[Show abstract][Hide abstract]ABSTRACT: Clinical and laboratory studies have shown that bismuth subsalicylate (BSS) is helpful in the healing of gastric ulcers because of the bactericidal effects of bismuth (Bi3+) on H. pylori. Bismuth or BSS has also been reported to possess other nonbactericidal or "gastroprotective" effects in the stomach. It is known in other cell types that the effects of extracellular divalent or trivalent cations (e.g., Ca2+) can activate a plasma membrane-bound calcium-sensing receptor (CaSR). In a previous study, we found the existence of a CaSR which was activated by extracellular Ca2+ and found to increase intracellular Ca2+ [Ca2+]i, MAP-kinase activity, and gastric epithelial cell proliferation. In the present study, we were interested in determining whether the effects of the trivalent cation Bi3+ (in the form of BSS) on [Ca2+]i, MAP-kinase activity, and proliferation of gastric cells. We found that BSS dose dependently increased [Ca2+]i, p44/p42 and p38 MAP-kinase activites, and gastric mucous epithelial cell growth. The addition of BAPTA to chelate intracellular Ca2+ blocked BSS-induced p44/p42 MAP-kinase activities but not p38 MAP-kinase activity. The p44/p42 MAP-kinase inhibitor PD98059 and the p38 MAP-kinase inhibitor SB203580 dose dependently decreased gastric mucous cell growth over a 24 hr. All of the BSS-induced changes in [Ca2+]i, MAP-kinase activity, and gastric cell proliferation could be reproduced with the CaSR-agonist gadolinium (Gd3+). Our data suggest that BSS may possess additional novel effects by increasing gastric mucous epithelial cell growth through a Ca2+/MAP-kinase-dependent pathway.
Article · Apr 2004 · Digestive Diseases and Sciences
[Show abstract][Hide abstract]ABSTRACT: In stomach, Helicobacter pylori (Hp) adheres to gastric mucous epithelial cells (GMEC) and initiates several different signal transduction events. Alteration of intracellular Ca2+ concentration ([Ca2+]i) is an important signaling mechanism in numerous bacteria-host model systems. Changes in [Ca2+]i induced by Hp in normal human GMEC have not yet been described; therefore, we examined effects of Hp on [Ca2+]i in normal human GMEC and a nontransformed GMEC line (HFE-145). Cultured cells were grown on glass slides, porous filters, or 96-well plates and loaded with fura 2 or fluo 4. Hp wild-type strain 60190 and vacA-, cagA-, and picB-/cagE- isogenic mutants were incubated with cells. Changes in [Ca2+]i were recorded with a fluorimeter or fluorescence plate reader. Wild-type Hp produced dose-dependent biphasic transient [Ca2+]i peak and plateau changes in both cell lines. Hp vacA- isogenic mutant produced changes in [Ca2+]i similar to those produced by wild type. Compared with wild type, cagA- and picB-/cagE- isogenic mutants produced lower peak changes and did not generate a plateau change. Preloading cultures with intracellular Ca2+ chelator BAPTA blocked all Hp-induced [Ca2+]i changes. Thapsigargin pretreatment of cultures to release Ca2+ from internal stores reduced peak change. Extracellular Ca2+ removal reduced plateau response. Hp-induced peak response was sensitive to G proteins and PLC inhibitors. Hp-induced plateau change was sensitive to G protein inhibitors, src kinases, and PLA2. These findings are the first to show that H. pylori alters [Ca2+]i in normal GMEC through a Ca2+ release/influx mechanism that depends on expression of cagA and picB/cagE genes.
Article · Aug 2003 · AJP Gastrointestinal and Liver Physiology
[Show abstract][Hide abstract]ABSTRACT: Helicobacter pylori may protect against the development of dysplasia in Barrett's epithelium of patients with gastroesophageal reflux disease. The aim of this study was to determine whether H. pylori preferentially induces apoptosis in Barrett's-derived cancer cells compared to normal cells. A Barrett's-derived adenocarcinoma cell line (OE33) was grown. H. pylori wild-type, isogenic vacA-, cagA(-), and picB-/cagE- mutant strains were grown on agar plates. Intact or sonicated bacteria were used to treat normal and OE33 cells for 24 hours, and Hoechst dye binding was performed to measure apoptosis. FAS protein expression was determined by Western immunoblotting. OE33 cells treated with intact H. pylori wild-type strains produced significant (P < 0.05) dose-dependent increases in apoptosis compared to normal esophageal cells. H. pylori wild-type and vacA- isogenic strains were more effective than cagA- and picB-/cage- isogenic strains in inducing apoptosis in OE33 cells. In OE33 cells, H. pylori sonicates produced lower levels of apoptosis than intact bacteria. Wild-type H. pylori strains increased Fas protein expression in OE33 cells at 18 hours. H. pylori induced apoptosis at a higher rate in the Barrett's-derived human esophageal adenocarcinoma cells than in normal esophageal cells. The H. pylori-induced apoptosis was primarily dependent on intact bacteria and the presence of the cagA and picB/cagE gene products. H. pylori-induced apoptosis may involve the Fas-caspase cascade.
