Prostaglandin D2 regulation of autophagy affects stemness of gastric cancer stem cells

Objective:To explore the effect and mechanism of prostaglandins D2(PGD2)on the stemness of gastric cancer stem cells(GCSCs).Methods:7901-GCSCs were enriched by serum-free culture method;then the positivity rate of CD44,a stemness marker,was detected by flow cytometry in serum-free cultured 7901-GCSCs;the sphere-forming ability was detected by the sphere-forming assay after stimulation with different concentrations of PGD2(2.5,5,10)μg/mL,and the expression of stemness-related indicators(OCT4,CD44)and autophagyrelated proteins(LC3,Beclin-1)after PGD2 stimulation was detected by the western blot assay in different concentrations.The expression of stemness-related indexes(OCT4,CD44)and autophagy-related proteins(LC3,Beclin-1)were detected by Western blot assay after stimulation with different concentrations of PGD2.The expression of autophagy-related proteins after stimulation with different concentrations of CQ(2.5,5,10)μM was detected by Western blot experiment.The protein expression of autophagy-related proteins(LC3,Beclin-1)and stemness-related indexes(OCT4,CD44)was detected by Western blot experiment after PGD2 as well as PGD2+CQ treatment.Results:Flow cytometry results showed that the expression of CD44 positivity was increased in serum-free cultured 7901-GCSCs compared with gastric cancer cells SGC-7901(P<0.05),which fulfilled the needs of subsequent experiments.The results of stem cell spheroid formation assay showed that the spheroid formation ability of 7901-GCSCs in the PGD2 group was significantly weakened compared with that of the DMSO group(P<0.05).Western blot results showed that the protein expression of stemness-related indexes(OCT4,CD44)was down-regulated in the 7901-GCSCs in the PGD2 group compared with that of the DMSO group(P<0.05),and the expression of autophagy-related proteins(LC3,Beclin-1)expression increased(P<0.05).Compared with the DMSO group,the expression of autophagy-related proteins(LC3,Beclin-1)was decreased in the CQ group(P<0.05).Western blot results also showed that the expression of cellular autophagy-related proteins and stemness-related indexes in the PGD2+CQ group was not significantly changed compared with that of the DMSO group(ns:the difference was not significant),suggesting that the CQ could block the effect of PGD2 on the expression of stemness markers in 7901-GCSCs.7901-GCSCs stemness inhibition.Conclusion:PGD2 may affect the stemness of 7901-GCSCs by regulating autophagy.

Immunosuppressive tumor microenvironment in gastric signet-ring cell carcinoma

Gastric signet-ring cell carcinoma(GSRCC)is a subtype of gastric cancer with distinct phenotype and high risk of peritoneal metastasis.Studies have shown that early GSRCC has a good prognosis,while advanced GSRCC is insensitive to radiotherapy,chemotherapy or immune checkpoint blockade therapy.With technological advancement of single-cell RNA sequencing analysis and cytometry by time of flight mass cytometry,more detailed atlas of tumor microenvironment(TME)in GSRCC and its association with prognosis could be investigated extensively.Recently,two single-cell RNA sequencing studies revealed that GSRCC harbored a unique TME,manifested as highly immunosuppressive,leading to high immune escape.The TME of advanced GSRCC was enriched for immunosuppressive factors,including the loss of CXCL13+-cluster of differentiation 8+-Tex cells and declined clonal crosstalk among populations of T and B cells.In addition,GSRCC was mainly infiltrated by follicular B cells.The increased proportion of SRCC was accompanied by a decrease in mucosaassociated lymphoid tissue-derived B cells and a significant increase in follicular B cells,which may be one of the reasons for the poor prognosis of GSRCC.By understanding the relationship between immunosuppressive TME and poor prognosis in GSRCC and the underlying mechanism,more effective immunotherapy strategies and improved treatment outcomes of GSRCC can be anticipated.

