Gastric Cancer Cell Line HGT-1 Research Articles

The role of fibroblasts in the modulation of integrin-dependent interactions between the gastric cell line HGT-1 and fibronectin.

Fibronectin plays an important role in gastric cancer progression. However, little is known about the microenvironmental factors modulating integrin-dependent interactions between gastric cancer cells and fibronectin. We therefore studied the regulation by fibroblasts of the integrin-dependent adhesion and migration of the gastric cancer cell line HGT-1 onto fibronectin. We first determined, by immunofluorescence, immunoblotting and flow cytometry, that HGT-1 cells expressed alpha3, alpha5, alpha6, alphaV and beta1 integrin chains, and the alphaVbeta3 and alphaVbeta5 dimers. We verified that HGT-1 cells xenografted to the immunosuppressed newborn rat retained the integrin repertoire detected in vitro and were able to induce the formation of tumors rich in fibronectin. By using an in vitro assay in the presence of neutralizing antibodies, we verified that HGT-1 adhesion and migration onto fibronectin involved beta1, alphaV and alpha5 integrin chains; we verified, by using an in situ adhesion test to rat gastric wall frozen sections, that in situ HGT-1 adhesion to fibronectin was integrin dependent. In coculture experiments, we showed that organ-specific fibroblasts from stomach, lung and dermis were able to induce, in a site-specific manner, the expression of beta1, alpha5 and alphaV integrin chains in HGT-1 cells, their integrin-dependent adhesion and migration on fibronectin and their capacity to secrete oncofetal fibronectin. In conclusion, our results show the capacity for tissue-derived fibroblasts to modulate the integrin-dependent interactions between the gastric cell line HGT-1 and fibronectin. They strongly suggest that, in gastric cancer, stromal fibroblasts contribute to promote fibronectin-mediated local invasion by tumor cells.

Prodigiosin induces cell death and morphological changes indicative of apoptosis in gastric cancer cell line HGT-1.

Gastric cancer is one of the most frequent malignancies and its treatment is far from satisfactory. The challenge to oncologists is the characterization of novel chemical entities with greater effectiveness. Prodigiosin is a red pigment produced by various bacteria including Serratia marcescens. Here we characterize the apoptotic action of prodigiosin in human gastric carcinoma cell line (HGT-1). Cells were assayed by the MTT assay, fragmentation pattern of DNA, Hoechst 33342 staining and study of actin microfilament architecture. Treatment of these cells with prodigiosin showed a constant decrease in viability by apoptosis. Morphological analysis of prodigiosin-treated cells demonstrated that prodigiosin induces cell shrinkage, chromatin condensation, reorganization of actin microfilament architecture, and detachment of cells from the cell culture substrate. Altogether these results suggest that prodigiosin induces apoptosis in HGT-1 human gastric cancer cells and raises the possibility of its use as a new chemotherapeutic drug.

Theα isoform of protein kinase C inhibits histamine-stimulated adenylate cyclase activity in a particulate fraction of the human gastric cancer cell line HGT-1

The isoform of protein kinase C responsible for the inhibition of histamine-stimulated adenylate cyclase by the phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA), has been investigated in a particulate fraction prepared from the human gastric cancer cell line HGT-1. The alpha and epsilon isoforms of protein kinase C were detected in HGT-1 cells and in a 40,000 x g particulate fraction by immunoblotting procedures. The inhibitory effect of TPA on histamine-stimulated adenylate cyclase was enhanced by the presence of Ca2+, but decreased in a concentration-dependent manner by anti-peptide antibody to protein kinase C alpha, but not to protein kinase C epsilon. Addition of Ca2+ and TPA to the 40,000 x g particulate fraction stimulated the phosphorylation of the protein kinase C substrate myelin basic peptide 4-14. Protein kinase C alpha is probably the isoform responsible for inhibition of histamine-stimulated adenylate cyclase in HGT-1 cells.

Endothelin-1 and ETA Receptor Subtype Are Expressed in the Gastric HGT-1 Cell Line

Recent studies have revealed that endothelin-1 (ET-1) may be produced by human cancer cell lines and have suggested that in vivo the peptide might play a modulatory role in the growth of stromal cells surrounding tumor cells and/or in the growth of the cancer cells themselves, through paracrine or autocrine mechanisms. Therefore, we investigated whether ET-1 and ET receptors could be expressed in the human gastric cancer cell line HGT-1. By applying the reverse transcriptase polymerase chain reaction (RT-PCR) to total RNA extracted from the cells, using oligonucleotides synthesized from the sequence of the prepro-ET-1 mRNA, we have amplified a cDNA at the expected size (453 bp), which hybridized with a labeled ET-1-specific probe. In addition, RT-PCR was carried out to test whether HGT-1 cells expressed mRNA for ETA and/or ETB receptor subtypes. The amplified products of cDNA were at the size predicted for the ETA receptor (368 bp), whereas no ETB receptor mRNA could be detected.

