kinase and ROS signaling in human gastric carcinoma cells.

Recent studies have suggested that the expression of interleukin-8 (IL-8) directly correlates with the vascularity of human gastric carcinomas. In this study, the effect of IL-1beta on IL-8 expression in human gastric cancer TMK-1 cells and the underlying signal transduction pathways were investigated. IL-1beta induced the IL-8 expression in a time- and concentration-dependent manner. IL-1beta induced the activation of extracellular signal-regulated kinases-1/2 and P38 mitogen-activated protein kinase (MAPK), but not the activation of c-jun amino-terminal kinse and Akt. Specific inhibitors of MEK-1 (PD980590) and P38 MAPK (SB203580) were found to suppress the IL-8 expression and the IL-8 promoter activity. Expression of vectors encoding a mutated-type MEK-1 and P38 MAPK resulted in decrease in the IL-8 promoter activity. IL-1beta also induced the production of reactive oxygen species (ROS). N-acetyl cysteine (NAC) prevented the IL-1beta-induced ROS production and IL-8 expression. In addition, exogenous H2O2 could induce the IL-8 expression. Deletional and site-directed mutagenesis studies on the IL-8 promoter revealed that activator protein-1 (AP-1) and nuclear factor (NF)-kappaB sites were required for the IL-1beta-induced IL-8 transcription. Electrophoretic mobility shift assay confirmed that IL-1beta increased the DNA-binding activity of AP-1 and NF-kappaB. Inhibitor (PD980590, SB203580) and ROS scavenger (NAC) studies revealed that the upstream signalings for the transcription factors AP-1 and NF-kappaB were MAPK and ROS, respectively. Conditioned media from the TMK-1 cells pretreated with IL-1beta could remarkably stimulate the in vitro growth of HUVEC and this effect was partially abrogated by IL-8-neutralizing antibodies. The above results suggest that MAPK-AP-1 and ROS-NF-kappaB signaling pathways are involved in the IL-1beta-induced IL-8 expression and that these paracrine signaling pathways induce endothelial cell proliferation.

EGCG blocks tumor promoter-induced MMP-9 expression via suppression of MAPK and AP-1 activation in human gastric AGS cells.

Overexpression of matrix metalloproteinases (MMPs) has been known to correlate closely with tumor cell invasion and strategies to down-regulate their expression may ultimately be of clinical utility. In this study, we investigated the effects of (-)-epigallocatechin gallate (EGCG), a major green tea catechin, on the cell invasiveness and MMP-9 induction in human gastric cancer AGS cells. EGCG inhibited the phorbol 12-myristate 13-acetate (PMA)-induced cell invasiveness and MMP-9 expression in a dose-dependent manner. EGCG treatment was found to reduce the MMP-9 transcriptional activity. To further study the mechanisms for the EGCG-mediated regulation of MMP-9, the effects of EGCG on transcription factor AP-1 and mitogen-activated protein kinase (MAPK) activities were examined. The results showed that EGCG suppressed the PMA-induced AP-1 activation. EGCG also abrogated the PMA-induced activation of extracellular-regulated protein kinase (Erk) and c-jun N-terminal kinase (JNK), which are upstream modulators of AP-1. These results suggest that EGCG may exert at least part of its anti-invasive effect in gastric cancer by controlling MMP expression through the suppression of MAPK and AP-1 activation.

Interleukin-1beta regulates angiopoietin-1 expression in human endothelial cells.

Angiopoietin (Ang)-1 is an important regulator of endothelial cell (EC) survival and stabilization. Ang-1 exerts its biological effects by binding to the EC-specific tyrosine kinase receptor Tie-2, and initiates intracellular signaling in ECs. However, regulatory mechanisms for endothelial Ang-1 expression have not been completely elucidated. In this study, we investigated the effects of angiogenic cytokines and growth factors on Ang-1 expression in human umbilical vein ECs (HUVECs). Northern blot analysis was performed after HUVECs were exposed to interleukin-1beta (IL-1beta), tumor necrosis factor-alpha, platelet-derived growth factor-BB, insulin-like growth factor-1, or vascular endothelial growth factor (VEGF). Both IL-1beta and tumor necrosis factor-alpha caused marked down-regulation of Ang-1 mRNA levels at 4 h with a further decrease observed at 24 h. Using signaling inhibitors, we identified the P38 pathway as the pathway that mediates IL-1beta down-regulation of Ang-1. Furthermore, treatment of cells with IL-1beta indirectly (via down-regulation of Ang-1) led to a decrease in Tie-2 autophosphorylation levels in HUVECs. We previously demonstrated that IL-1beta regulates VEGF expression in tumor cells. This observation was confirmed in ECs in the present study. Because pericytes play a role in regulating EC function, we also determined whether IL-1beta would also down-regulate Ang-1 in human vascular smooth muscle cells. Similar to our findings in HUVECs, we found that IL-1beta decreased Ang-1 expression in human vascular smooth muscle cells. Direct effects of IL-1beta on angiogenesis were investigated by use of an in vivo Gelfoam angiogenesis assay in which IL-1beta produced a significant increase in vessel counts (P = 0.0189). These results suggest that IL-1beta indirectly regulates angiogenesis by modulating the expression of Ang-1. IL-1beta may trigger a proangiogenic response by decreasing Ang-1 levels in ECs and pericytes and up-regulating VEGF in ECs and tumor cells.