Effect of acacetin on inhibition of apoptosis in Helicobacter pyloriinfected gastric epithelial cell line

BACKGROUND Helicobacter pylori(H.pylori)infection can cause extensive apoptosis of gastric epithelial cells,serving as a critical catalyst in the progression from chronic gastritis,gastrointestinal metaplasia,and atypical gastric hyperplasia to gastric carcinoma.Prompt eradication of H.pylori is paramount for ameliorating the pathophysiological conditions associated with chronic inflammation of the gastric mucosa and the primary prevention of gastric cancer.Acacetin,which has multifaceted pharmacological activities such as anti-cancer,anti-inflammatory,and antioxidative properties,has been extensively investigated across various domains.Nevertheless,the impact and underlying mechanisms of action of acacetin on H.pylori-infected gastric mucosal epithelial cells remain unclear.AIM To explore the defensive effects of acacetin on apoptosis in H.pylori-infected GES-1 cells and to investigate the underlying mechanisms.METHODS GES-1 cells were treated with H.pylori and acacetin in vitro.Cell viability was assessed using the CCK-8 assay,cell mortality rate via lactate dehydrogenase assay,alterations in cell migration and healing capacities through the wound healing assay,rates of apoptosis via flow cytometry and TUNEL staining,and expression levels of apoptosis-associated proteins through western blot analysis.RESULTS H.pylori infection led to decreased GES-1 cell viability,increased cell mortality,suppressed cell migration,increased rate of apoptosis,increased expressions of Bax and cle-caspase3,and decreased Bcl-2 expression.Conversely,acacetin treatment enhanced cell viability,mitigated apoptosis induced by H.pylori infection,and modulated the expression of apoptosis-regulatory proteins by upregulating Bcl-2 and downregulating Bax and cleaved caspase-3.CONCLUSION Acacetin significantly improved GES-1 cell viability and inhibited apoptosis in H.pylori-infected GES-1 cells,thereby exerting a protective effect on gastric mucosal epithelial cells.

Multifaceted role of microRNAs in gastric cancer stem cells: Mechanisms and potential biomarkers

MicroRNAs(miRNAs)have received much attention in the past decade as potential key epigenomic regulators of tumors and cancer stem cells(CSCs).The abnormal expression of miRNAs is responsible for different phenotypes of gastric cancer stem cells(GCSCs).Some specific miRNAs could be used as promising biomarkers and therapeutic targets for the identification of GCSCs.This review summarizes the coding process and biological functions of miRNAs and demon-strates their role and efficacy in gastric cancer(GC)metastasis,drug resistance,and apoptosis,especially in the regulatory mechanism of GCSCs.It shows that the overexpression of onco-miRNAs and silencing of tumor-suppressor miRNAs can play a role in promoting or inhibiting tumor metastasis,apart from the initial formation of GC.It also discusses the epigenetic regulation and potential clinical applications of miRNAs as well as the role of CSCs in the pathogenesis of GC.We believe that this review may help in designing novel therapeutic approaches for GC.

Effects of simulated carbon dioxide and helium peumoperitoneum on proliferation and apoptosis of gastric cancer cells

AIM:To investigate the effects of carbon dioxide (CO2) and helium insufflation administered at different pressures on the growth and apoptosis of cultured human gastric cancer cells. METHODS:The gastric cancer cells MKN-45 were exposed to a CO2 and helium environment maintained at different pressures (0, 5, 10 and 15 mmHg). The cells were exposed to simulated pneumoperitoneum environment for 4 h, and pH of the culture media was measured after it was moved to normal conditions for 0, 2, 4, 6 and 8 h. Proliferation viability of MKN-45 was examined by 3-4,5Dimethylthiazol-2-yl,5-diphenyltetrazolium bromide or triazolyl blue (MTT) assay after it was moved to normal conditions. Apoptotic ratio was measured by Annexin V-FITC/PI double labelled staining. RESULTS:The pH of media was acid and recovered to normal after 4 h in the CO2 group while it was basic in the helium group. There was no difference between CO2 groups (under 10 mmHg ) and control group (P > 0.05) in the proliferative viability of the cells. The cultured cells exposed to 15 mmHg CO2 environment grew more slowly than control group from 4 to 7 d (P < 0.01 ) while there was no difference from 1 to 3 d (P > 0.05). The proliferative viability in helium group was not obviously different from the control group (P > 0.05). The apoptotic ratio of the cultured cells was markedly higher than that of the control group (P < 0.01) at 10 and 15 mmHg CO2 insufflation pressure. In helium group, the apoptotic ratio was not obviously different from the control group (P > 0.05). CONCLUSION:There is no obvious effect in the proliferation and apoptosis of MKN-45 cells under 10 mmHg CO2 insufflation pressure and helium in any pressure. Fifteen mmHg CO2 insufflation pressure can inhibit the proliferation of the cells and improve apoptosis.

