Sequence analysis of the HBV S protein in Chinese patients with coexisting HBsAg and anti-HBs antibodies.

The coexistence of hepatitis B surface antigen (HBsAg) and antibodies to HBsAg (anti-HBs) in patients with hepatitis B virus (HBV) infection has been discovered and explained for several decades, but debate still exists. This study was to explore the relationship between this special serological pattern and mutations in S gene region. Fifteen patients with coexisting HBsAg and anti-HBs were selected as the experimental group, and 27 patients with HBsAg positive only were selected as the control group. The S gene region was amplified and sequenced. No significant differences were observed between the two groups with regard to age, gender, alanine aminotransferase level, HBsAg titer, genotype, and HBV DNA level. The patients from the two groups were infected with HBV of the genotype B and C. Compared with the control group, the experimental group showed a higher variability in amino acid within the N-terminal region and the MHR, especially the "a" determinant. The most frequent change in patients from the experimental group was located at positions s126. The coexistence of HBsAg and anti-HBs might be associated with the increased amino acid mutations in the "a" determinant. Further studies should be performed to determine the clinical implication of this serological pattern, including the binding of anti-HBs to HBsAg, escape from immune system, and efficacy of antiviral therapy.

[The expression and role of PTEN in doxorubicin induced gastric cancer cell apoptosis].

OBJECTIVE: To study the expression of PTEN and its significance in doxorubicin-treated gastric cancer cells. METHODS: (1) Gastric cancer BGC-823 cells were treated with doxorubicin. Cell proliferation and apoptosis were evaluated by MTT and flow cytometry. The expression of PTEN at the mRNA and protein level were determined by RT-PCT and Western blot, respectively. (2) The gastric cancer xenografts model was constructed. The apoptosis of gastric cancer xenografts cells was determined by TUNEL. The expression of PTEN at the mRNA and protein level were detected using RT-PCR and Western blot, respectively. (3)BGC-823 cells were transfected with PTEN siRNA before addition of doxorubicin. The proliferation and apoptosis of these cells as well as the expression level of PTEN protein were determined. RESULTS: (1) After administration of doxorubicin, the proliferation of BGC-823 cells was inhibited in a time-dependent manner. (2) Doxorubicin significantly induced apoptosis of BGC-823 cells. (3) Doxorubicin treated BGC-823 cells showed a significant increase in the expression of PTEN at the mRNA and protein level in a time-dependent manner. TUNEL assay also showed a significant increase of apoptosis rate in gastric cancer xenografts treated with doxorubicin compared with control group [(28.11 ± 1.05)% vs (2.78 ± 1.63)%]. The expression of PTEN at the mRNA and protein level in the gastric cancer xenografts were significantly increased after administration of doxorubicin (0.5667 ± 0.0043 vs 0.2217 ± 0.0063, 0.14 ± 0.26 vs 0.04 ± 0.15, P < 0.05). (4) After treated with doxorubicin, the expression of PTEN in siRNA-transfected BGC-823 cells was significantly higher than that in non-transfected BGC-823 cells (P < 0.0001). The apoptosis of PTEN siRNA-transfected BGC-823 cells was significantly decreased compared with non-transfected BGC-823 cells [(10.35 ± 1.04)% vs (31.37 ± 3.58)%, P < 0.05]. CONCLUSION: Doxorubicin can effectively inhibit the growth and induce the apoptosis of BGC-823 gastric cancer cells. Increasing PTEN protein may be one of the main mechanism involved in this effect.

Phosphoinositide 3-kinase/Akt pathway plays an important role in chemoresistance of gastric cancer cells against etoposide and doxorubicin induced cell death.

The major obstacle to successful treatment of gastric cancer is chemotherapy resistance. Our study was designed to investigate the role of phosphoinositide 3-kinase (PI3K)/Akt pathway in the development of chemoresistance in gastric cancer. In the present study, elevated Akt expression and Akt phosphorylation (Ser 473), as well as decreased PTEN expression were observed in 28 cases of gastric cancer tissues. Etoposide and doxorubicin stimulated Akt and PI3K activities in 2 gastric cancer cell lines (BGC-823 and SGC-7901), and the activities were concentration and time-dependent. Up-regulation of PTEN expression in BGC-823 cells by PEAK8-PTEN transient transfection obviously decreased the basal and anticancer drugs induced Akt activities, then sensitized BGC-823 cells to etoposide and doxorubicin. Pretreatment of BGC-823 and SGC-7901 cells with wortmannin, a PI3K inhibitor, attenuated cells's resistance to etoposide and doxorubicin. In addition, pretreatment of wortmannin blocked etoposide and doxorubicin induced IkappaB-alpha degradation, NFkappaB activation, phosphorylation of Akt, MDM-2 and forkhead transcription factors. Wortmannin pretreatment also promoted the accumulation of p27/Kip, but inhibited the Mcl-1 expression. Furthermore, wortmannin promoted etoposide and doxorubicin induced caspase-3, caspase-9 activation and poly ADP-ribose polymerase cleavage. Taken together, the observations indicate the PI3K/Akt pathway plays an important role in the chemoresistance of gastric cancer cells. A new strategy for combined chemotherapy of gastric cancer should be designed to more specifically block PI3K/Akt pathway and then decrease the amount of resistant cells.