Regulation of urokinase plasminogen activator by epigallocatechin-3-gallate in human fibrosarcoma cells.

(-)-Epigallocatechin-3-gallate (EGCG), a main flavanol of green tea, potently suppressed the urokinase-type plasminogen activator (uPA) expression in human fibrosarcoma HT 1080 cells. EGCG induced not only the suppression of the uPA promoter activity but also the destabilization of uPA mRNA. EGCG inhibited the phosphorylation of extracellular signal-regulated kinases 1 and 2 (Erk-1/2) and P38 mitogen-activated protein kinase (MAPK), but not the phosphorylation of c-jun N-terminal kinase (JNK) and Akt. Specific inhibitors of Erk-1/2 (2'-amino-3'-methoxyflavone, PD98059) and P38 MAPK (pyridinylimidazole, SB203580) were found to suppress the uPA expression and the uPA promoter activity. However, the specific inhibitors did not affect the uPA mRNA stability. These results suggest that EGCG could regulate the uPA expression by at least two different mechanisms: EGCG may inhibit the Erk-1/2 and P38 MAPK, leading to suppression of the uPA promoter activity, and EGCG may destabilize the uPA mRNA in an Erk-1/2- and p38 MAPK-independent way.

Involvement of NF-kappaB and caspases in silibinin-induced apoptosis of endothelial cells.

Silibinin, the flavonoid found in the milk thistle, has been shown to suppress cell growth and exhibit anti-cancer effects. Some flavonoids were reported to inhibit angiogenesis which is essential for tumor growth and metastasis. In this study, to clarify the underlying mechanisms for the anti-cancer effect of silibinin, we examined the effects of silibinin on human endothelial ECV304 cells. Silibinin was found to suppress the growth and induce the apoptosis of ECV304 cells. The induction of apoptosis by silibinin was confirmed by ladder-patterned DNA fragmentation, cleaved and condensed nuclear chromatin and DNA hypoploidy. Silibinin could effectively inhibit constitutive NF-kappaB activation as revealed by electrophoretic mobility shift assay and NF-kappaB-dependent luciferase reporter study. Consistent with this, silibinin treatment resulted in a significant decrease in the nuclear level of p65 subunit of NF-kappaB. In addition, silibinin treatment caused a change in the ratio of Bax/Bcl-2 in a manner that favors apoptosis. Silibinin also induced the cytochrome c release, activation of caspase-3 and caspase-9 and cleavage of PARP. These results suggest that silibinin may exert, at least partly, its anti-cancer effect by inhibiting angiogenesis through induction of endothelial apoptosis via modulation of NF-kappaB, Bcl-2 family and caspases.

Angiogenesis and antiangiogenic therapy of colon cancer liver metastasis.

The fact that tumor growth and metastatic spread relies on angiogenesis has been widely proven and accepted. The understanding of cancer biology and metastasis formation has led to the development of new therapeutic approaches that target tumor biology. The survival and establishment of metastatic lesions depend on a shift in the normal balance of proangiogenic and antiangiogenic factors that favor angiogenesis. Colorectal cancer is one of the leading cancer deaths worldwide. Angiogenesis has been associated with colon cancer progression and metastatic spread, thereby significantly affecting patient survival. New experimental approaches that inhibit angiogenic processes have demonstrated promising antineoplastic effects on metastatic colorectal cancer and are partially being investigated in clinical trials. This review focuses on angiogenesis in colorectal cancer metastasis formation as a target for antiangiogenic therapy, describing the experience from experimental studies and current clinical trials.

P38 MAPK pathway is involved in the urokinase plasminogen activator expression in human gastric SNU-638 cells.

