Helicobacter pylori inhibits activity of cdc2 kinase and delays G2/M to G1 progression in gastric adenocarcinoma cell line

BACKGROUND: Helicobacter pylori is a bacterial pathogen strongly associated with ulcer diseases and gastric cancer. The bacterial-induced alteration of cell-cycle control in host cells may play a role in the pathogenetic mechanisms. The aims of this study were to define the effect of H. pylori on the G2/M to G1 transition in a gastric cell line. METHODS: Cultured gastric cells, AGS, were synchronized in the S/early G2 phase and treated with intact H. pylori. The cell-cycle distribution of AGS cells was determined by flow cytometry. The activity of cdc2 kinase, as well as of some parameters that affect the kinase activity, was also examined. RESULTS: H. pylori delays cell-cycle progression at the G2/M phase in AGS cells. The G2/M delay was associated with reduced activity of cdc2 kinase. Both down-regulation of cell-cycle regulators (p34cdc2, cyclin B1 and cdc25C) and decreased association between p34cdc2 and cyclin B1 were found to be associated with the activity of cdc2 kinase abated after the H. pylori infection. In addition, the H. pylori-induced G2/M delay required direct contact between the bacteria and host cells. CONCLUSIONS: H. pylori inhibits G2/M to G1 progression and causes a reduction of cell division in gastric epithelial cells.     

Upregulated Cyclooxygenase-2 Inhibits Apoptosis of Human Gastric Epithelial Cells Infected with Helicobacter pylori

Helicobacter pylori induces apoptosis and alters the proliferation of gastric mucosal epithelial cells. Cyclooxygenase-2 (COX-2), the inducible form of prostaglandin (PG) synthesis, is known to cause alteration in epithelial cell growth. The goal of this study was to determine whether COX-2 gene expression by H. pylori infection could influence gastric epithelial cell apoptosis. Expression of COX-2 mRNA and proteins was up-regulated in Hs746T gastric epithelial cell lines infected with H. pylori, when assessed by quantitative RT-PCR and western blot. Inhibition of COX-2 expression using NS-398, a specific COX-2 inhibitor, showed a significant increase of gastric epithelial cell apoptosis and caspase-3 activation in Hs746T cells infected with H. pylori. Moreover, the effect of NS-398 on H. pylori-induced apoptosis was reversed by the addition of PGE₂. These results suggest that up-regulated COX-2 expression by H. pylori infection can inhibit apoptosis of gastric epithelial cells.     

Duodenogastric Reflux and Helicobacter pylori Infection Synergistically Increase Gastric Mucosal Cell Proliferative Activity in Mongolian Gerbils

Background: Helicobacter pylori and duodenogastric reflux (DGR) are both recognized as aetiological factors in chronic gastritis and gastric carcinogenesis. In this study, a Mongolian gerbil (MG) model was used to investigate the histopathological changes in the gastric mucosa resulting from DGR and/or H. pylori infection. Methods: One-hundred-and-eleven 7-week-old, specific-pathogen-free, male MGs were divided into four groups: normal controls, gerbils with surgically induced DGR, and H. pylori-infected gerbils with and without DGR. Gerbils were killed 4, 12 and 26 weeks after DGR surgery, their stomachs removed and sections prepared. Sections were fixed immediately in 20% phosphate-buffered formalin and subjected to haematoxylin and eosin staining, Alcian blue at pH 2.5/periodic acid-Schiff staining, and immunostaining for smooth muscle cells, H. pylori and 5′-bromo-2′-deoxyuridine (BrdU). Results: The gastric mucosa of H. pylori-infected gerbils showed chronic active gastritis irrespective of DGR throughout the experimental period. The gastric mucosa of H. pylori-infected gerbils with DGR demonstrated higher BrdU labelling than in the other groups. Conclusions: In MGs, DGR and H. pylori infection synergistically increased gastric mucosal cell proliferative activity. DGR and H. pylori infection may be involved synergistically in gastric carcinogenesis by increasing cell proliferative activity.     