[Impact of PI3K /Akt /mdm2 signaling pathway on the sensitivity of gastric cancer cell line SGC7901 to doxorubicin].

OBJECTIVE: To explore whether PI3K/Akt/mdm2 signalling pathway affect the sensitivity of gastric cancer cell line SGC7901 cells to doxorubicin. METHODS: Gastric cancer cell line SGC7901 cells were exposed to doxorubicin and specific PI3K inhibitor wortmannin. Cell apoptosis was detected using flow cytometry. PI3K activity was detected by radioactive immunoprecipitation-kinase assay. Western blotting was employed to evaluate the expressions of PI3K-p85, pAkt-S473, Akt, pmdm2-S166 and p53. RESULTS: The level of apoptosis in gastric cancer SGC7901 cells treated with doxorubicin was gradually increasing. wortmannin enhanced its effects significantly. PI3K activity and the expression of pAkt-S473 increased in a time-dependent manner, pmdm2-S166, p53 were also increased wortmannin inhibited phosphorylation of mdm2 and improved the p53 expression. CONCLUSION: PI3K/Akt/mdm2 signalling pathway can be activated by doxorubicin and suppress apoptosis by promoting phosphorylation of mdm2. PI3K inhibitor wortmannin can enhance sensitivity of gastric cancer cells to chemotherapy.

Effect of fluoxetine on depression-induced changes in the expression of vasoactive intestinal polypeptide and corticotrophin releasing factor in rat duodenum.

AIM: To investigate changes in vasoactive intestinal polypeptide (VIP) and corticotrophin releasing factor (CRF) in the plasma and duodenum of chronic stress-induced depressed rats and the effects of fluoxetine hydrochloride (fluoxetine) treatment on depression-induced changes in VIP and CRF. METHODS: A Sprague-Dawley rat model of chronic stress-induced depression was produced. Thirty experimental rats were randomly divided into the following groups: control group, saline-treated depressed group, and fluoxetine-treated depressed group. Open-field testing was performed to assess the rats' behavior. VIP and CRF levels in plasma were measured by ELISA. Immunofluorescence techniques combined with laser scanning confocal microscopy (LSCM) were used to investigate VIP and CRF expression in the duodenum. RESULTS: The open-field behavior, both crossing and rearing, of depression model rats, decreased significantly compared with those of normal control rats over 5 min. Defecation times increased significantly. Compared to the control group, FITC fluorescence of duodenal CRF expression and plasma CRF levels in the depressed rats increased significantly (fluorescence intensity of duodenal CRF: 11.82 +/- 2.54 vs 25.17 +/- 4.63; plasma CRF: 11.82 +/- 2.54 ng/L vs 25.17 +/- 4.63 ng/L, P < 0.01), whereas duodenal VIP expression and plasma VIP levels decreased significantly (fluorescence intensity of duodenal VIP: 67.37 +/- 18.90 vs 44.51 +/- 16.37; plasma VIP: 67.37 +/- 18.90 ng/L vs 44.51 +/- 16.37 ng/L, P < 0.01). Fluoxetine improved depressed behavior, increased VIP expression and decreased CRF expression in plasma and the duodenal tissue of depressed rats. CONCLUSION: Chronic stress can induce injury to the duodenum, accompanied by increasing CRF and decreasing VIP in the plasma and duodenum. Treatment with fluoxetine can ameliorate pathological changes in the duodenum of depressed rats, which suggests that antidepressants are an effective therapeutic agent for some duodenal diseases caused by chronic stress. VIP is a potential therapeutic strategy.

[The role of p38 MAPK in gastrin-induced u-PA expression in human colon cancer cells].