Article · Feb 2003 · Journal of Gastrointestinal Surgery
[Show abstract][Hide abstract]ABSTRACT: The purpose of the present study was to determine whether human gastric mucous epithelial cells express a functional Ca2+-sensing receptor (CaR). Human gastric mucous epithelial cells were isolated from surgical tissues and cultured on glass coverslips, plastic dishes, or porous membrane filters. Cell growth was assessed by the MTT assay, CaR localization was detected by immunohistochemistry and confocal microscopy, CaR protein expression was assessed by Western immunoblotting, and intracellular Ca2+ concentration ([Ca2+]i) was determined by fura 2 spectrofluorometry. In paraffin sections of whole stomach, we found strong CaR immunohistochemical staining at the basolateral membrane, with weak CaR-staining at the apical membrane in mucous epithelial cells. Confocal microscopy of human gastric mucous epithelial cell cultures showed abundant CaR immunofluorescence at the basolateral membrane and little to no CaR immunoreactivity at the apical membrane. Western immunoblot detection of CaR protein in cell culture lysates showed two significant immunoreactive bands of 140 and 120 kDa. Addition of extracellular Ca2+ to preconfluent cultures of human gastric mucous epithelial cells produced a significant proliferative response. Changes in [Ca2+]i were also observed in response to graded doses of extracellular Ca2+ and Gd3+. The phospholipase C inhibitor U-73122 specifically inhibited Gd3+-induced changes in [Ca2+]i in the gastric mucous epithelial cell cultures. In conclusion, we have identified the localization of a functional CaR in human gastric mucous epithelial cells.
Article · Oct 1999 · The American journal of physiology
[Show abstract][Hide abstract]ABSTRACT: The specific paclitaxel dose or time course in the treatment of colon carcinoma without the disruption of normal colonic cell proliferation is currently not known. The aim of this study was to determine the effects of paclitaxel on the growth of human colonic epithelial cells using cultures of normal, polyposis, and cancerous cells.
Normal, polyposis, and cancerous human colonic cells (Caco-2, T-84, and LoVo cell lines) were cultured, then treated with paclitaxel (10(-9)-10(-5) M) for 0-7 days.[AU: Please verify all dosages throughout.] Cell proliferation was assayed using either a Coulter-Counter or MTT-growth assay. Immunofluorescence and Western immunoblotting measured P-glycoprotein.
Low paclitaxel doses (1 x 10(-9)-10(-8) M) were more effective than higher paclitaxel doses (>1 x 10(-8) M) in the growth inhibition of polyposis, Caco-2, and LoVo cancer (but not T-84) cell lines. Low paclitaxel doses had little effect on normal colonic cell growth over 7 days. Higher paclitaxel doses (>1 x 10(-8)-10(-5) M) produced a dose-dependent inhibitory effect on the growth of normal human colonic epithelial cells over 7 days but had no effect on the growth of polyposis, Caco-2, and LoVo cells over 3-7 days of treatment. Immunofluorescence and Western immunoblotting of cultures showed that 1 x 10(-6) M paclitaxel increased P-glycoprotein expression in Caco-2 and LoVo cells. There was no effect of paclitaxel on P-glycoprotein expression in T-84 cancer cells, which were found to have high endogenous basal levels of P-glycoprotein. P-glycoprotein expression in Caco-2 cells was found on plasma membranes and in perinuclear areas.
Lower paclitaxel doses are more effective over time for the growth inhibition of polyposis and cancerous colonic cells, with minimal effects on the growth of normal colonic epithelial cells. Increased P-glycoprotein expression appears to be correlated with paclitaxel resistance in polyposis and cancerous colonic cells.