RING Finger Proteins Are Involved in the Progression of Barrett Esophagus to Esophageal Adenocarcinoma: A Preliminary Study

BACKGROUND/AIMS: To investigate the differential expression of RING finger (RNF) proteins in Barrett esophagus (BE) and esophageal adenocarcinoma (EAC). METHODS: The differential expression of RNFs in normal esophagus (NE), BE, and EAC was screened using microarray assay. Real-time quantitative polymerase chain reaction (PCR), tissue microarray assay, and Western blot analysis were independently performed to detect the mRNA and protein expression of screened RNFs. RESULTS: The expression of nine RNFs in the BE or EAC was 2-fold higher than those in NE. Among these proteins, the RNF32 and RNF121 expression in BE was 20.3-fold and 16.4-fold higher, respectively, than that in NE, and the expression of RNF24, RNF130, RNF141, RNF139, RNF11, RNF14, and RNF159 was upregulated more than 2-fold compared with NE. The expression of nine RNFs was not only upregulated in the EAC but was also positively related to the RNF expression in BE. The PCR results also indicated increased expression of these RNFs in BE and EAC compared to NE. Furthermore, the mRNA expression of all RNFs, except for RNF141 in EAC, was dramatically higher than those in the BE. Similar results were also obtained from the Western blot analysis. CONCLUSIONS: A total of nine RNFs play critical roles in the progression of BE to EAC.

Mechanisms of human telomerase reverse transcriptase RNAi which increases hepatocellular carcinoma cell apoptosis induced by TRAIL / 中华肝脏病杂志

<p><b>OBJECTIVES</b>To investigate the mechanisms for human telomerase reverse transcriptase (hTERT) RNA interference (RNAi) in increasing hepatocellular carcinoma cell apoptosis induced by TNF-related apoptosis-inducing ligand (TRAIL).</p><p><b>METHODS</b>Cell apoptosis was identified by flow cytometry analysis after annexin V/PI double staining. Expression of apoptosis-related proteins, procaspase-8, -9, -3, Bax, Bcl-2 and hTERT, were identified by Western blotting analysis; telomerase activity and telomere length were detected by telomeric repeat amplification protocol (TRAP) and telomere amount and length assay (TALA) methods.</p><p><b>RESULTS</b>Hepatocellular carcinoma cell apoptosis induced by TRAIL were all significantly increased by hTERT RNAi (P less than 0.05). For example, apoptosis rates were enhanced from 5.53% (untransformed) to 10.35% (transformed) in HepG 2 cells and from 14.73% to 77.24% in SMMC 7721 cells after being treated by 100 ng/ml TRAIL for 24 h. Moreover, activation of procaspase-8, -9 and -3 in transformed cells after being treated by TRAIL were all significantly raised (P less than 0.05) in a dose-dependent manner. The expression of procaspase-8, -9 and Bcl-2 were effectively augmented (P less than 0.05), but expressions of Bax and hTERT were strikingly decreased (P less than 0.05). Meanwhile, telomerase activity was apparently suppressed and telomere length was markedly shortened (P less than 0.05). There were no remarkable differences in these effects between control cells and the untransformed cells (P more than 0.05).</p><p><b>CONCLUSION</b>Enhanced cell apoptosis induced by TRAIL through hTERT RNAi may be related to up-regulation of procaspase-8 and -9 expressions. However the down-regulation of hTERT expression, reduced telomerase activity and shortened telomere length may not be related to expressions of Bcl-2 and Bax.</p>

Pharmacological action and mechanisms of ginkgolide B / 中华医学杂志(英文版)

