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Biomedical subjects

G E Gallick

Publications and source records attributed to G E Gallick.

At least 19 recordsLinked to original sources
PubMed2006

Vascular endothelial growth factor receptor-1 mediates migration of human colorectal carcinoma cells by activation of Src family kinases.

Vascular endothelial growth factor (VEGF) is the predominant pro-angiogenic cytokine in human malignancy, and its expression correlates with disease recurrence and poor outcomes in patients with colorectal cancer. Recently, expression of vascular endothelial growth factor receptors (VEGFRs) has been observed on tumours of epithelial origin, including those arising in the colon, but the molecular mechanisms governing potential VEGF-driven biologic functioning in these tumours are not well characterised. In this report, we investigated the role of Src family kinases (SFKs) in VEGF-mediated signalling in human colorectal carcinoma (CRC) cell lines. Vascular endothelial growth factor specifically activated SFKs in HT29 and KM12L4 CRC cell lines. Further, VEGF stimulation resulted in enhanced cellular migration, which was effectively blocked by pharmacologic inhibition of VEGFR-1 or Src kinase. Correspondingly, migration studies using siRNA clones with reduced Src expression confirmed the requirement for Src in VEGF-induced migration in these cells. Furthermore, VEGF treatment enhanced VEGFR-1/SFK complex formation and increased tyrosine phosphorylation of focal adhesion kinase, p130 cas and paxillin. Finally, we demonstrate that VEGF-induced migration is not due, at least in part, to VEGF acting as a mitogen. These results suggest that VEGFR-1 promotes migration of tumour cells through a Src-dependent pathway linked to activation of focal adhesion components that regulate this process.

Adenocarcinoma↗
PubMed2005

Overexpression of neuropilin-1 promotes constitutive MAPK signalling and chemoresistance in pancreatic cancer cells.

Neuropilin-1 (NRP-1) is a novel co-receptor for vascular endothelial growth factor (VEGF). Neuropilin-1 is expressed in pancreatic cancer, but not in nonmalignant pancreatic tissue. We hypothesised that NRP-1 expression by pancreatic cancer cells contributes to the malignant phenotype. To determine the role of NRP-1 in pancreatic cancer, NRP-1 was stably transfected into the human pancreatic cancer cell line FG. Signal transduction was assessed by Western blot analysis. Susceptibility to anoikis (detachment induced apoptosis) was evaluated by colony formation after growth in suspension. Chemosensitivity to gemcitabine or 5-fluorouracil (5-FU) was assessed by MTT assay in pancreatic cancer cells following NRP-1 overexpression or siRNA-induced downregulation of NRP-1. Differential expression of apoptosis-related genes was determined by gene array and further evaluated by Western blot analysis. Neuropilin-1 overexpression increased constitutive mitogen activated protein kinase (MAPK) signalling, possibly via an autocrine loop. Neuropilin-1 overexpression in FG cells enhanced anoikis resistance and increased survival of cells by > 30% after exposure to clinically relevant levels of gemcitabine and 5-FU. In contrast, downregulation of NRP-1 expression in Panc-1 cells markedly increased chemosensitivity, inducing > 50% more cell death at clinically relevant concentrations of gemcitabine. Neuropilin-1 overexpression also increased expression of the antiapoptotic regulator, MCL-1. Neuropilin-1 overexpression in pancreatic cancer cell lines is associated with (a) increased constitutive MAPK signalling, (b) inhibition of anoikis, and (c) chemoresistance. Targeting NRP-1 in pancreatic cancer cells may downregulate survival signalling pathways and increase sensitivity to chemotherapy.

Antineoplastic Agents, Alkylating↗
PubMed2001

In vivo intracellular signaling as a marker of antiangiogenic activity.