OBJECTIVE: To study the effect of gastrin on the mRNA and protein expression of urokinase-type plasminogen activator (u-PA) in human colon cancer cells and detect the role of p38 MAPK in this process. METHODS: Lipofectin method was used to transfect pCR3. 1/CCK2R vector expressing gastrin receptor into a colon cancer cell line colo320. Gastrin and gastrin antagonist were used to up-regulate and down-regulate the signaling pathway, respectively. Human colon cancer colo320 cells and colo320/ CCK2,R cells were cultured and then stimulated with gastrin for different time; SB203580 was added into culture medium to prevent p38 kinase pathway before incubating with gastrin; Western blot and RT-PCR were used to examine the u-PA expression. Western blot was employed to detect p38 kinase phosphorylation. RESULTS: Gastrin increased evidently the mRNA and protein expressions of u-PA and induced p38 kinase phosphorylation in colo320/CCK,R cells time-dependently. However, the extent of enhancement of u-PA and p38 MAPK expression in colo320 cells was much less than that in colo320/CCK2R cells. The gastrin antagonist L-365, 260 showed an effect of competitive inhibition on gastrin-induced u-PA expression and p38 kinase phosphorylation. The inhibitor SB203580 could sufficiently suppress gastrin-induced p38 kinase phosphorylation and significantly attenuate gastrin-induced u-PA mRNA and protein expressions in colo320/ CCK2 R cells in a dose-dependent manner. CONCLUSION: Gastrin-gastrin receptor signal transduction pathway can obviously induce u-PA expression in human colon cancer cells via activating the phosphorylation of p38 kinase.

[Effects of hFRNK on E-cadherin/beta-catenin in colon cancer cells in vitro].

OBJECTIVE: To explore the effects of human FRNK gene on E-cadherin/beta-catenin complex in colon cancer cell line Colo320WT cells stimulated with extrinsic gastrinl7. METHODS: AdEasy system was used to construct pAdhFRNK expressing human FRNK gene by recombination in E. coli. BJ5283. pCR3.1/GR plasmid expressing gastrin receptor CCK-2 was transfected into colon cancer cell line Colo320 cells by Lipofectamine 2000 and expressing stably CCK-2R clones were screened by G418 (500 pg/ml). The expression levels of gastrin receptor in Colo320 cells and the transfected Colo320WT cells were assayed by RT-PCR. Colo320WT cells were treated by 10(-8) mol/L gastrinl7 for 12 h; and after Colo320WT cells were infected by pAdhFRNK (MOI: 100) for 2 d the cells were treated by gastrin17 for 12 h again. The expression levels of E-cadherin and beta-catenin in TX-100 soluble fraction and TX-100 insoluble fraction of Colo320WT cells were assayed by co-immunoprecipation and Western blot. E-cadherin and beta-catenin's distribution in Colo320WT cells were detected by immunocytochemistry. RESULTS: When 10(-8) mol/L gastrin17 stimulated Colo320WT cells for 12 h, the expression levels of E-cadherin and beta-catenin in TX-100-soluble fraction decreased apparently, while the expression levels of E-cadherin and beta-catenin in TX-100-insoluble fraction increased markedly. When pAdhFRNK infected Colo320WT cells for 2 d and 10(-8) mol/L gastrin17 treated the cells for 12 h, the expression levels of E-cadherin and beta-catenin in TX-100-soluble fraction increased apparently again, and the expression levels of E-cadherin and beta-catenin in TX-100-insolutble fraction decreased markedly. Immunocytochemistry showed that the distribution of E-cadherin and beta-catenin was translocated from plasma membrane into cytoplasm and nucleus in the cells stimulated with gastrinl7, and after the cells were infected with pAdhFRNK and stimulated by gastrinl7 again. beta-catenin was mainly observed in cytoplasm and little nuclear immunoreactivity. CONCLUSION: An adenovirus vector pAdhFRNK can inhibit abnormal distribution of E-cadherin and beta-catenin in the gastrin17-stimulated cells. The mechanism is probably that hFRNK can disphosphorylate phosphorylated FAK and block FAK pathway.

[Molecular mechanism of gastrin increasing colon cancer cells' invasion].