Overexpression of urokinase plasminogen activator (uPA) is known to correlate closely with tumor cell invasion and metastasis. In gastric cancer, however, the mechanism for induction of uPA remains to be elucidated. In this study, we investigated the intracellular signaling for uPA expression in human gastric carcinoma cells (AGS, SNU-1, SNU-5, and SNU-638). SNU-638 cells which expressed a high level of uPA was found to be highly invasive on a matrigel, while AGS, SNU-1, and SNU-5 cells with low levels of uPA expression were only slightly invasive. SNU-638 cells showed a much higher P38 MAPK activity than the 3 other cell lines. However, there was no significant difference in the activities of P44/42 MAPK (Erk-1/2), JNK and Akt among the above cell lines. Treatment of SNU-638 cells with SB203580, a specific P38 MAPK inhibitor, reduced both the promoter activity and mRNA expression of uPA. Expression of a vector encoding a mutated-type P38alpha MAPK resulted in decrease in the uPA promoter activity in SNU-638 cells. These results suggest that P38 MAPK signaling pathway is important for uPA expression in gastric SNU-638 cells by enhancing the promoter activity of uPA.

Cyclooxygenase-2 is up-regulated by interleukin-1 beta in human colorectal cancer cells via multiple signaling pathways.

Overexpression of cyclooxygenase-2 (COX-2) has been observed in human colorectal cancer. COX-2 expression in human tumors can be induced by growth factors, cytokines, oncogenes, and other factors. The mechanisms regulating COX-2 expression in human colon cancer have not been completely elucidated. We hypothesized that the proinflammatory cytokine interleukin-1 beta (IL-1 beta) mediates COX-2 expression in HT-29 human colon cancer cells. Treatment of HT-29 cells with IL-1 beta induced expression of COX-2 mRNA and protein in a time- and dose-dependent manner. Inhibitors of the extracellular signal-regulated kinase 1/2, c-Jun NH(2)-terminal kinase, P38 mitogen-activated protein kinase, and nuclear factor-kappa B (NF-kappa B) signaling pathways blocked the ability of IL-1 beta to induce COX-2 mRNA. In contrast, Wortmannin, a phosphoinositide 3-kinase inhibitor upstream of protein kinase B/Akt, led to a slight increase in COX-2 mRNA expression after IL-1 beta treatment. Electrophoretic mobility shift assay on nuclear extracts demonstrated that IL-1 beta induced NF-kappa B DNA binding activity in HT-29 cells, and the activated NF-kappa B complex was eliminated after treatment with an inhibitor of NF-kappa B. Supershift assay indicated that the two NF-kappa B subunits, p65 and p50, were involved in activation of NF-kappa B complex by IL-1 beta stimulation. The stability of COX-2 mRNA was not altered by IL-1 beta treatment. These data demonstrate that IL-1 beta induces COX-2 expression in HT-29 cells through multiple signaling pathways and NF-kappa B.

Blockade of insulin-like growth factor I receptor function inhibits growth and angiogenesis of colon cancer.

PURPOSE AND EXPERIMENTAL DESIGN: Insulin-like growth factors (IGFs) I and II and their principle receptor, IGF-I receptor (IGF-IR), are frequently expressed in human colon cancers and play a role in preventing apoptosis, enhancing cell proliferation, and inducing expression of vascular endothelial growth factor (VEGF). To elucidate the in vitro and in vivo effects of IGF-IR in human colon cancer growth and angiogenesis, HT29 cells were transfected with a truncated dominant-negative (DN) form of IGF-IR or vector alone. RESULTS: IGF-I increased VEGF expression in parental and vector-transfected cells, whereas IGF-I induction of VEGF mRNA and protein was abrogated in IGF-IR DN cells. The IGF-IR DN cells demonstrated inhibited growth in both monolayer culture and soft agar (P < 0.05). s.c. injections of IGF-IR DN cells in nude mice led to significantly decreased tumor growth (P < 0.05). Immunohistochemical analyses revealed that IGF-I DN tumors demonstrated decreased tumor cell proliferation, VEGF expression, and vessel count and increased tumor cell apoptosis (P < 0.05 for all parameters compared with controls). Furthermore, IGF-IR DN-transfected cells yielded significantly decreased tumorigenicity and growth in the liver. CONCLUSIONS: These studies demonstrate that the IGF ligand-receptor system plays an important role in multiple mechanisms that mediate human colon cancer growth including regulation of VEGF and angiogenesis.