幽门螺杆菌对人上皮细胞的吸附

幽门螺杆菌对人胃粘膜的吸附是实现其感染的第一步。本研究以人上皮细胞株ＨＥｐ—２为材料对幽门螺杆菌吸附上皮细胞的性能进行了探讨，结果显示，幽门螺杆菌ＹＣ—１１Ａ株具有特异吸附于ＨＥｐ—２细胞表面的性能，其吸附能力在与ＨＥｐ—２细胞共孵育３ｈ后达到高峰，吸附率达８１％，此后吸附率虽无显著提高，但单个ＨＥｐ—２细胞吸附的幽门螺杆菌数量却随孵育时间的延长而有明显增加。在大气环境中与在含５％氧气的微氧环境中幽门螺杆菌对ＨＥｐ—２的吸附无显著差异。抗幽门螺杆菌优势抗原单克隆抗体不能有效阻断幽门螺杆菌对ＨＥｐ—２的吸附。     

Ammonia Inhibits Proliferation and Cell Cycle Progression at S-Phase in Human Gastric Cells

Helicobacter pylori (Hp) has strong ureaseactivity and produces a large amount of ammonia in thestomach. In animal studies, ammonia was shown toaccelerate cell kinetics of gastric mucosa, andlong-term exposure of the stomach to ammonia leads tomucosal atrophy. To understand this process, we examinedthe effects of ammonia on the growth and cell cycleprogression of human gastric cancer cell lines (HGC-27, MKN1, MKN45) using flowcytometric analysis. Ineach cell line, ammonia inhibited the cell growth in adose-dependent manner and caused significantaccumulation of S-phase cells at a cytostatic dose. DNA synthesis of HGC-27 cells treated with ammoniawas also suppressed to about 50% of that of theuntreated cells. Similar effects were observed onaddition of ammonium chloride at the same concentration, while adjusting the pH of the media with NaOHalone to that with the cytostatic dose of ammonia didnot affect the cell cycle progression. Theseobservations indicate that ammonia induces S-phasearrest in gastric cells independently of pH.     

LY294002对人胃腺癌细胞株BGC-823糖酵解水平的影响及其机制探讨

背景:有氧糖酵解是肿瘤细胞的特征性表型之一,靶向糖酵解有望成为一种有效的肿瘤治疗策略。M2型丙酮酸激酶(PKM2)在肿瘤组织中呈高表达,参与肿瘤细胞的有氧糖酵解。缺氧诱导因子-1α(HIF-1α)是调控肿瘤细胞糖酵解相关基因表达的重要转录因子,而PI3K信号在肿瘤细胞中可上调HIF-1α表达。目的:探讨PI3K特异性抑制剂LY294002对人胃腺癌细胞增殖和糖酵解水平的影响及其可能机制。方法:以不同浓度LY294002(10～100μmol/L)在不同条件下(常氧或缺氧)处理人胃腺癌细胞株BGC-823,CCK-8实验和流式细胞分析检测细胞增殖和细胞凋亡,蛋白质印迹法检测p-Akt、p-mTOR、HIF-1α、PKM2表达,比色法检测反映糖酵解水平的胞内乳酸脱氢酶活性和胞外乳酸浓度。结果:LY294002可呈浓度和时间依赖性地抑制BGC-823细胞增殖,在较高浓度时可诱导细胞早期凋亡。LY294002可呈浓度依赖性地抑制p-Akt、p-mTOR、HIF-1α表达,在较高浓度时可抑制PKM2表达,同时降低糖酵解水平。缺氧诱导可消除LY294002对HIF-1α、PKM2表达和糖酵解水平的抑制作用。结论:LY294002可通过阻断PI3K/Akt/mTOR信号通路抑制人胃腺癌细胞增殖及其糖酵解水平,而HIF-1α介导了糖酵解的抑制过程。     

Effects of Helicobacter pylori Infection and Gastritis on Gastric Alcohol Dehydrogenase Activity

Sex and age differences in gastric alcohol dehydrogenase activity in relation to abnormalities of gastric histology and Helicobacter pylori infection were determined in 63 patients (32 men and 31 women) undergoing upper endoscopy for gastrointestinal symptoms. No sex difference was found in gastric alcohol dehydrogenase activity. Males older than 50 years had lower enzyme activity than younger males. Patients with H. pylori and/or moderate to severe chronic and acute inflammation and epithelial mucin depletion had lower alcohol dehydrogenase activity in the antrum, but not in the fundus. H. pylori was found more frequently in the older male patients. Antral alcohol dehydrogenase was most decreased in older patients of both sexes with H. pylori infection. In conclusion, H. pylori infection and/or chronic active gastritis are important causes of low gastric alcohol dehydrogenase activity.     