OBJECTIVE: To explore the molecular mechanism of increasing the invasion of colon cancer cells by gastrin 17. METHODS: The plasmid pCR 3.1/GR expressing the gastrin receptor cholecystokinin-2 receptor (CCK-2R) was transfected into colonic carcinoma cells of the line Colo320 by Lipofectamine 2000. The clones expressing stably CCK-2R were screened by G418 and named as Colo320WT cells. The expression levels of gastrin receptor of the Colo320 and Colo320WT cells were assayed by RT-PCR and Western blotting. The Colo320WT cells were treated by gastrin-17, and the expression levels of phosphorylated FAKTyr397 and total focal adhesion kinase (FAK) in the Colo320WT cells at the time points 0, 1, 6, 12, 24, and 48 h were detected by Western blotting. Another Colo320WT cells were treated by L365, 260, gastrin17 receptor blocker, for 30 minutes firstly and then treated by gastrin17 again for 12 hours, and then Western blotting was used to detect the expression levels of phosphorylated FAKTyr397 and total focal adhesion kinase (FAK) at the time points 0, 1, 6, 12, 24, and 48 h. Confocal microscopy was used to observe the phosphorylated FAKTyr397 localizing in the lamellipodia. The information of FAK-Src-p130(Cas)-Dock180 signaling complex was assayed by coimmuniprecipation and immunity blotting. The level of Rac-GTPase was tested by pull down assay. RESULTS: The level of phosphorylated FAKTyr397 expression in the Colo320WT cells after the gastrin17 intervention increased time-dependently and peaked at the time point of 12 h, and the phosphorylated FAKTyr397 expression in the Colo320WT cells treated by L365, 260 decreased remarkably, but the level of total FAK remained unchanged. The phosphorylated FAKTyr397/FAK levels were 2.82%, 9.28%, 22.62%, 38.59%, 28.41%, and 14.94%, 0, 1, 6, 12, 24, and 48 h after the gastrin17 treatment respectively, and the level was 7.21% after L365, 260 treatment. The amount of phosphorylated FAKTyr397 localizing in the lamellipodia of the Colo320WT cells that were treated by gastrin17 increased time-dependently and peaked at the time-point 12 h. FAK-Src-p130(Cas)-Dock180 signaling complex was formed in the Colo320WT cells stimulated with gastrin17. Gastrin17 activated Rac, but did not affect the total Rac expression. CONCLUSION: The mechanism of increasing the colon cancer cells' invasion by gastrin17 is probably that gastrin17 makes FAK-Tyr397 phosphorylated and be localized to lamellipodia, causes the forming of FAK-Src-p130(Cas)-Dock180 signaling complex when it is bound to its receptor CCK-2, and activation of Rac.

Effects of gastrin 17 on beta-catenin/Tcf-4 pathway in Colo320WT colon cancer cells.

AIM: To explore the effect of gastrin 17 (G17) on beta-catenin/T cell factor-4 (Tcf-4) signaling in colonic cancer cell line Colo320WT. METHODS: The pCR3.1/GR plasmid, which expresses gastrin receptor, cholecystokinin-2 receptor (CCK-2R), was transfected into a colonic cancer cell line Colo320 by Lipofectamine (TM)2000 and the stably expressing CCK-2R clones were screened by G418. The expression levels of gastrin receptor in the Colo320 and the transfected Colo320WT cell line were assayed by RT-PCR. Colo320WT cells were treated with G17 in a time-dependent manner (0, 1, 6, 12, 24 and 48 h), then with L365,260 (Gastrin(17) receptor blocker) for 30 min, and with G17 again for 12 h or L365,260 for 12 h. Expression levels of beta-catenin in a TX-100 soluble fraction and TX-100 insoluble fraction of Colo320WT cells treated with G17 were detected by co-immuniprecipation and Western blot. Immunocytochemistry was used to examine the distribution of beta-catenin in CoLoWT320 cells. Expression levels of c-myc and cyclin D1 in Colo320WT cells treated with G17 were assayed by Western blot. RESULTS: Expression levels of beta-catenin in the TX-100 solution fraction decreased apparently in a time-dependent fashion and reached the highest level after G17 treatment for 12 h, while expression levels of beta-catenin in the TX-100 insoluble fraction were just on the contrary. Immunocytochemistry showed that beta-catenin was translocated from the cell membranes into the cytoplasm and nucleus under G17 treatment. Expression levels of c-myc and cyclin D1 in the G17-treated Colo320WT cells were markedly higher compared to the untreated Colo320WT cells. In addition, the aforementioned G17-stimulated responses were blocked by L365,260. CONCLUSION: Gastrin17 activates beta-catenin/Tcf-4 signaling in Colo320WT cells, thereby leading to over-expression of c-myc and cyclin D1.

Endoscopic findings and pathologic characteristics of gastric eosinophilic granuloma: a report of 18 patients.

AIM: To investigate the endoscopic findings and patholo-gic characteristics of gastric eosinophilic granuloma (GEG). METHODS: A retrospective study of 18 cases of gastric eosinophilic granulomas was conducted. Gastroscopy was performed and all specimens of biopsies were stained by HE and observed under light microscopy. RESULTS: Ulcer was the most frequent endoscopic appearance. The others included deformed pylorus and/or duodenal bulb, esophagitis, mucous hyperemia and/or mucosal erosion. Eosinophilic cell infiltration and generous hyperplasia of arterioles, venules and lymph vessels were found in the lesions of the patients. Interstitium had massive eosinophilic infiltrates and was made up of collagen fibers and fibroblasts. Lymphoid follicles were revealed in some sections of biopsies. CONCLUSION: GEG is lack of specific symptoms and physical signs. It can be misdiagnosed as gastric ulcer in most cases before biopsies. Endoscopy and endoscopic multiple deep biopsies in suspected areas are indispensable for correct diagnosis of GEG.