Protein kinase C alpha is the isoform responsible for inhibition of histamine H2 receptor mediated stimulation of adenylate cyclase in the human gastric cancer cell line HGT-1.

Protein kinase C alpha is the isoform responsible for inhibition of histamine H2 receptor mediated stimulation of adenylate cyclase in the human gastric cancer cell line HGT-1.

Protein kinase C inhibits histamine H2 receptor-mediated stimulation of cyclic AMP content in the human gastric cancer cell line HGT-1.

Protein kinase C inhibits histamine H2 receptor-mediated stimulation of cyclic AMP content in the human gastric cancer cell line HGT-1.

Pharmacology of histamine H 2 receptor antagonists in the human gastric cancer cell line HGT-1 : Structure-activity relationship of isocytosine-furan and imidazole derivatives related to cimetidine

Imidazole and isocytosine-furan derivatives inhibited H 2 receptor activity in HGT-1 cells, in accordance with the following relative potencies ( ic 50 = 2.3 μ M cimetidine as reference): SKF 93479 = cimetidine = 100 > metiamide = 62 > SKF 92408 = 2 > SKF 91581 = 0.07) . The Schild plot for cimetidine was linear (slope = 0.97) with a pA 2 value of 6.72 ± 0.12 ( K i = 0.18 μM cimetidine), suggesting competitive inhibition. Preincubation of HGT-1 cells for 10 min with H 2 antagonists at 2μM concentration resulted in 90–100% inactivation (SKF 93479 and oxmetidine) and 65% inactivation (ranitidine) which persisted for 30 min, even after a washout period. Accordingly, the kinetics of 2μM [ 3H] SKF 93479 binding to HGT-1 cells revealed a half-time for association of 10 min and a dissociation half-time of 120 min. There was a good correlation between the kinetics and relative potencies of cimetidine and SKF 93479 in inhibiting H 2 receptor activity in purified plasma membranes (40 nM) as well as in intact HGT-1 cells preincubated for 2 hr with SKF 93479 before histamine addition (45 nM). Chronic treatment of HGT-1 cells for 6 days with 2 μM SKF 93479 specifically blocked H 2 receptor activity since cyclic AMP generation induced by other hormones and agents such as VIP, glucagon, GIP and sodium fluoride was unaltered. In contrast, short term and chronic treatment by cimetidine was readily reversible. The isocytosine-furan derivative SKF 93479 differs from the imidazole analogue cimetidine by its apparent irreversible action, due to the slow onset of association from HGT-1 cells. The isocytosine ring in SKF 93479 and oxmetidine seems to play a preponderant role in their apparent long-lasting, irreversible actions.

Selective disappearance of histamine H2-receptor activity in the human gastric cancer cell line HGT-1 after short-term or chronic treatment by histamine or its H2-antagonists.

Homologous loss of histamine H2-receptor activity (cAMP generation) was observed after short-term (10-20 min) or chronic treatment (6 days) of cultured HGT-1 cells with histamine (desensitization) or the H2-receptor antagonist SKF 93479. This inactivation process was not observed when HGT-1 cells were exposed to the classical H2-antihistamine cimetidine. The data show: (1) that the compound SKF 93479 has a very prolonged inhibitory action on histamine receptor activity, suggesting an irreversible interaction between the antagonist and the receptor; (2) that cimetidine is a reversible H2-receptor antagonist which can be removed without changing the the efficacy and the potency of histamine on gastric cells; (3) that the H2-receptor antagonists cimetidine and SKF 93479 specifically block histamine H2-receptor activity in HGT-1 cells since cAMP generation induced by other hormones such as vasoactive intestinal peptide (VIP), glucagon or gastric inhibitory peptide (GIP) was unchanged after treatment; (4) the first evidence for time-dependent (half-life: 20 min) desensitization of gastric H2-receptors.