Effects of tachyplesin and n-sodium butyrate on proliferation and gene expression of human gastric adenocarcinoma cell line BGC-823

AIM： To investigate the effects of tachyplesin and n-sodium butyrate on proliferation and gene expression of human gastric adenocarcinoma cell line BGC-823. METHODS： Effects of tachyplesin and n-sodium butyrate on proliferation of BGC-823 cells were determined with trypan blue dye exclusion test and HE staining. Effects of tachyplesin and n-sodium butyrate on cell cycle were detected by flow cytometry. Protein levels of c-erbB-2, c-myc, p53 and p16 were examined by immunocytochemistry. RESULTS： The inhibiting effects were similar after 2.0 mg/L tachyplesin and 2.0 mmol/L n-sodium butyrate treatment, the inhibitory rate of cellular growth was 62.66% and 60.19% respectively, and the respective maximum mitotic index was decreased by 49.35% and 51.69% respectively. Tachyplesin and n-sodium buD/rate treatment could markedly increase the proportion of cells at G0/G1 phase and decrease the proportion at S phase. The expression levels of oncogene c-erbB-2, c-myc, and mtp53 proteins were down-regulated while the expression level of tumor suppressor gene p16 protein was up-regulated after the treatment with tachyplesin or n-sodium buD/rate. The effects of 1.0 mg/L tachyplesin in combination with 1.0 mmol/L n-sodium butyrate were obviously superior to their individual treatment in changing cell cycle distribution and expression of c-erbB-2, c-myc, mtp53 and p16 protein. The inhibitory rate of cellular growth of BGC-823 cells after combination treatment was 62.29% and the maximum mitotic index wasdecreased by 51.95%. CONCLUSION： Tachyplesin as a differentiation inducer of tumor cells has similar effects as n-sodium butyrate on proliferation of tumor cells, expression of correlative oncogene and tumor suppressor gene. It also has a synergistic effect on differentiation of tumor cells.

Effect of Helicobacter pylori on gastric epithelial cells

The gastrointestinal epithelium has cells with features that make them a powerful line of defense in innate mucosal immunity. Features that allow gastrointestinal epithelial cells to contribute in innate defense include cell barrier integrity, cell turnover, autophagy, and innate immune responses. Helicobacter pylori (H. pylori) is a spiral shape gram negative bacterium that selectively colonizes the gastric epithelium of more than half of the world&#x02019;s population. The infection invariably becomes persistent due to highly specialized mechanisms that facilitate H. pylori&#x02019;s avoidance of this initial line of host defense as well as adaptive immune mechanisms. The host response is thus unsuccessful in clearing the infection and as a result becomes established as a persistent infection promoting chronic inflammation. In some individuals the associated inflammation contributes to ulcerogenesis or neoplasia. H. pylori has an array of different strategies to interact intimately with epithelial cells and manipulate their cellular processes and functions. Among the multiple aspects that H. pylori affects in gastric epithelial cells are their distribution of epithelial junctions, DNA damage, apoptosis, proliferation, stimulation of cytokine production, and cell transformation. Some of these processes are initiated as a result of the activation of signaling mechanisms activated on binding of H. pylori to cell surface receptors or via soluble virulence factors that gain access to the epithelium. The multiple responses by the epithelium to the infection contribute to pathogenesis associated with H. pylori.