Therapeutic effects and molecular mechanisms of Ginkgo biloba extract on liver fibrosis in rats.

Oxidative stress can be implicated as a cause of liver fibrosis. In this sense, Ginkgo Biloba Extract (EGB), an antioxidant, may be beneficial in restraining liver fibrosis. The aim of this study was to evaluate the effects of EGB on experimental liver fibrosis. Rat liver fibrosis was induced by intraperitoneal injection of carbon tetrachloride (CCl4) twice a week for 8 weeks. Three groups of rats received EGB (0.25, 0.5 and 1.0 g/kg, respectively) by stomach everyday. CCl4 administration induced liver fibrosis, which was inhibited by EGB in a dose-dependent manner. The histopathologic score of fibrosis, liver function and the levels of plasma hyaluronic acid (HA) and laminin (LN) were significantly improved in rats treated with CCl4 + EGB, compared with those treated with CCl4 only (p < 0.01 or p < 0.05). The activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were notably elevated, while malondialdehyde (MDA) content was significantly decreased in the rats treated with CCl4 + EGB (p < 0.01 or p < 0.05). Inhibition of hepatic stellate cell (HSC) activation and nuclear factor kappaBP65 (NF-kappaBP65) expression was demonstrated in the livers of EGB-treated rats. The activation of NF-kappaB was significantly suppressed in EGB-treated rats determined by electrophoretic mobility shift assay (EMSA). Furthermore, EGB reduced expressions of transforming growth factor-beta1 (TGF-beta1) and collagen I mRNA. In conclusion, EGB is able to ameliorate liver injury and prevent rats from CCl4-induced liver fibrosis by suppressing oxidative stress. This process may be related to inhibiting the induction of NF-kappaB on HSC activation and the expression of TGF-beta1.

Expression of cellular FLICE-inhibitory protein and its association with p53 mutation in colon cancer.

AIM: To investigate the expression of cellular FLICE (Fas associated death domain-like IL-1beta-converting enzyme)-inhibitory protein (c-FLIP) and its association with p53 mutation in colon cancer. METHODS: Immunohistochemical staining of c-FLIP and mutant p53 by using specific antibodies was performed by the standard streptavidin-peroxidase technique for 45 colon cancer tissue samples with matched normal tissues. Semi-quantitative reverse transcriptional (RT)-PCR was used to measure c-FLIP mRNA levels. t-test statistical method was used in data analyses. RESULTS: c-FLIP mRNA was expressed in all colon cancer tissues and its level (0.63+/-0.12) was significantly higher than that in normal tissues (0.38+/-0.10, P<0.01). Immuno-histochemically, c-FLIP protein was also expressed in all colon cancers (45/45) and 71.1% (32/45) showed an intense immunostaining, in contrast, 93.3% (42/45) of normal colonic mucosa showed positive staining and none of them immunostained intensely. The quantity of c-FLIP protein was significantly higher in cancer tissues than in normal mucosa (7.04+/-1.20 vs 5.21+/-0.86, P<0.01). Positive staining of mutant p53 protein was found in 60% (27/45) colon cancers. c-FLIP mRNA level was decreased in p53 positive group compared with p53 negative cancer tissues (0.59+/-0.13 vs 0.69+/-0.14, P<0.01), but c-FLIP protein had a significantly higher level in p53 positive cancer tissues than in negative ones (7.57+/-1.30 vs 6.25+/-1.27, P<0.01). CONCLUSION: c-FLIP is specially overexpressed in colon cancers and it might contribute to carcinogenesis of normal colonic mucosa. p53 may exert transcriptional upregulation effects on c-FLIP gene and more potent effects on promoting the degradation of c-FLIP protein.

Evaluation of contrast-enhanced helical hydro-CT in staging gastric cancer.

AIM: To discuss the helical computed tomography (CT) characteristics of gastric cancer and evaluate the diagnostic value of contrast-enhanced helical hydro-CT (HHCT) in staging gastric cancer. METHODS: A total of 50 patients with gastric cancer were included in this study. The CT findings in them were retrospectively analyzed and correlated with pathologic findings at surgery. All patients were preoperatively imaged by plain and contrast-enhanced helical CT after orally ingesting 1,000-1,500 mL water. Peristalsis was minimized by intra-venous administration of spasmolytics. RESULTS: The foci of gastric cancer became more prominent in all the 50 patients and showed strong enhancement in contrast-enhanced HHCT. The tumor was located at the gastric cardia in 14 cases, at the gastric fundus in 3 cases, at the gastric body in 8 cases, at the gastric antrum in 4 cases, at the gastric fundus and the body in 8 cases, at the gastric body and antrum in 11 cases, and at three segments of the stomach in 2 cases. The CT features of gastric cancer were focal or diffuse mural thickening, soft tissue mass, cancerous ulcer, stenosis of stomach, infiltration to adjacent tissues, lymph node and distant metastases. Strong contrast enhancement of the gastric wall was closely related to gastric cancer. The accuracy rate of contrast-enhanced HHCT in staging gastric cancer was 86% (43/50). The detection rate of lymph node metastases by CT was 60% (12/20). CONCLUSION: Contrast-enhanced HHCT is a reliable method to diagnose and stage gastric cancer.