Helicobacter pylori and Cell Proliferation of the Gastric Mucosa: Possible Implications for Gastric Carcinogenesis

Objectives: Helicobacter pylori infection is recognized as a risk factor for gastric adenocarcinoma. "Mitogenesis increases mutagenesis," so the effects of H. pylori infection on the gastric mucosal proliferative compartment have been investigated. Methods: In 25 H. pylori-positive and 19 H. pylori-negative subjects, epithelial cell proliferative activity and the pattern of the proliferative compartment were separately evaluated in relation to both the different type of mucosa (antrum and corpus) and the H. pylori positivity/negativity after ³H-thymidine labeling. Results: Both mucosal cell kinetics and the pattern of the proliferative compartment in the antrum appeared different from those of the corpus. Comparing H. pylori -positive and H. pylori -negative subjects, differences were detected only in the total number of cells in the antrum, whereas all of the cell kinetics parameters, except the labeling index, were greater in the corpus of the former group. A superficialization of the proliferative compartment was shown in H. pylori -positive subjects. Changes were more evident in subjects with more severe gastritis but were also present in H. pylori -positive subjects without corpus gastritis. Conclusions: These results show that H. pylori infection is associated with modifications in the proliferative compartment of the gastric mucosa. Both infection per se and chronic gastritis seem to be relevant for such changes.     

Progression of Atrophic Gastritis and Intestinal Metaplasia Drives Helicobacter pylori Out of the Gastric Mucosa

This study was performed to evaluate the implication of anti-H. pylori IgG positivity when CLOtest, histological test, and culture in the antrum and body are all negative, and to find out the specific disease category that is more affected by the hostile relationship of atrophic gastritis and intestinal metaplasia (IM) with H. pylori. Four hundred thirty-six patients (84 controls, 69 with duodenal ulcer, 96 with benign gastric ulcer, 43 with dysplasia, 144 with gastric cancer), who had not received any eradication therapy, were divided into three groups according to H. pylori test: CLOtest or histological H. pylori-positive group (group A; 294 cases), only anti-H. pylori IgG-positive group (group B; 62 cases), and anti-H. pylori IgG-negative group (group C; 80 cases). The grade of neutrophil and monocyte infiltration, atrophic gastritis, and IM was compared according to the updated Sydney system classification. Neutrophil and monocyte infiltrations were significantly severe in the group A. In contrast, the grade of atrophic gastritis and IM in the antrum was significantly higher in group B than the other two groups, A or C. When patients were divided according to the disease outcome in each group, the grade of IM in the body was statistically higher only in the patients with cancer or dysplasia in group B. These results suggest that anti-H. pylori IgG positivity with all negative invasive H. pylori tests represents past infection with H. pylori rather than a false negative, especially in the case of dysplasia and gastric cancer.     

粪便肿瘤型M2丙酮酸激酶在胃肠道肿瘤检测中的意义

肿瘤型M2丙酮酸激酶（M2-PK）是近年发现的一种新型肿瘤标志物。目的：评价粪便肿瘤型M2-PK在胃肠道肿瘤和癌前状态的检测以及肿瘤分期中的意义。方法：以酶联免疫吸附测定（ELISA）检测34例胃癌、31例结直肠癌、19例胃肠道息肉、26例慢性萎缩性胃炎和19例对照者的粪便肿瘤型M2-PK值，胃癌和结直肠癌患者同时行传统外周血肿瘤标志物癌胚抗原（CEA）、糖链抗原（CA）19-9和CA24-2水平检测。结果：胃癌组和结直肠癌组的粪便肿瘤型M2-PK检测值和阳性率均较对照组显著增高（P〈0．01），胃肠道息肉组的检测值亦较对照组显著增高（P〈0．05）。随着肿瘤临床病理分期的进展和转移的发生，粪便肿瘤型M2-PK检测值逐渐增高（胃癌组：P〈0．05；结直肠癌组：P〈0．01）。胃癌组和结直肠癌组粪便肿瘤型M2-PK检测的阳性率均显著高于血清CEA、CA19．9和CA24．2（P〈0．01）。结论：粪便肿瘤型M2-PK对胃肠道肿瘤和癌前状态之一的胃肠道息肉的诊断有一定临床意义。     