<p><b>OBJECTIVE</b>To review the recent research progress in pharmacological actions and mechanisms of ginkgolide B. Data sources Information included in this article was identified by searching of PUBMED (1987 - 2006) online resources using the key terms "ginkgolide B", "platelet activating factor", and "pharmacological". Study selection Mainly original milestone articles and critical reviews written by major pioneer investigators of the field were selected.</p><p><b>RESULTS</b>The key issues related to the pharmacological actions and mechanisms of ginkgolide B were summarized. The ginkgolide B possesses a number of beneficial effects such as anti-inflammatory, anti-allergic, antioxidant, and neuroprotective effects. Meantime, their mechaniams were discussed.</p><p><b>CONCLUSIONS</b>The Ginkgolide B is the most potent antagonist of platelet activating factor (PAF) and exhibits therapeutic action in a variety of diseases mainly by the PAF receptor.</p>

Effects of hTERT RNAi on apoptosis of hepatocellular carcinoma cells induced by TRAIL / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To study the effects of human telomerase reverse transcriptase (hTERT) RNA interference (RNAi) on biological characteristics of hepatocellular carcinoma cell lines HepG2 and SMMC-7721 and on apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL).</p><p><b>METHODS</b>Small hairpin hTERT (shTERT) sequence was identified by PCR method; hTERT expressions, morphological features, cell proliferation and replicative senescence were respectively determined using RT-PCR, hematoxylin-eosin (HE) staining, growth curve and beta-galactosidase (b-Gal) staining; cell cycle and apoptosis were identified using flow cytometry after propidium iodide (PI) staining and annexin V/PI double staining.</p><p><b>RESULTS</b>shRNA were found in 6/8 HepG2 and 6/6 SMMC-7721 cell clones transformed by the recombined plasmid pSilencer 3.1-H1 neo-shTERT. The interference rates of hTERT on HepG2 and SMMC-7721 were 100% and 43.3% respectively. Cells in G2-M phases increased from 7.1% to 10.6% and from 6.9% to 7.9% respectively; and the percentage of replicative senenscence cells increased from 0 to 20.4% and from 3.6% to 10.0% respectively. The nucleus/cytoplasm ratios of the cells were obviously decreased after hTERT RNAi treatment. Moreover, apoptosis of hepatocellular carcinoma cells and apoptosis induced by TRAIL were strikingly increased by hTERT RNAi (P < 0.05). For example, apoptosis rates were increased from 3.5% to 5.2% in HepG2 cells and from 4.8% to 7.9% in SMMC-7721 cells after hTERT RNAi treatment. Apoptosis rates were increased from 5.3% to 10.4% in HepG 2 cells and from 13.9% to 77.2% in SMMC-7721 cells after being treated by 100 ng/ml TRAIL for 24 h. However, there were no remarkable changes between control cells and untransformed cells.</p><p><b>CONCLUSION</b>hTERT RNAi not only has a significant effect on biological characteristics of hepatocellular carcinoma cells, but also obviously can increase cell apoptosis induced by TRAIL.</p>

A preliminary study on anti-liver cancer immunity of the virus-like particulate peptide-nucleic acid vaccine / 中华肝脏病杂志

<p><b>OBJECTIVES</b>To construct a novel virus-like particulate peptide-nucleic acid vaccine (VPNV) of human telomerase reverse transcriptase (hTERT), and to study its anti-liver cancer immunity.</p><p><b>METHODS</b>A cationic antigenic peptide was synthesized and purified, and then human granulocyte macrophage colony stimulating factor (hGM-CSF) and TERT gene were cloned into the eukaryotic expression vector pTCAE. The peptide was combined with the nucleic acid vaccine to make a VPNV, which was transfected into eukaryotic cell COS-7. The immunogenicity of hGM-CSF and hTERT were detected using ELISA and Western blot. The efficacy of VPNV for inducing antigen specific CTL response was determined using the lactate dehydrogenase release method.</p><p><b>RESULTS</b>VPNV was verified capable to trigger specific CTL responses and has shown a specific cytolytic activity to liver cancer cell HepG2.</p><p><b>CONCLUSION</b>A VPNV which can stimulate antigen specific CTL response was successfully constructed. This paves the way for our further investigation of anti-liver cancer immunity in mice.</p>

The relationship between methylenetetrahydrofolate reductase gene polymorphism and microsatellite instability in gastric cancer / 中华流行病学杂志