Adenovirus expressing p27kip1 suppresses growth of established esophageal carcinoma xenografts.

AIM: To investigate the growth suppression of adenovirus expressing p27(kip1) on established esophageal tumors in nude mice. METHODS: Esophageal carcinoma xenografts in nude mice were established by tumor tissue mass transplantation. The successfully constructed recombinant adenoviral vectors carrying p27(kip1) gene (Ad-p27(kip1)) were directly injected into the esophageal tumors in nude mice. Compared to control group, the growth curve of tumor was drawn and the growth inhibition rate of tumor was calculated. The histology of tumors was examined by hematoxylin and eosin (H&E) staining. The expression of p27(kip1) and survivin was detected in tumors by immunohistochemical technique. RESULTS: The growth of tumors in gene therapy group with Ad-p27(kip1) was obviously suppressed compared to control group (0.42+/- 0.08 g vs 1.17+/- 0.30 g, t=6.39, P< 0.01), the inhibition rate of tumor growth reached 64.1%. Pathological detection showed that the tumors in nude mice were poorly differentiated esophageal squamous carcinoma. In addition, the expression of p27(kip1) was increased, while the expression of survivin was decreased in tumors after being transfected with Ad-p27(kip1). CONCLUSION: p27(kip1) gene therapy mediated by adenovirus vector has a significant inhibitory effect on esophageal carcinoma in vivo. Up-regulated p27(kip1) expression and down-regulated survivin expression may be its important mechanisms.

Expression and significance of nuclear factor kappaB p65 in colon tissues of rats with TNBS-induced colitis.

AIM: To investigate the role of NF-kappaB in the pathogenesis of TNBS-induced colitis in rats. METHODS: Thirty-two healthy adult Sprague-Dawley (SD) rats were randomly divided into four groups of eight each: normal, NS, model I, model II groups in our study. Rat colitis model was established through 2-,4-,6-trinitrobenzene sulfonic acid (TNBS) enema. At the end of four weeks, the macroscopical and histological changes of the colon were examined and mucosa myeloperoxidase (MPO) activities assayed. NF-kappaB p65 expression was determined by Western blot assessment in cytoplasmic and nuclear extracts of colon tissue, and the expressions of TNF-alpha and ICAM-1 protein in colon tissue were examined by immunohistochemistry. The relativities between expression of NF-kappaB p65 and other parameters were analyzed. RESULTS: TNBS enema resulted in pronounced pathological changes of colonic mucosa in model II group (macroscopic and histological injury indices 6.25+/-1.39 and 6.24+/-1.04, respectively), which were in accordance with the significantly elevated MPO activity (1.69+/-0.11). And the nuclear level of NF-kappaB and expression of TNF-alpha, ICAM-1 in rats of model II group were higher than that of normal control (9.7+/-1.96 vs 1.7+/-0.15, 84.09+/-14.52 vs 16.03+/-6.21, 77.69+/-8.09 vs 13.41+/-4.91 P<0.01), Linear correlation analysis revealed that there were strong correlations between the nuclear level of NF-kappaB and the tissue positive expression of TNF-alpha and ICAM-1, MPO activities, macroscopical and histological indices in TNBS-induced colitis, respectively (r = 0.8235, 0.8780, 0.8572, 0.9152, 0.8247; P<0.05). CONCLUSION: NF-kappaB plays a pivotal role in the pathogenesis of ulcerative colitis, which might account for the up-regulation the expression of TNF-alpha and ICAM-1.

Protective effect of Radix Acanthopanacis Senticosi capsule on colon of rat depression model.