胃液总胆汁酸和幽门螺杆菌感染与胃黏膜肠化生的相关性研究

背景：幽门螺杆菌（Hp）感染和胃内胆汁反流与胃黏膜肠化生密切相关,但其机制尚不完全清楚。目的：研究胃液总胆汁酸（TBA）和Hp感染与肠化生指标CDX2、MUC2的相关性。方法：收集81例有胃内胆汁反流且无腹部手术史者和40例无胆汁反流对照者的胃液标本检测TBA浓度,于胃窦部取活检标本行HE染色、HID—AB—PAS染色肠化生分型和CDX2、MUC2免疫组化染色。同时行血清HpIgG抗体检测。结果：胆汁反流组慢性非萎缩性胃炎（CNAG）37例,慢性萎缩性胃炎（CAG）44例（均伴完全型肠化生）,对照组40例均为CNAG。胆汁反流组胃液TBA浓度和胃黏膜CDX2、MUC2蛋白阳性表达率显著高于对照组（P〈0．01）,其中CAG组显著高于CNAG组（P〈0．01）。胆汁反流组与对照组间血清HpIgG抗体阳性率无明显差异。胆汁反流组CDX2、MUC2蛋白阳性表达率随TBA浓度的增高呈上升趋势（P〈0．01）,Hp感染阳性者CDX2、MUC2蛋白阳性表达率显著高于Hp感染阴性者（P〈0．05）。TBA〉3000μmol／L者的CDX2、MUC2蛋白阳性表达率显著高于Hp感染阳性者（91．2％对63．0％和91．2％对60．9％,P〈0．01）。结论：除Hp感染外,高浓度胆汁酸亦为引起胃黏膜肠化生和CDX2、MUC2表达的重要因素且其作用可能大于Hp感染。     

Gastric adaptation to aspirin and Helicobacter pylori infection in man

Abstract The relationship between Helicobacter pylori infection and aspirin (ASA)-induced gastropathy and gastric adaptation to ASA remains unclear. We compared gastric damage and adaptation after repeated exposures to ASA in the same subjects without H. pylori infection and those infected by H. pylori before and after eradication of this H. pylori. Twenty-four volunteers in two groups (A and B), without H. pylori infection (group A) and with H. pylori infection (group B) before and after H. pylori eradication, were given ASA 2 g/day or placebo for 14 days. Mucosal damage was evaluated by endoscopy and gastric microbleeding; mucosal prostaglandin (PG) E₂ generation and luminal transforming growth factor (TGF)α were determined on days 0,3,7 and 14 of the ASA course. In all subjects, ASA-induced gastric damage reached a maximum on day 3. In H. pylori-positive subjects this damage was maintained at a similar level up to the 14th day of observation. Following H. pylori eradication, the damage was significantly lessened at day 14, as revealed by both endoscopy and microbleeding, and was accompanied by increased mucosal release of TGFα. Prostaglandin E₂ generation was significantly higher in H. pylori-positive subjects than after H. pylori eradication, but ASA treatment resulted in greater than 90% reduction of this generation independent of H. pylori status. Gastric adaptation to ASA is impaired in H. pylori-positive subjects but eradication of this bacterium restores this process.     

Staurosporine-induced G<Subscript>2</Subscript>/M arrest in primary effusion lymphoma BCBL-1 cells

Staurosporine, an inhibitor of protein kinase C, is a potential antitumor drug and its derivatives are used as anticancer drugs in clinical trials. Human herpesvirus 8 (HHV-8) is implicated in all forms of Kaposis sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castlemans disease (MCD), indicating it to be a DNA tumor virus. It is difficult to culture cell lines derived from KS patients; we therefore used a cell line derived from PEL (BCBL-1) to investigate whether staurosporine affects the HHV-8-related tumors. Our results show that staurosporine treatment reduces the cell viability of BCBL-1 cells and causes cell cycle arrest in the G₂/M phase. The G₂/M arrest was associated with the decrease in the expression of Cdc2 and cyclin B. Furthermore, the induction of the HHV-8 lytic cycle was not observed under the staurosporine treatment.     

Eosinophil Cationic Protein and Phospholipase A2 Activity in Human Gastric Juice: With Emphasis on Helicobacter pylori Status and Effects of Antacids

To elucidate possible new effects of antacids, gastric juice from 15 volunteers with known Helicobacter pylori status were analysed for eosinophil cationic protein (ECP), phospholipase A₂ (PLA₂) activity, phosphatidylcholine (PC), and bile acids (BA) before and after administration of one tablet of antacid or placebo in a double blind cross-over design. Geomtric mean ECP concentrations were more than 13 times higher in gastric juice from H. pylori-positive (12.9 μg/l) than from H. pylori-negative (0.97 μg/l) subjects (p = 0.0032). Geometric mean PLA₂ activity was 1.31 U/l for the negative subjects and 4.02 U/l for the positive subjects (p = 0.13). There were no differences between positive and negative subjects with regard to either PC or BA concentration. Regardless of H. pylori status, mean PC concentration increased significantly after antacids as compared with placebo (p = 0.024). The effect of antacids did not differ significantly from placebo for ECP, PLA₂ activity, or BA concentration. Hence, antacids may not act by binding ‘toxic’ H. pylori-associated gastric juice components like ECP or PLA₂. Increased concentration of PC may indicate an increased protective capacity induced by antacids.     