Alterations in endothelial cell (EC) signaling could serve as a marker of effective antiangiogenic therapy. We determined the effect of an antiangiogenic tyrosine kinase inhibitor, SU6668, on tumor EC signaling in liver metastases in mice. In vitro immunofluorescence verified that pretreatment of ECs with SU6668 before exposure to VEGF decreased in vitro phosphorylation of Erk and Akt. Using double-fluorescence immunohistochemistry, phosphorylated Erk and Akt were constitutively expressed in ECs in liver metastases in untreated mice, but SU6668 blocked activation of these signaling intermediates. Determining the activation status of the Erk and Akt signaling pathways in tumor ECs may serve as a surrogate marker for the effectiveness of antiangiogenic regimens.

Androstadienes↗
PubMed2001

EGCG, a major component of green tea, inhibits tumour growth by inhibiting VEGF induction in human colon carcinoma cells.

Catechins are key components of teas that have antiproliferative properties. We investigated the effects of green tea catechins on intracellular signalling and VEGF induction in vitro in serum-deprived HT29 human colon cancer cells and in vivo on the growth of HT29 cells in nude mice. In the in vitro studies, (-)-epigallocatechin gallate (EGCG), the most abundant catechin in green tea extract, inhibited Erk-1 and Erk-2 activation in a dose-dependent manner. However, other tea catechins such as (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECG), and (-)-epicatechin (EC) did not affect Erk-1 or 2 activation at a concentration of 30 microM. EGCG also inhibited the increase of VEGF expression and promoter activity induced by serum starvation. In the in vivo studies, athymic BALB/c nude mice were inoculated subcutaneously with HT29 cells and treated with daily intraperitoneal injections of EC (negative control) or EGCG at 1.5 mg day(-1)mouse(-1)starting 2 days after tumour cell inoculation. Treatment with EGCG inhibited tumour growth (58%), microvessel density (30%), and tumour cell proliferation (27%) and increased tumour cell apoptosis (1.9-fold) and endothelial cell apoptosis (3-fold) relative to the control condition (P< 0.05 for all comparisons). EGCG may exert at least part of its anticancer effect by inhibiting angiogenesis through blocking the induction of VEGF.

Animals↗
PubMed2001

Vascular endothelial growth factor is upregulated by interleukin-1 beta in human vascular smooth muscle cells via the P38 mitogen-activated protein kinase pathway.

Small tumor vessels are composed of endothelial cells (ECs) and vascular smooth muscle cells (VSMCs). These cells have been shown to communicate with each other via cytokine signaling during neovascularization. We previously demonstrated that interleukin-1 beta (IL-1 beta) leads to induction of vascular endothelial growth factor (VEGF) in human colon carcinoma cells. As pericytes play a role in regulating EC function, we hypothesized that IL-1 beta may mediate EC survival by induction of VEGF in a paracrine manner. We investigated the effects of IL-1 beta on VEGF expression in human VSMCs (hVSMCs) and the signal transduction pathways that may be involved. Treatment of hVSMCs with IL-1 beta induced VEGF expression in a time- and concentration-dependent manner and increased both the VEGF promoter activity and the mRNA half-life. Treatment with IL-1 beta induced the expression of P38 mitogen-activated protein kinase (MAPK) within 5 min but did not activate extracellular signal-regulated kinases (Erk)-1/2, c-jun amino terminal kinase (JNK), or Akt. SB203580, a specific P38 MAPK inhibitor, blocked the ability of IL-1 beta to induce VEGF mRNA and promoter activity. Conditioned media from hVSMCs pretreated with IL-1 beta prevented apoptosis of ECs, an effect that was partially abrogated by VEGF-neutralizing antibodies. These data demonstrate that IL-1 beta may induce VEGF in hVSMCs, and suggest that this paracrine signaling pathway, may prevent, in part, apoptosis of ECs.

Cell Survival↗
PubMed2001

Differential effects of phosphatidylinositol-3/Akt-kinase inhibition on apoptotic sensitization to cytokines in LNCaP and PCc-3 prostate cancer cells.