AIM: To investigate the abnormity of rat colon caused by depression and the ameliorative effects of Radix Acanthopanacis Senticosi (RAS) capsule on colon and their mechanisms in rat depression model. METHODS: Chronic stress-induced model of depression of Wistar rats was produced. The experimental animals were randomly divided into model control, 5-aminosalicylic acid (5-ASA) therapy group and three RAS capsule therapy groups. These five groups were intracolonically treated daily (8:00 a.m.) for 2 wk with normal saline, 5-ASA (100 mg/kg) and RAS capsule at the doses of 300, 600 and 900 mg/kg, respectively. A normal control group of rats was also included in the study. Colonic activities of nitric oxide (NO) and superoxide dismutase (SOD), levels of malondialdehyde (MDA) and inducible nitric oxide synthase (iNOS) were determined by ultraviolet spectrophotometry. The expression of cyclooxygenase-2 (COX-2) in colonic tissue was detected by immunohistochemistry. RESULTS: Enhanced colon inflammatory response and oxidative stress were observed in the chronic stress-induced rat depression model, which manifested as the significant increase of MDA, iNOS and NO levels, as well as the expressions of COX-2 in the colon tissue, but the colonic SOD activity was significantly decreased compared with the normal control (MDA: 10.34+/-2.77 vs 2.55+/-0.70; iNOS: 1.11+/-0.44 vs 0.25+/-0.16; COX-2: 53.26+/-8.16 vs 4.87+/-1.65; NO: 11.28+/-5.66 vs 4.76+/-1.55; SOD: 53.39+/-11.15 vs 84.45+/-22.31; P < 0.01). However, these parameters were significantly ameliorated in rats treated locally with RAS capsule at the doses of 300, 600 and 900 mg/kg (iNOS: 0.65+/-0.31, 0.58+/-0.22 and 0.64+/-0.33; NO: 5.99+/-2.73, 6.87+/-1.96 and 6.50+/-1.58; MDA: 2.92+/-0.75, 3.19+/-1.08 and 3.26+/-1.24; SOD: 70.81+/-12.36, 73.30+/-15.30 and 69.09+/-11.03, respectively). The expressions of COX-2 in the colon were significantly ameliorated (28.83+/-9.48 and 27.04+/-9.56, respectively) when RAS capsule was administered at the doses of 600 and 900 mg/kg. CONCLUSION: Administration of RAS capsule intracolonically may have significant therapeutic effects on the colon of rat depression model, which are probably due to its antioxidative action and inhibition of arachidonic acid metabolism.

Effects of garlicin on apoptosis in rat model of colitis.

AIM: To investigate the effects of garlicin on apoptosis and expression of bcl-2 and bax in lymphocytes in rat model of ulcerative colitis (UC). METHODS: Healthy adult Sprague-Dawley rats of both sexes, weighing 180+/-30 g, were employed in the present study. The rat model of UC was induced by 2,4,6-trinitrobenzene sulfonic acid (TNBS) enema. The experimental animals were randomly divided into garlicin treatment group (including high and low concentration), model control group, and normal control group. Rats in garlicin treatment group and model control group received intracolic garlicin daily at doses of 10.0 and 30.0 mg/kg and equal amount of saline respectively 24 h after colitis model was induced by alcohol and TNBS co-enema. Rats in normal control group received neither alcohol nor only TNBS but only saline enema in this study. On the 28th d of the experiment, rats were executed, the expression of bcl-2 and bax protein was determined immunohistochemically and the apoptotic cells were detected by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate fluorescence nick end labeling (TUNEL) method. At the same time, the rat colon mucosal damage index (CMDI) was calculated. RESULTS: In garlicin treatment group, the positive expression of bcl-2 in lymphocytes decreased and the number of apoptotic cells was more than that in model control group, CMDI was lower than that in model control group. The positive expression of bax in lymphocytes had no significant difference. CONCLUSION: Garlicin can protect colonic mucosa against damage in rat model of UC induced by TNBS enema.

Overexpression of cyclooxygenase-2 in gastric cancer correlates with the high abundance of vascular endothelial growth factor-C and lymphatic metastasis.

Cyclooxygenase-2 (COX-2) and vascular endothelial growth factor (VEGF)-C are closely related with the development and metastasis of tumors. The gene expression of COX-2 and VEGF-C in gastric cancer and the correlation between them were investigated; 64 paraffin-embedded gastric cancer samples and 22 flesh gastric cancer samples were tested by using immunohistochemistry and the reverse transcription polymerase chain reaction (RT-PCR) technology, respectively. The mean expressive density of COX-2 and VEGF-C mRNA in gastric cancer, with beta-actin coamplified as an internal standard, were both significantly higher than those in non-cancerous gastric mucosa (1.363 +/- 0.351 vs 0.763 +/- 0.304, 0.972 +/- 0.331 vs 0.314 +/- 0.215, p < 0.001). The positive rates of COX-2 and VEGF-C in 64 gastric cancer samples were 72% and 64% respectively. Their expression in the lymph-node metastasis groups were higher than that of the non-lymph-node metastasis groups (p < 0.05). Moreover, there was a close correlation between COX-2 and VEGF-C expression levels (p < 0.05). The study indicates gastric tumor tissues that produce COX-2 and VEGF-C may have a higher lymphatic invasion and metastatic potential. COX-2 may participate in VEGF-C lymphangiogenic pathway and the high expression of them may play an important role in the lymphatic proliferation and spread in gastric cancer.