Hsp90 inhibitors cause G2/M arrest associated with the reduction of Cdc25C and Cdc2 in lung cancer cell lines

Purpose: Hsp90, a molecular chaperone, is involved in folding, assembly, maturation, and stabilization of the client proteins which regulate survival of cancer cells, and thus Hsp90 inhibitors may be potential molecular targeting agents for cancer treatment. We investigated whether Hsp90 inhibitors have therapeutic value in lung cancer. Methods: First, expression levels of Hsp90 in lung cancer cells were examined by western blotting and immunohistochemical analyses. Next, the effect of Hsp90 inhibitors, geldanamycin and 17-allylaminogeldanamycin (17-AAG), on lung cancer cell growth was examined. Results: Remarkable high expression of Hsp90 protein in lung cancer cell lines and a more intense signal for Hsp90 by immunohistochemistry in males, patients with smoking index over 600, and squamous cell carcinoma were observed. Both Hsp90 inhibitors dose dependently inhibited the growth of lung cancer cell lines and induced G2/M arrest concomitant with decreased protein levels of Cdc25C and Cdc2. Moreover, combination of an Hsp90 inhibitor and irradiation had an additive effect on cell growth inhibition and reduction of Cdc25C and Cdc2 protein levels. Conclusion: Hsp90 inhibitor is thus a therapeutic tool for lung cancer based on its target proteins, which are involved in tumor progression and antiproliferative activity in lung cancer cells.     

幽门螺杆菌相关性胃癌患者局部Th1/Th2细胞免疫应答变化

背景:幽门螺杆菌(Hp)感染与胃癌发生相关,免疫应答可能是其主要致病机制之一。目的:探讨Hp相关性胃癌患者黏膜局部Th1/Th2细胞免疫应答变化。方法:选取2011年1～6月27例胃癌患者和28例对照者,采用14C-尿素呼气试验、Hp抗体检测Hp感染,免疫组化法检测胃黏膜局部干扰素(IFN)-γ和白细胞介素(IL)-4表达情况。结果:Hp总体感染率为61.8%。Hp阳性患者中,胃癌组IFN-γ表达明显低于对照组(P<0.05),IL-4表达明显高于对照组(P<0.05);Hp阴性患者中,胃癌组IFN-γ、IL-4表达与对照组均无明显差异(P>0.05)。Hp阳性胃癌患者IL-4表达明显高于Hp阴性胃癌患者(P<0.05),Hp阳性对照组IFN-γ表达明显高于Hp阴性对照组(P<0.05)。结论:Hp阳性对照者以Th1细胞介导的免疫应答为主,Hp阳性胃癌患者以Th2细胞为主。黏膜局部Th1/Th2细胞免疫应答变化可能与Hp感染相关疾病的进展,特别是胃癌的发生密切相关。     

慢性萎缩性胃炎患者幽门螺杆菌根除后胃黏膜G细胞数量和血清胃泌素含量的变化

背景:幽门螺杆菌(H.pylori)感染是慢性萎缩性胃炎(CAG)最重要的致病因素,根除H.pylori能否阻止或逆转胃黏膜萎缩目前尚不清楚.目的:通过观察CAG患者H.pylori根除前后胃黏膜G细胞数量和血清胃泌素含量的变化,探讨H.pylori感染对胃黏膜G细胞数量及其分泌功能的影响.方法:60例H.pylori阳性的CAG患者进行了根除治疗,在治疗前和治疗结束3个月后分别行胃镜检查.采用免疫组织化学法和放射免疫分析法测定H.pylori根除前后胃窦黏膜G细胞数量和血清胃泌素含量.结果:31例H.pylori感染的CAG患者在根除治疗3个月后进行了复查,根除率为77.4%.G细胞数量和血清胃泌素含量随胃黏膜萎缩程度的加重而逐渐显著减少(P<0.01).轻度萎缩组H.pylori根除后G细胞数量与治疗前相比无显著差异(P<0.05),而升高的血清胃泌素含量显著降低(P<0.01);中、重度萎缩组H.pylori根除后减少的G细胞数量显著增加(P<0.05),血清胃泌素含量呈上升趋势(P<0.05).结论:CAG患者根除H.pylori后胃黏膜G细胞数量及其合成、分泌胃泌素的功能可出现恢复性变化,可能有助于阻断CAG的进一步发展.