Alterations in phosphatidylinositol 3'-kinase (PI3'-kinase) and Akt activation frequently occur in prostate cancer and may disrupt apoptotic induction by such cytokines as tumor necrosis factor (TNF) and TNF-related apoptosis-inducing ligand (TRAIL). To examine the role of PI3' phosphorylation in the cellular response to cytokines, two prostate cancer cell lines with constitutively activated PI3'-kinase cascades (LNCaP and PC-3) were examined for direct sensitivity to cytokines. TNF or TRAIL alone failed to activate apoptosis in either LNCaP or PC-3 cells, and drug-mediated inhibition of the PI3k/Akt cascade caused only minimal activation of apoptosis in either cell line. Suppression of PI3'-kinase/Akt signaling markedly enhanced the apoptotic activity of both TNF and TRAIL in LNCaP cells but not in PC-3 cells. Adenovirus-mediated PTEN/MMAC1 expression in LNCaP cells reduced Akt activation, activated apoptosis, and sensitized cells to TNF but not to TRAIL. Together, these results suggest that PI3'-kinase signaling inhibits both TNF-mediated and TRAIL-mediated apoptosis but may represent one of several apoptotic resistance mechanisms that inhibit cytokine-mediated killing of prostate cancer cells.

Androstadienes↗
PubMed1999

Extracellular signal-regulated kinase activation is required for up-regulation of vascular endothelial growth factor by serum starvation in human colon carcinoma cells.

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor important for colon cancer neovascularization. In previous studies, serum starvation led to induction of VEGF in human colon carcinoma cells. We investigated the possible participation of mitogen-activated protein kinases in serum starvation induction of VEGF in the HT29 human colon carcinoma cell line. The extracellular signal-regulated kinases (Erks) 1 and 2 were activated after 3-6 h of serum starvation. Using transient transfection of VEGF promoter-reporter constructs, serum starvation led to an increase in VEGF promoter activity. An inhibitor of phosphorylation of Erk-1/2 blocked the increase of VEGF expression and promoter activity induced by serum starvation. Serum starvation activates several mitogen-activated protein kinases, but activation of Erk-1/2 is critical for the up-regulation of VEGF mRNA in colon carcinoma cells.

Cell Line↗
PubMed1999

Transcriptional induction of the urokinase receptor gene by a constitutively active Src. Requirement of an upstream motif (-152/-135) bound with Sp1.

Since c-src overexpression increases colonic cell invasiveness and because both Src activity and urokinase receptor protein are elevated in invasive colon cancers, the present study was undertaken: 1) to determine if a constitutively active Src regulates urokinase receptor expression and 2) to identify required cis-elements and trans-acting factors. SW480 colon cancer cells transfected with an expression plasmid (c-srcY527F) encoding a constitutively active Src protein manifested increased urokinase receptor gene expression and Src activity. Treatment of the src transfectants with a Src-inhibitor (PD173955) reduced urokinase receptor protein levels and laminin degradation. Inasmuch as we recently implicated an upstream region of the urokinase receptor promoter (-152/-135) in constitutive urokinase receptor expression, we determined its role for the induction by src. Whereas the activity of a CAT reporter driven by this region was stimulated by c-srcY527F, the u-PAR promoter mutated at the Sp1-binding motif in the -152/-135 region was not. Nuclear extracts from the src transfectants demonstrated increased Sp1 binding to region -152/-135 compared with those from SW480 cells. Finally, endogenous urokinase receptor protein amounts in 10 colon cancers and corresponding normal colon correlated with Src specific activity. These data suggest that urokinase receptor gene expression is regulated by Src partly via increased Sp1 binding.

Electrophoresis, Polyacrylamide Gel↗
PubMed1999

Decreased Src tyrosine kinase activity inhibits malignant human ovarian cancer tumor growth in a nude mouse model.