<p><b>OBJECTIVE</b>To explore the relationship between methylenetetrahydrofolate reductase (MTHFR) gene polymorphism and microsatellite instability (MSI) in patients with gastric cancer.</p><p><b>METHODS</b>MTHFR gene C677T and A1298C polymorphism were detected by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and MSI was examined with PCR.</p><p><b>RESULTS</b>MTHFR gene C677T and A1298C polymorphisms were analyzed on 122 gastric cancers and 110 normal controls The genotype frequencies of MTHFR 677CC, 677CT and 677TT were 47.5%, 39.3% and 13.1% on patients with gastric cancer, and 48.5%, 42.6%, 8.9% in the controls respectively. There was no significant difference of genotype frequency between the two groups (P > 0.05). The individuals with 677CT genotype, 677TT genotype and 677CT + TT genotype exhibited significantly reduced risk (OR = 0.38,95% CI: 0.15-0.98; OR = 0.26,95% CI: 0.03-2.18 and OR = 0.36,95% CI: 0.07-0.98) of developing gastric cardia cancer compared with those harboring the wild-type(677CC). The individuals with 677TT genotype having a 3.03-fold (95% CI: 1.07-8.65) increased risk of developing gastric corpus cancer. The genotype frequency of MTHFR 1298AA, 1298AC and 1298CC were 59.8%, 36.1% and 4.1% in gastric cancer patients, and 57.4%, 7.6%, 5.0% in the controls, respectively. The distribution of MTHFR A1298C genotype was not significantly different between gastric cancer and controls (P > 0.05). The individuals with 1298CC genotype had a reduced risk of developing gastric antrum cancer (OR = 0.41- fold, 95% CI: 0.03-2.18, 0.05-3.72) when comparing with those having 1298AA genotype. Patients with MSI+ gastric cancer had an increased frequency of the MTHFR 677TT genotype when comparing with those suffering from MSI- gastric cancer (P = 0.009) and with controlled subjects (P = 0.008). There was no significant association found between MTHFR A1298C polymorphism and MSI (P>0.05).</p><p><b>CONCLUSION</b>Polymorphism of MTHFR C677T was associated with increased risk on gastric corpus cancer and reduced risk on gastric cardia cancer. The polymorphism of MTHFR A1298C was associated with reduced risk for gastric antrum cancer while MSI pathway was possibly involved in the development of gastric cancer with MTHFR 677TT genotype.</p>

Antisense DNMT1 gene fragment in the sensitivity change of SMMC-7721 cells to tumor necrosis factor related apoptosis inducing ligand and its mechanism / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To observe the sensitivity change of SMMC-7721 cells transfected with antisense DNMT1 gene fragment to tumor necrosis factor related apoptosis inducing ligand (TRAIL) and its mechanism.</p><p><b>METHODS</b>Cell survival rate was measured by trypan blue, apoptosis rate by TUNEL method and the expression of bcl-2, bax and bad by flow cytometry.</p><p><b>RESULTS</b>Cell survival rate of SMMC-7721 cells transfected with antisense DNMT1 gene fragment was markedly lower than that transfected with sense DNMT1 gene fragment or empty vector (P < 0.05 and 0.01), but the apoptosis rate was on the contrary (P < 0.05 or 0.01). The expression of bax and bad (especially the former), but not bcl-2 of SMMC-7721 cells transfected with antisense DNMT1 gene fragment was markedly higher than those of SMMC-7721 cells transfected with sense DNMT1 gene fragment or empty vector.</p><p><b>CONCLUSION</b>The sensitivity of SMMC-7721 cells to TRAIL can be enhanced by the transfection of antisense DNMT1 gene fragment, which may be related to the increase of bax and bad expression.</p>