Expression of E-selectin, integrinβ_1 and immunoglobulin superfamily member in human gastric carcinoma cells and its clinicopathologic significance

AIM： To study the expression levels of E-selectin, integrin β1 and immunoglobulin supperfamily memberintercellular adhesion molecule-1 （ICAM-1） in human gastric carcinoma cells, and to explore the relationship between these three kinds of cell adhesion molecules and gastric carcinoma. METHODS： The serum contents of E-selectin, integrin β1 and ICAM-1 were detected by enzyme-linked immunosorbent assay （ELISA）, in 47 healthy individuals （control group） and in 57 patients with gastric carcinoma （gastric carcinoma group） respectively prior to operation and 7 d after operation. RESULTS： The serum E-selectin, ECAM-1 and integrin β1 were found to be expressed in both control and gastric carcinoma groups. However, they were highly expressed in patients with gastric carcinoma patients before operation or with unresectable tumours. The expression levels of ICAM-1 and integrin β1 were significantly higher in gastric carcinoma patients than in controls （P 〈 0.01）. A comparison of the E-selectin levels between the two groups showed statistically insignificant differnce （P = 0.64）. In addition, the expression levels were all decreased substantially in the postoperative patients subjected to radical resection of the tumours, indicating that the high level expressions of these compounds might be the important factor for predicting the prognosis of these patients. CONCLUSION： Serum E-selectin, ICAM-1 and integrin β1 expression levels are probably related to the metastasis and relapse of gastric cancer.

Effects of small interfering RNA inhibit Class Ⅰ phosphoinositide 3-kinase on human gastric cancer cells

AIM: To investigate the effects of small interfering RNA (siRNA)-mediated inhibition of Class?I?phosphoinositide 3-kinase (Class?I?PI3K) signal transduction on the proliferation, apoptosis, and autophagy of gastric cancer SGC7901 and MGC803 cells.METHODS: We constructed the recombinant replication adenovirus PI3K(I)-RNA interference (RNAi)-green fluorescent protein (GFP) and control adenovirus NC-RNAi-GFP, and infected it into human gastric cancer cells. MTT assay was used to determine the growth rate of the gastric cancer cells. Activation of autophagy was monitored with monodansylcadaverine (MDC) staining after adenovirus PI3K(I)-RNAi-GFP and control adenovirus NC-RNAi-GFP treatment. Immunofluorescence staining was used to detect the expression of microtubule-associated protein 1 light chain 3 (LC3). Mitochondrial membrane potential was measured using the fluorescent probe JC-1. The expression of autophagy was monitored with MDC, LC3 staining, and transmission electron microscopy. Western blotting was used to detect p53, Beclin-1, Bcl-2, and LC3 protein expression in the culture supernatant.RESULTS: The viability of gastric cancer cells was inhibited after siRNA targeting to the Class?I?PI3K blocked Class?I?PI3K signal pathway. MTT assays revealed that, after SGC7901 cancer cells were treated with adenovirus PI3K(I)-RNAi-GFP, the rate of inhibition reached 27.48% ± 2.71% at 24 h, 41.92% ± 2.02% at 48 h, and 50.85% ± 0.91% at 72 h. After MGC803 cancer cells were treated with adenovirus PI3K(I)-RNAi-GFP, the rate of inhibition reached 24.39% ± 0.93% at 24 h, 47.00% ± 0.87% at 48 h, and 70.30% ± 0.86% at 72 h (P < 0.05 compared to control group). It was determined that when 50 MOI, the transfection efficiency was 95% ± 2.4%. Adenovirus PI3K(I)-RNAi-GFP (50 MOI) induced mitochondrial dysfunction and activated cell apoptosis in SGC7901 cells, and the results described here prove that RNAi of Class?I?PI3K induced apoptosis in SGC7901 cells. The results showed that adenovirus PI3K(I)-RNAi-GFP transfection induced punctate distribution of LC3 immunoreactivity, indicating increased formation of autophagosomes. The results showed that the basal level of Beclin-1 and LC3 protein in SGC7901 cells was low. After incubating with adenovirus PI3K(I)-RNAi-GFP (50 MOI), Beclin-1, LC3, and p53 protein expression was significantly increased from 24 to 72 h. We also found that Bcl-2 protein expression down-regulated with the treatment of adenovirus PI3K(I)-RNAi-GFP (50 MOI). A number of isolated membranes, possibly derived from ribosome-free endoplasmic reticulum, were seen. These isolated membranes were elongated and curved to engulf a cytoplasmic fraction and organelles. We used transmission electron microscopy to identify ultrastructural changes in SGC7901 cells after adenovirus PI3K(I)-RNAi-GFP (50 MOI) treatment. Control cells showed a round shape and contained normal-looking organelles, nucleus, and chromatin, while adenovirus PI3K(I)-RNAi-GFP (50 MOI)-treated cells exhibited the typical signs of autophagy.CONCLUSION: After the Class?I?PI3K signaling pathway has been blocked by siRNA, the proliferation of cells was inhibited and the apoptosis of gastric cancer cells was enhanced.