The Src protein tyrosine kinase is overexpressed and activated in a number of human cancers, including some human ovarian cancers. To determine whether Src activity plays a role in ovarian tumor growth, stable derivatives of the SKOv-3 human ovarian cancer cell line that exhibited reduced Src tyrosine kinase activity were generated by transfection with an antisense c-src construct. Comparison of these cell lines with parental SKOv-3 cells and stable sense c-src vector-transfected control lines revealed no phenotypic alterations in anchorage-dependent proliferation, adherence, density saturation, or wound migration. However, reduction in Src activity was associated with altered cellular morphology, dramatically reduced anchorage-independent growth, and, when assessed for tumor development in a xenograft nude mouse model, diminished tumor growth. Furthermore, reduction of Src activity in the antisense c-src cell lines was associated with reduced vascular endothelial growth factor mRNA expression in vitro, and tumors derived from these cell lines displayed a phenotype indicative of abortive microvessel vascularization. These results strongly suggest that Src is involved in critical oncogenic pathways that modulate tumor growth from this ovarian cell line. Furthermore, this evidence suggests that as in other tumor systems, Src activity is required for vascular endothelial growth factor induction and angiogenic development.

Animals↗
PubMed1998

Stimulation of the protein tyrosine kinase c-Yes but not c-Src by neurotrophins in human brain-metastatic melanoma cells.

The c-Yes proto-oncogene (pp62c-Yes) encodes a non-receptor-type protein tyrosine kinase (NRPTK) of the Src family. c-Yes activities and protein levels are elevated in human melanoma and melanocyte cell lines. Because the neurotrophins (NT) are important in the progression of melanoma to the brain-metastatic phenotype, we determined whether NT stimulate c-Yes activity in human MeWo melanoma cells and two variant sublines with opposite metastatic capabilities, 3 S 5 and 70W. The highly brain-metastatic 70W subline had an intrinsically higher c-Yes activity than parental MeWo or poorly metastatic 3 S 5 cells. c-Yes kinase was further induced by the prototypic human NT, nerve growth factor (NGF) in a dose and time-dependent manner. In contrast, c-Src activity (pp60-Src) was similar in all these cells and unaffected by NGF exposure. Additionally, human NGF and neurotrophin-3 stimulated c-Yes in brain-metastatic 70W cells. The magnitude of c-Yes activation correlated with the degree of invasion of 70 W cells following incubation of these neurotrophins. To further examine NT stimulation of c-Yes in melanoma cells, three additional cell lines were examined. Metastatic TXM-13 and TXM-18 increased c-Yes activity in response to NGF. In contrast, no increase was observed in low-metastatic TXM-40 cells. Together, these data suggest that altered c-Yes expression may play a role in the malignant progression of the human melanocyte towards the brain-metastatic phenotype and that NT enhance the activity of c-Yes in signaling penetration into the matrix of NT-rich stromal microenvironments such as the brain.

Brain Neoplasms↗
PubMed1998

Down-regulation of vascular endothelial growth factor in a human colon carcinoma cell line transfected with an antisense expression vector specific for c-src.

Vascular endothelial growth factor (VEGF) is implicated in the angiogenesis of human colon cancer. Recent evidence suggests that factors that regulate VEGF expression may partially depend on c-src-mediated signal transduction pathways. The tyrosine kinase activity of Src is activated in most colon tumors and cell lines. We established stable subclones of the human colon adenocarcinoma cell line HT29 in which Src expression and activity are decreased specifically as a result of a transfected antisense expression vector. This study determined whether VEGF expression is decreased in these cell lines and whether the smaller size and reduced growth rate of antisense vector-transfected cell lines in vivo might result, in part, from reduced vascularization of tumors. Northern blot analysis of these cell lines revealed that VEGF mRNA expression was decreased in proportion to the decrease in Src kinase activity. Under hypoxic conditions, cells with decreased Src activity had a <2-fold increase in VEGF expression, whereas parental cells had a >50-fold increase. VEGF protein in the supernatants of cells was also reduced in antisense transfectants compared with that from parental cells. In nude mice, subcutaneous tumors from antisense transfectants showed a significant reduction in vascularity. These results suggest that Src activity regulates the expression of VEGF in colon tumor cells.

Animals↗
PubMed1997

Regulation of vascular endothelial growth factor expression in human colon carcinoma cells by activity of src kinase.