Cooperative anti-tumor effect of aspirin and TNF-related apoptosis-inducing ligand / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To observe the anti-tumor effect of combination TNF-related apoptosis-inducing ligand (TRAIL) with aspirin on liver cancer cell line, SMMC-7721.</p><p><b>METHODS</b>The survival fraction of SMMC-7721 cells was measured by MTT assay, apoptosis rate and cell cycle was determined by flow cytometry, and the expression of apoptosis-related gene was identified by western blot.</p><p><b>RESULTS</b>The survival fraction of SMMC-7721 cells treated with 300 ng/ml TRAIL, 3 mmol/L or 10 mmol/L aspirin alone was 82.76%, 81.34% and 71.29% respectively, and the survival fractions of SMMC-7721 cells treated with TRAIL and 3 mmol/L or 10 mmol/L aspirin were 43.54% and 37.8% respectively. The apoptosis rates of SMMC-7721 cells induced by TRAIL and 3 mmol/L or 10 mmol/L aspirin were higher than that induced by TRAIL or aspirin alone (34.76% and 38.56% vs 21.25%, 1.89% and 6.08%), and G0/G1 arrest was observed under TRAIL and aspirin. The expression of Bcl-2 in SMMC-7721 cells treated by 3 mmol/L or 10 mmol/L aspirin decreased markedly, but no effect on Bax.</p><p><b>CONCLUSION</b>The cooperative anti-tumor effect of aspirin and TRAIL may be related to the inhibition of the expression of Bcl-2 by aspirin</p>

Relationship between mitochondrial DNA instability and interleukin-8 activity in gastric mucosa / 中华病理学杂志

<p><b>OBJECTIVE</b>To evaluate the relationship between mitochondrial DNA instability (mtMSI) and interleukin-8 (IL-8) activity in gastric mucosa of various lesions.</p><p><b>METHODS</b>IL-8 level in gastric mucosa was assayed using ELISA method. The mtMSI was detected by PCR-SSCP techniques.</p><p><b>RESULTS</b>mtMSI was observed in 11 out of 30 (36.7%) gastric cancers, 2 of 15 (13.3%) intestinal metaplasia, 2 of 10 dysplasia and 1 of 10 chronic atrophic gastritis. IL-8 level in mtMSI+ group [(76.8 +/- 3.8) pg/mg] was significantly higher than that in mtMSI- group [(48.3 +/- 3.6) pg/mg, P < 0.05].</p><p><b>CONCLUSION</b>mtMSI closely correlates with IL-8 level in gastric mucosa and is involved in gastric carcinogenesis.</p>

Rapid induction of mRNAs for liver regeneration genes by hepatopoietin and partial hepatectomy / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the effect of recombinant human hepatopoietin (rhHPO) and partial hepatectomy on rapidly induced expression of immediate early gene.</p><p><b>METHODS</b>We investigated the different gene expression within 1 hour after 2/3 partial hepatectomy by representational difference analysis and in primary cultured hepatocytes system.</p><p><b>RESULTS</b>In the expressed sequence tag (EST) library, we identified that most of these genes were immediate early gene, and found one new gene PC3 that might be associated to liver regeneration in the EST library. Moreover, PC3 gene was rapidly induced after 2/3 partial hepatectomy and the expressing peak was within 1~2 hours after operation. HPO can rapidly induce the expression of these genes (c-fos, LRF-1, and PC3, etc.) in primarily cultured rat hepatocyte, which might be one of HPO molecular mechanism on stimulating hepatocyte proliferation.</p><p><b>CONCLUSIONS</b>rhHPO and partial hepatectomy can rapidly induce the expression of immediate early gene. PC3 gene is immediate early gene related to liver regeneration.</p>

Effects of esophageal mucosai acid exposure on expression of c-fos protein in central nervous system in ovaibumin sensitized rats / 中华消化杂志