A study on arsenic trioxide inducing in vitro apoptosis of gastric cancer cell lines

INTRODUCTION Cell apoptosis,which involves the biologic regulation of the numbers and vital activity of cells,is an important metaboloc process in both normal cells and tumor cells.

Xiaotan Sanjie decoction attenuates tumor angiogenesis by manipulating Notch-1-regulated proliferation of gastric cancer stem-like cells

AIM: To determine the underlying mechanisms of action and influence of Xiaotan Sanjie (XTSJ) decoction on gastric cancer stem-like cells (GCSCs).

Inhibitory effect of Polo-like kinase 1 depletion on mitosis and apoptosis of gastric cancer cells

AIM： Polo-like kinase 1 （PLK1） serine/threonine kinase plays a vital role in multiple phases of mitosis in gastric cancer cells. To investigate the effect of PLK1 depletion on mitosis and apoptosis of gastric cancer cells. METHODS： PLK1 expression was blocked by small RNA interference（siRNA）. The expression levels of PLK1, cdc2, cyclin B and caspase 3 were detected by Western blotting. Then, PLK1 depletion, cdc2 activity, cell proliferation, cell cycle phase distribution, mitotic spindle structure, and the rate of apoptosis of the PLK1 knockdown cells were observed. RESULTS： PLK1 gene knockdown was associated with increased cyclin B expression, increased cdc2 activity （but not with the expression levels）, accumulation of gastric cancer cells at G2/M, improper mitotic spindle formation, delayed chromosome separation and delayed or arrested cytokinesis. Moreover, PLK1 depletion in gastric cancer cells was associated with decreased proliferation, attenuated pro-caspase 3 levels and increased apoptosis. CONCLUSION： Blockage of to decreased mitosis or even PLK1 expression may lead apoptosis in gastric cancer cells, indicating that PLK1 may be a valuable therapeutic target for gastric cancer.

Sox2 enhances the tumorigenicity and chemoresistance of cancer stem-like cells derived from gastric cancer

Gastric cancer stem-like cells（GCSCs） have been identified to possess the ability of self-renewal and tumor initi-ation.However,the mechanisms involved remain largely unknown.Here,we isolated and characterized the GCSCs by side population（SP） sorting procedure and cultured sphere cells（SC） from human gastric cancer cell lines SGC-7901,BGC-823,MGC-803,HGC-27 and MKN-28.The sorting and culture assay revealed that SP cells proliferated in an asymmetric division manner.In addition,SP cells exhibited a higher potential of spheroid colony formation and greater drug resistance than non-SP cells（NSP）.Moreover,the SC were found with enhanced capabilities of drug resistance in vitro and tumorigenicity in vivo.Sox2 mRNA and protein was highly and significantly overex-pressed in the SP cells and SC.Importantly,downregulation of Sox2 with siRNA obviously reduced spheroid colony formation and doxorubicin efflux,as well as increased apoptosis rate in sphere cells in vitro and suppressed tumori-genicity in vivo.These results suggest that both SP cells and cultured SC enrich with GCSCs and that Sox2 plays a pivotal role in sustaining stem cell properties and might be a potential target for gastric cancer therapy.