BACKGROUND: The c-src protooncogene encodes a protein tyrosine kinase, pp60c-src, that is a mediator in many signal transduction pathways. One pathway in which pp60c-src protein tyrosine kinase activity is implicated involves regulation of vascular endothelial growth factor (VEGF), an angiogenic factor important to neovascularization of growing tumors. Recently we demonstrated that decreased activity of pp60c-src in colon tumor cells contributes to decreased expression of VEGF. This study examined the relationship between pp60c-src activation, cell density, and VEGF production in a colon tumor cell line. METHODS: Parental HT-29 colon adenocarcinoma cells and stable subclones created by transfection with c-src antisense and sense (control) expression vectors were plated under sparse (2 x 10(4) cells/cm2) and confluent (20 x 10(4) cells/cm2) conditions and grown for 36 hours. Protein and RNA were extracted from cells to determine pp60c-src levels, c-Src tyrosine kinase activity, and VEGF mRNA expression. RESULTS: The pp60c-src kinase activity of HT-29 cells and control sense-transfected clones grown under confluent conditions was increased threefold to fivefold compared with cells grown under sparse conditions. In contrast, the ability of confluent culture conditions to increase pp60c-src activity was blunted in antisense transfectants. By regression analysis, VEGF expression was found to vary directly with pp60c-src levels (r2 = 0.886). CONCLUSIONS: Cell density contributes to the regulation of c-src kinase activity and VEGF expression in HT-29 cells. When the steady-state level of pp60c-src is reduced in antisense transfectants, not only is the steady-state level of VEGF reduced, but the ability of confluence to stimulate pp60c-src activity and VEGF production is too. These data suggest that c-src may be an intermediary of both constitutive and inducible pathways for VEGF production in colon tumor cells.

Adenocarcinoma↗
PubMed1997

Decreased tumorigenicity of a human colon adenocarcinoma cell line by an antisense expression vector specific for c-Src.

In greater than 80% of colon tumors and established cell lines, the specific activities of the protein tyrosine kinases pp60(c-src) and pp62(c-yes) are increased with respect to normal colonic epithelial cells. However, no mutations in either gene have been identified in colon tumors. Therefore, the possible biological consequences of activations of these protein tyrosine kinases in colon tumors have been unclear. To determine if pp60(c-src) activation affects growth and tumorigenicity of established colon tumor cell lines, an antisense expression vector that specifically reduces pp60(c-src) expression was constructed. The vector was transfected into HT 29 cells, an established colon tumor cell line in which both pp60(c-src) and pp62(c-yes) are activated. Two stable subclones were isolated in which pp60(c-src) but not pp62(c-yes) expression and activity were reduced. These established cell lines proliferated more slowly than parental cells proportionately to reduction in pp60(c-src) expression. When injected into nude mice, antisense transfected cells formed slow-growing tumors; however, the rate of tumor growth was reduced far greater than would be predicted from decreased proliferation rates in tissue culture. In contrast, stable subclones transfected with a comparable "sense" expression vector were unaltered in growth rates in tissue culture and in nude mice with respect to parental HT 29 cells. These data demonstrate that the activation of pp60(c-src) alone contributes to the tumorigenicity of HT 29 cells, a cell line widely used as a model for biological properties of colon carcinoma. Furthermore, because pp60(c-src) and pp62(c-yes) appear redundant to the growth regulation of normal colonic epithelial cells, the data suggest that src-specific inhibitors might be of therapeutic value for colon cancer.

Adenocarcinoma↗
PubMed1996

Differential activation of pp60(c-src) and pp62(c-yes) in human colorectal carcinoma liver metastases.