[Effect of gastrin on invasiveness of human colon cancer cells].

OBJECTIVE: To investigate the effect of gastrin on invasiveness of human colon cancer cells and the role of gastrin receptor-focal adhesion kinase (FAK) signal transduction pathway in this proess. METHODS: pCR3.1/GR vector expressing gastrin receptor was transfected into a colorectal cancer cell line Colo320 with lipofectamine 2000, and screened by G418. The expression levels of gastrin receptor of the parental cell line Colo320 and the transfected cell line Colo320/GR were assayed by RT-PCR. On the other hand, antisense oligonucleotide of FAK was used to block its expression. The mock transfected Colo320 and sense oligonucleotide Colo320 cells were used as controls. Colo320 and Colo320/GR cells were treated with increasing doses (0 approximately 100 nmol/L) of gastrin. Invasiveness of Colo320 and Colo320/GR cells was determined by Boyden chamber. Phosphorylation of focal adhesion kinase (FAK) tyr-397 was examined by immunoprecipitation and Western-blot. RESULTS: RT-PCR results showed that the Colo320/GR cells had an mRNA level four times as high as that of Colo320 cells. Western blot showed that FAK tyr397 phosphorylation of Colo320 cells was apparently decreased. Colo320 and Colo320/GR cells showed a dose-dependent response to gastrin on invasiveness and phosphorylation of FAK tyr-397. Invasiveness of Colo320 cells reached its climax when concentration of gastrin was 100 nmol/L, and FAK tyr-397 phosphorylation was marked when concentration of gastrin was 10 nmol/L, but the latter decreased when gastrin concentration was increased to 100 nmol/L. Colo320/GR cells had the same tendency as Colo320 cells, but showed an even stronger invasiveness and a higher level of FAK tyr-397 phosphorylation than Colo320 cells. Before gastrin stimulation, the invasiveness of Colo320 cells transfected with antisense oligonucleotides and the controls showed no difference. After gastrin stimulation, the increase in invasiveness was much less than that in the controls. CONCLUSION: Gastrin can evidently promote invasiveness of Colo320 cells via gastrin-gastrin receptor-FAK signal transduction pathway.

[A study of gastrin-focal adhesion kinase signal pathway in human colon cancer cells].

OBJECTIVE: The study investigated the effect of gastrin on tyrosine phosphorylation and protein expression of focal adhesion kinase (FAK) in human colon cancer cells. METHODS: The eukaryotic plasmid that expresses cholecystokinin 2 receptor (CCK2R) stably, named pCR3.1/CCK2R, was transfected into Colo320 to construct an up-regulated gastrin-CCK2R signal pathway; the gastrin antagonist was used to down-regulate the signal pathway. Thus, including the normal control, there were three signal pathways with different CCK2R levels. Different doses of gastrin were used to stimulate FAK in the different time. FAK tyrosine phosphorylation and FAK expression in different groups were detected by immunoprecipitation and Western-blotting assays, and analyzed with Labimage software. RESULTS: RT-PCR result showed that Colo320 transfected with CCK2R had a mRNA level four times higher than that normal Colo320 did, and which suggested that up-regulated signal transduction pathway was constructed. After stimulated by gastrin with 0, 0.1, 1, 10 and 100 nmol/L, tyrosine phosphorylation levels of Colo320 were 24.0%, 39.7%, 46.2%, 50.4% and 44.5%, and those of Colo320 transfected with CCK2R were 24.6%, 70.7%, 90.1%, 100% and 88.6%. When incubated with gastrin at 0, 2.5, 5, 10 and 20 min, the tyrosine phosphorylation levels of Colo320 were 23.9%, 63.6%, 58.6%, 45.5% and 40.9%, and those of Colo320 transfected with CCK2R were 24.5%, 84.6%, 100%, 98.6% and 97.9%. The increases of tyrosine phosphorylation in both Colo320 and Colo320 transfected with CCK2R were dose dependence of gastrin. In Colo320, the time of phosphorylation had a tendency of exhaustion at 2.5 min; but in Colo320 transfected with CCK2R, it was at 10 min. Gastrin had no effects on FAK protein expression in different cell groups. An up-regulated level of CCK2R could enhance the effect of gastrin on FAK tyrosine phosphorylation. The gastrin antagonist showed an effect of competitive inhibition on tyrosine phosphorylation of FAK. CONCLUSIONS: FAK is a signal transducer in downstream of CCK(2)R; FAK exerts its functions by tyrosine phosphorylation, but dose not increase FAK protein. Gastrin-CCK2R-FAK signal pathway is a pivotal one in the cell growth and proliferation caused by gastrin.