Low intensity ultrasound-induced apoptosis in human gastric carcinoma cells

AIM： To investigate the low intensity ultrasound （US）- induced apoptosis in human gastric carcinoma cells and its potential mechanism and to suggest a new therapeutic approach to gastric carcinoma. METHODS： Human SGC-7901 gastric carcinoma cells were cultured in vitro and irradiated by low intensity US for 10 min at different intensities with different incubation times after irradiation. Morphologic changes were examined under microscope with trypan blue staining and then the percentage of early apoptotic cells was detected by flow cytometry （FCM） with double staining of fiuorescein isothiocyanate （FITC）- Annexin V/propidium iodide （PI）. Two-dimensional electrophoresis （2DE） was used to get the protein profile and some proteins differently expressed after US irradiation were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry （MALDI-TOF-MS）. Functional analysis was performed to investigate the mechanism of US-induced cell apoptosis. RESULTS： The percentage of apoptotic cells increased about 10% after US irradiation （12.0 W/cm^2, 12 h culture）, The percentage of early apoptosis and secondary necrosis in the US-irradiated cells increased with the increased US intensity. Moreover, apoptotic cells increased with the increased culture time after US irradiation and reached its maximum at about 12 h.Several new proteins appeared after US irradiation and were up or down regulated more than 2 times. Some heat shock proteins （HSPs） were found to be associated with the signal process simulating the apoptosis of cells. CONCLUSION： Low intensity US could induce apoptosis in human gastric carcinoma cells. US-induced apoptosis is related to US intensity/culture time. US-induced apoptosis may be caspases-dependent and endoplasmic reticulum （ER） stress-triggered apoptosis may also contribute to it. Proteomic experimental system is useful in finding the protein alteration in carcinoma cells after US irradiation, helping to develop a new cancer therapy.

Enhanced expression of epidermal growth factor receptor gene in gastric mucosal cells by the serum derived from rats treated with electroacupuncture at stomach meridian acupoints

AIM: To investigate the effect of serum derived from rats treated with electroacupuncture at stomach meridian acupoints on the expression of epidermal growth factor receptor (EGFR) gene in gastric mucosal cells. METHODS: The stress-induced gastric mucosal injury in rat model was established by water-immersion and restrained stress methods. 52 rats were randomly divided into: normal group (n = 8), model group (n = 8), model serum group (n = 12), stomach serum group (n = 12), and gallbladder serum group (n = 12). The gastric mucosal cells were separated by pronase-EDTA digestion method and incubated with serum. The EGFR gene expression in gastric mucosal cells was detected by reverse transcription-polymerase chain reaction (RT-PCR) method. RESULTS: Compared with normal group (0.6860 ± 0.0594), the serum derived from rats of the stomach group (1.2272 ± 0.0813, P = 0.00 < 0.01) and gallbladder group (0.9640 ± 0.0387, P = 0.00 < 0.01) had a tendency to enhance the EGFR gene expression in gastric mucosal cells. Such tendency existed in the model group (0.7104 ± 0.0457) but with no signifi cant difference (P = 0.495 > 0.05) and in model serum group (0.8516 ± 0.0409) with an extremely obvious difference (P = 0.001 < 0.01). Furthermore, the EGFR gene expression in stomach serum group was significantly higher than that in gallbladder serum group (P = 0.00 < 0.01). CONCLUSION: The present study shows that serumderived from rats treated with electroacupuncture at stomach meridian acupoints can distinctly increase the EGFR gene expression of gastric mucosal cells. Therefore, there is certain meridian specificity in the serum, which could provide a proof for the TCM theory “particular relation between meridian and internal organ”.

Quercetin exerts anti-inflammatory effects via inhibiting tumor necrosis factor-α-induced matrix metalloproteinase-9 expression in normal human gastric epithelial cells