pp60(c-src) and pp62(c-yes) are protein tyrosine kinases whose specific activities are increased in primary colorectal carcinomas. Activity of pp60(c-src) is further increased in colorectal liver metastases. This study was undertaken to compare pp60(c-src) and pp62(c-yes) expression and activity in human colorectal carcinoma liver metastases and to determine the potential prognostic significance of differences in activation of these two kinases. The pp60(c-src) and pp62(c-yes) tyrosine kinase activities and protein levels relative to those in normal colonic mucosa were determined using an immune complex kinase assay and immunoblot analysis in tissue specimens from 22 patients with primary colorectal carcinoma and synchronous metastatic liver disease and from 9 patients with metachronous colorectal carcinoma liver metastases. Of the primary colon tumors, 64% of the tumors contained elevated activities of both pp60(c-src) and pp62(c-yes). For liver metastases, however, only 10% had activation of both tyrosine kinases, 61% had elevated pp60(c-src) activity only, and 23% had elevated pp62(c-yes) activity only. Analysis of synchronous metastases from primary tumors with elevated activities in both kinases demonstrated that in 71% of these patients, the activity of either pp60(c-src) or pp62(c-yes) decreases relative to the primary tumor. Protein levels of pp60(c-src) and pp62(c-yes) in primary carcinomas and metastases remained unchanged from levels in normal colonic mucosa. These results demonstrate that differential regulation of the activities of pp60(c-src) and pp62(c-yes) occurs during tumor progression. Patients with either synchronous or metachronous liver metastases and elevated pp62(c-yes) kinase activity have biologically more aggressive disease and a worse prognosis than patients without elevated pp62(c-yes) activity in their liver metastases (median survival, 13 months versus 30 months, P < 0.005, Wilcoxon signed rank test). Analysis of patients with synchronous liver metastases also demonstrated a worse prognosis for those with elevated pp62(c-yes) kinase activity (P < 0.05, Wilcoxon signed rank test).

Colorectal Neoplasms↗
PubMed1995

A tyrphostin-derived inhibitor of protein tyrosine kinases: isolation and characterization.

We recently reported that tyrphostin 23 (3,4-dihydroxybenzylidene malononitrile) is unstable in solution and that some of the degradation products are better inhibitors of the tyrosine kinase activity of Src and the EGF-receptor kinase than the parent compound itself (Ramdas et al., Cancer Res. 54, 867-868, 1994). In this study, the tyrphostin 23-derived compound designated P3, which is a more stable and potent protein tyrosine kinase inhibitor, was isolated. P3 was purified from oxidized tyrphostin 23 by solvent extraction, silica-gel flash chromatography, and reverse-phase high-pressure liquid chromatography. The physical characteristics of the isolated compound were determined and its chemical structure elucidated by 1H and 13C NMR spectroscopy. The proposed structure of this new inhibitor is that of a tyrphostin 23 dimer joined at the benzylidene carbon. P3 was evaluated in vitro as an inhibitor of four different protein tyrosine kinases (Src, Csk, EGF-receptor, and FGF-receptor) and two protein serine kinases (PK-A and PK-C). This compound exhibited the most inhibitory activity against Src with a Ki value of 6 microM and was less inhibitory toward the other protein kinases with Ki values ranging from 35 to 300 microM. P3 did not inhibit other nucleotide-utilizing enzymes such as lactate dehydrogenase and hexokinase. The growth and colony formation of HT-29 colon adenocarcinoma cells that contain activated Src was inhibited by P3 with an IC50 value of approximately 10 microM.

Catechols↗
PubMed1993

Bacterial expression of an active tyrosine kinase from a protein A/truncated c-src fusion protein.

The carboxy-terminal half of the c-src protein fused to the protein A moiety was expressed in bacteria. The protein A/truncated c-src fusion protein, which does not have SH2 and SH3 domains, is found in the periplasmic space allowing for a simple one-step purification and demonstrated high efficiency in autophosphorylation and exogeneous substrate phosphorylation. The missense mutation at codon 294 (Ile-->Thr), which is located in the ATP-binding domain of the c-src, resulted in dramatic reduction of tyrosine kinase activity of the fusion protein. Using the fusion protein, we also revealed that staurosporin, a well-known kinase inhibitor, directly affects autophosphorylation of the C-terminal half of the c-src protein. This truncated c-src expression system provides a good source of enzyme for diverse experiments and is an ideal model for understanding the implication of structural alterations in the catalytic activity of the c-src kinase by site-directed mutagenesis experiments.

Alkaloids↗