Desensitization by histamine of H 2 receptor-mediated adenylate cyclase activation in the human gastric cancer cell line HGT-1

Short-term treatment of cultured HGT-1 cells with histamine produced a time-dependent (half-life: 20 min) and homologous desensitization of histamine H 2, receptor activity mediating cAMP generation in HGT-1 cells and gastric acid secretion in normal gastric mucosa. Histamine treatment resulted in loss of response of the adenylate cyclase to histamine in purified plasma membranes, but had no effect on basal, vasoactive intestinal peptide (VIP)- or NaF-stimulated enzyme activities. We propose that the desensitization of gastric histamine H 2 receptor by histamine evidenced in cellular or subcellular preparations from HGT-1 cells could be involved in the physiological regulation and pharmacological control of gastric cell function in man. Desensitization Histamine H 2receptor Adenylate cyclase Acid secretion HGT-1 cell

Gastric inhibitory peptide (GIP), pancreatic glucagon and vasoactive intestinal peptide (VIP) are cAMP-inducing hormones in the human gastric cancer cell line HGT-1. Homologous desensitization of VIP receptor activity

GIP (EC 50 = 8 × 10 −9 M, 5-fold stimulation), pancreatic glucagon (EC 50 = 10 −8M, 13-fold) and porcine or chicken VIP (EC 50 = 2.5 × 10 −9 M, 10-fold) are shown to activate the cAMP generating system in HGT-1 cells. Combinations of GIP, pancreatic glucagon and VIP indicate the occurence of 3 separate sets of recognitions sites for these 3 peptides. Accordingly, chronic treatment of cultured HGT-1 cells by VIP (10 −8 M) during 6 days resulted in homologous desensitization of VIP receptor activity. Other peptides structurally related to the secretin-glucagon family, to neurotensin, or to gastrin are either ineffective or very weak agonist (hpGRF). GIP or pancreatic glucagon are inactive on the human colonic cell line HT-29, indicating the gastric specificity of the effect of GIP and glucagon in transformed epithelial cells originating from the human gastrointestinal tract. This implies that GIP and (pancreatic-entero) glucagon peptides may regulate gastric secretions directly, under similar mechanisms that those we evidenced in the rat.

Irreversible and specific inactivation by AH 22216 of histamine H 2 receptors in the human gastric cancer cell line HGT-1

We compared the interaction of AH 22216 (a new histamine H 2 receptor antagonist) and cimetidine on the receptor-cAMP systems sensitive to histamine and to Vasoactive Intestinal Peptide (VIP) in the human gastric cancer cell line HGT-1. When added simultaneously with histamine (10 −4 M), the potency of AH 22216 is similar to that of cimetidine (IC 50 = 4–6.6×10 −6 M, respectively). Schild plot analysis indicated a non-competitive inhibition by AH 22216 (pA 2=6.22, slope=1.4±0.03). Preincubations of AH 22216 (10 min, 10 −5 M) with HGT-1 cells (even after a washout period) resulted in a complete and persistent (60 min) inactivation of the subsequent histamine effect, without changing the kinetics of the VIP-induced stimulation in the system. Under these conditions, the potency of AH 22216 increased from 6.6 to 0.7 × 10 −6 M. This inactivation was not observed with cimetidine. The data indicate that AH 22216 is an irreversible and specific inhibitor of the gastric histamine H 2 receptor.

Histamine and VIP interactions with receptor-cyclic AMP systems in the human gastric cancer cell line HGT-1

In HGT-1 cells incubated at 20°C for 15 min with mM 3-isobutyl-1-methylxanthine (IBMX), histamine (10 −4M) increased basal cAMP levels from 2.12 ± 0.14 to 22.9 ± 2 pmol per 10 6 cells, with a potency of 6.4 × 10 −6M. IBMX was added in order to inhibit cAMP degradation by low and high Km cAMP-phosphodiesterases (cAMP-PDE). The use of specific H 1, H 2 agonists or antagonists indicated that the histamine effect was due to an interaction with typical H 2 -receptors that are involved in gastric acid secretion. Cyclic AMP levels were also increased (10-fold) by vasoactive intestinal peptide VIP (3 × 10 − II − 10 −8M). Porcine peptide having N-terminal histidine and C-terminal isoleucine amide (PHI) and secretin were respectively 80 and 3600 times less potent than VIP and did not produce additive effect when tested in combinations with VIP. This observation indicates that these two peptides, structurally related to VIP, are acting through the recognition sites for VIP. Combination of VIP and Histamine results in additive stimulation on intact cells as well as on membrane-bound adenylate cyclase, suggesting the existence of two cell populations bearing respectively the two sets of receptors. Two other human cancer cell lines originating from nongastric tumors (HT-29 and HL-60) possess only VIP or histamine receptors, respectively, indicating the gastric cellular originality of the HGT-1 cells. It is concluded that HGT-1 cells possess both VIP and histamine H 2 receptors with similar pharmacological properties to those characterized in normal human fundic glands (1,2). Therefore, this cell line can be a good model to study drugs used therapeutically during the treatment of patients for gastric ulcer or cancer.