BACKGROUND Gastric injury is the most common digestive system disease worldwide and involves inflammation,which can lead to gastric ulcer or gastric cancer(GC).Matrix metallopeptidase-9[MMP-9(gelatinase-B)]plays an important role in inflammation and GC progression.Quercetin and quercetin-rich diets represent potential food supplements and a source of medications for treating gastric injury given their anti-inflammatory activities.However,the effects and mechanisms of action of quercetin on human chronic gastritis and whether quercetin can relieve symptoms remain unclear.AIM To assess whether tumor necrosis factor-α(TNF-α)-induced MMP-9 expression mediates the anti-inflammatory effects of quercetin in normal human gastric mucosal epithelial cells.METHODS The normal human gastric mucosa epithelial cell line GES-1 was used to establish a normal human gastric epithelial cell model of TNF-α-induced MMP-9 protein overexpression to evaluate the antiinflammatory effects of quercetin.The cell counting Kit-8 assay was used to evaluate the effects of varying quercetin doses on cell viability in the normal GES-1 cell line.Cell migration was measured using Transwell assay.The expression of proto-oncogene tyrosine-protein kinase Src(cSrc),phospho(p)-c-Src,extracellular-signal-regulated kinase 2(ERK2),p-ERK1/2,c-Fos,p-c-Fos,nuclear factor kappa B(NF-κB/p65),and p-p65 and the effects of their inhibitors were examined using Western blot analysis and measurement of luciferase activity.p65 expression was detected by immunofluorescence.MMP-9 m RNA and protein levels were measured by quantitative reverse transcription polymerase chain reaction(q RT–PCR)and gelatin zymography,respectively.RESULTS q RT-PCR and gelatin zymography showed that TNF-αinduced MMP-9 m RNA and protein expression in a dose-and time-dependent manner.These effects were reduced by the pretreatment of GES-1 cells with quercetin or a TNF-αantagonist(TNFR inhibitor)in a dose-and timedependent manner.Quercetin and TNF-αantagonists decreased the TNF-α-induced phosphorylation of c-Src,ERK1/2,c-Fos,and p65 in a dose-and time-dependent manner.Quercetin,TNF-αantagonist,PP1,U0126,and tanshinone IIA(TSIIA)reduced TNF-α-induced c-Fos phosphorylation and AP-1–Luciferase(Luc)activity in a dose-and time-dependent manner.Pretreatment with quercetin,TNF-αantagonist,PP1,U0126,or Bay 11-7082 reduced TNF-α-induced p65 phosphorylation and translocation and p65–Luc activity in a dose-and timedependent manner.TNF-αsignificantly increased GES-1 cell migration,and these results were reduced by pretreatment with quercetin or a TNF-αantagonist.CONCLUSION Quercetin significantly downregulates TNF-α-induced MMP-9 expression in GES-1 cells via the TNFR-c-Src–ERK1/2 and c-Fos or NF-κB pathways.

Dynamic functional and ultrastructural changes of gastric parietal cells induced by water immersion-restraint stress in rats

AIM： To investigate the dynamic functional and ultrastructural changes of gastric parietal cells induced by water immersion-restraint stress （WRS） in rats.METHODS： WRS model of Sprague-Dawley （SD） rats was established. Fifty-six male SD rats were randomly divided into control group, stress group and post-stress group. The stress group was divided into 1, 2 and 4 h stress subgroups. The post-stress group was divided into 24, 48 and 72 h subgroups. The pH value of gastric juice, ulcer index （UI） of gastric mucosa and H^＋, K^＋- ATPase activity of gastric parietal cells were measured. Ultrastructural change of parietal cells was observed under transmission electron microscope （TEM）.RESULTS： The pH value of gastric juice decreased time-dependently in stress group and increased in post-stress group. The H^＋, K^＋-ATPase activity of gastric parietal cells and the UI of gastric mucosa increased time-dependently in stress group and decreased in poststress group. Compared to control group, the pH value decreased remarkably （P = 0.0001）, the UI and H^＋, K^＋- ATPase activity increased significantly （P = 0.0001, P = 0.0174） in 4 h stress subgroup. UI was positively related with stress time （r = 0.9876, P 〈 0.01） but negatively with pH value （r = -0.8724, P 〈 0.05）. The parietal cells became active in stress group, especially in 4 h stress subgroup, in which plenty of intracellular canalicular and mitochondria were observed under TEM. In post-stress group, the parietal cells recovered to resting state.CONCOUSION： The acid secretion of parietal cells is consistent with their ultrastructural changes during the development and healing of stress ulcer induced by WRS and the degree of gastric mucosal lesions, suggesting gastric acid play an important role in the development of stress ulcer and is closely related with the recovery of gastric mucosal lesions induced by WRS.

H pylori stimulates proliferation of gastric cancer cells through activating mitogen-activated protein kinase cascade

AIM: To explore the mechanism by which H pylori causes activation of gastric epithelial cells. METHODS: A VacA (+) and CagA (+) standard H pylori line NCTC 11637 and a human gastric adenocarcinoma derived gastric epithelial cell line BGC-823 were applied in the study. MTT assay and 3H-TdR incorporation test were used to detect the proliferation of BGC-823 cells and Western blotting was used to detect the activity and existence of related proteins. RESULTS: Incubation with H pylori extract increased the proliferation of gastric epithelial cells, reflected by both live cell number and DNA synthesis rate. The activity of extracellular signal-regulated protein kinase (ERK) signal transduction cascade increased within 20 min after in- cubation with H pylori extract and appeared to be a sus- tained event. MAPK/ERK kinase (MEK) inhibitor PD98059 abolished the action of H pylori extract on both ERK activity and cell proliferation. Incubation with H pylori extract increased c-Fos expression and SRE-dependent gene expression. H pylori extract caused phosphorylation of several proteins including a protein with molecular size of 97.4 kDa and tyrosine kinase inhibitor genistein inhibited the activation of ERK and the proliferation of cells caused by H pylori extract. CONCLUSION: Biologically active elements in H pylori extract cause proliferation of gastric epithelial cells through activating tyrosine kinase and ERK signal trans- duction cascade.

Localization and translocation of RhoA protein in the human gastric cancer cell line SGC-7901

AIM： To elucidate the localization of RhoA in gastric SGC-7901 cancer cells and its translocation by lysophosphatidic acid （LPA） and/or 8-chlorophenylthio- cAMP （CPT-cAMP）.METHODS： Immunofluorescence microscopy was used to determine the localization of RhoA. Western blotting was used to detect both endogenous and exogenous RhoA in different cellular compartments （membrane, cytosol, nucleus） and the translocation of RhoA following treatment with LPA, CPT-cAMP, or CPT-cAMP ＋ LPA.RESULTS： Immunofluorescence staining revealed endogenous RhoA to be localized in the membrane, the cytosol, and the nucleus, and its precise localization within the nucleus to be the nucleolus. Western blotting identified both endogenous and exogenous RhoA within different cellular compartments （membrane, oltosol, nucleus, nucleolus）. After stimulation with LPA, the amount of RhoA within membrane and nuclear extracts increased, while it decreased in the cytosol fractions. After treatment with CPT-cAMP the amount of RhoA within the membrane and the nuclear extracts decreased, while it increased within the cytosol fraction. Treatment with a combination of both substances led to a decrease in RhoA in the membrane and the nucleus but to an increase in the cytosol.CONCLUSION： In SGC-7901 cells RhoA was found to be localized within the membrane, the cytosol, and the nucleus. Within the nucleus its precise localization could be demonstrated to be the nucleolus. Stimulation with LPA caused a translocation of RhoA from the cytosol towards the membrane and the nucleus; treatment with CPT-cAMP caused the opposite effect. Furthermore, pre-treatment with CPT-cAMP was found to block the effect of LPA.

Effects of H pylori infection on gap-junctional intercellular communication and proliferation of gastric epithelial cells in vitro

AIM： To explore the effects of H pylori infection on gap-junctional intercellular communication （GJIC） and proliferation of gastric epithelial cells in vitro. METHODS： A human gastric epithelial cell line （SGC- 7901） cultured on coverslips was exposed overnight to intact H pylori （CagA^＋ or CagA^- strains） and sonicated extracts, respectively. GJIC between the cells was detected by fluorescence redistribution after photobleaching （FRAP） technique. Proliferation of SGC cells was determined by methylthiazolyl tetrazolium （MTT） assay. RESULTS： When compared with control in which cells were cultured with simple medium alone, both CagA^＋ and CagA^- H pylori isolates could inhibit GJIC （CagA^＋： F = 57.98, P 〈 0.01; CagA^-： F = 29.59, P 〈 0.01） and proliferation （CagA^＋： F = 42.65, P 〈 0.01; CagA^-： F = 58.14, P 〈 0.01） of SGC-7901 cells. Compared with CagA^- strains, CagA^＋ H pylori more significantly downregulated GJIC of gastric cells （intact Hpylori： t = 13.86, P 〈 0.01; sonicated extracts： t = 11.87, P 〈 0.01） and inhibited proliferation gastric cells to a lesser extent in vitro （intact H pylori： t = 3.06, P 〈 0.05; sonicated extracts： t = 3.94, P 〈 0.01）. CONCLUSION： Compared with CagA^- H pylori strains, CagA^＋ strains down-regulate GJIC of gastric epithelial cells more significantly and inhibit proliferation of gastric cells to a lesser extent in vitro. H pylori, especially CagA^＋ strains, may play an important role in gastric carcinogenesis.