Objective To investigate the distribution of neurons expressing Fos protein in central nervous system (CNS) following esophageal mucosal acid exposure,and to map the contribution of spe- cific brain areas in sensitizing responsivity and emphasize the coding change of CNS to the esophageal acid stimulation.Methods Thirty-six healthy Sprague-Dwley rats were randomly divided into five groups. Group A (n=6) was normal group of home cage control animals to which no stimulation was given. Group B (n=7) was saline group which received esophageal perfusion with normal saline solution (0.9% NaCl).Group C (n=8) was treated with esophageal mucosal acid exposure containing 0.1 mol/L HCl. Group D (n=7) was sensitized by ovalbumin.Group E (n=8) received basal ovalbumin-sensitization plus esophageal mucosal acid exposure.The rat model of esophageal visceral hypersensitivity was estab- lished by the basic ovalbumin-sensitization combined with intra-esophageal mucosal acid exposure.The neuronal expressions of c-fos proto-oncogene were detected with immunohistochemical counter-staining and computerized color image analyzer under various conditions.Results The rats in model group with basic ovalbumin-sensitization plus esophageal acid perfusion initiated a high density expression of c-fos- immunoreactive(Fos-IR) neurons in multineuronal networks.A significantly higher number of Fos positive neurons was found in the model group than those in the corresponding regions of other groups (P＜0.05) in the following brain areas:frontal and parietal cortex,insular cortex,cingulated cortex,central amyg- daloid nucleus,the K(?)lliker-Fuse nucleus,the nucleus ambiguus,parabrachial nucleus,hypothalamic paraventricular nucleus,paraventricular thalamic nucleus,paratrigeminal nucleus,the nucleus of solitary tract,area postrema,reticular nucleus of medulla,whereas no significant difference was found in the dorsal motor nucleus of the vagus,supraoptic nucleus,periaqueductal gray matter or orbital part of infe- rior frontal gyrus.The values of Fos-IR neurons were also increased in the central amygdala,parabrachi- al nucleus,paraventricular nucleus,the paratrigeminal nucleus and NTS in the model group than that in the corresponding regions of other groups (P＜0.05).Conclusion The basic ovalbumin-sensitization fa- cilitated dramatically the c-fos expression evoked by esophageal acid perfusion,suggesting that visceral hypersensitivity induced by ovalbumin may alter cortical reactivity processing of esophageal acid stimula- tion in brain areas.

Cerebral evoked potentials response to esophageal mucosal acid exposure and distention in patients with non-erosive gastro-oesophageal reflux disease / 中华消化杂志

Objective To investigate characteristics and alternation of cerebral evoked potentials (CEP) response to esophageal mucosal acid exposure and distention in patients with non-erosive gastro-oesoph- ageal reflux disease (NERD) and in healthy subjects,and to study the mechanism of visceral hypersensitivity in NERD.Methods Twenty-one NERD patients and 10 volunteers were recruited.Mechanical distention stimulation and acid perfusion of the esophagus were performed using the balloon-affixed and polyvinyl multi- lumen catheter.First,maximally tolerated pain thresholds of all subjects were recorded,then esophageal mechanical stimulation with a 75% of maximal tolerated intensity and a frequency of 0.2 Hz was performed altogether 64 times by means of a computer-controlled barostat.The alternation of esophageal CEP was recorded before and after acid perfusion with a multichannel international 10-20 system of electroencephalography. Experimental data was analyzed by student's t-test and one way analysis of variance.Results Esophageal mu- cosal distention may evoke recognizable and reproducible and multi-peak CEP.The latencies for N1,P1 and N2 in volunteers were (246?77),(388?84)and (502?78) ms,CEP morphology of NERD patients was charac- terized by randomly distributed patterns,and the latencies for N1 ,P1 and N2 were (192?46),(293?76) and (440?79)ms,significantly shorter for mechanical stimulation compared with those of control group respectively (all P value

Mucin gene expression in Barrett's oesophagus / 中华消化内镜杂志

Objective To assess mucin gene expression in Barrett's esophagus.Methods Mucin core protein-MUC1,MUC2,MUC3,MUCSAC and MUC6 were detected by immunohistochemistry.The re- lationship between mucin expression and magnification-endoscopic characteristics,pathohistologic epithelial types of Barrett's esophagus was analyzed.Results Mild expression of MUC1 was predominantly found in the superficial epithelium of both gastric and specialised intestinal metaplasia.In a small number of specimens, mild expression of MUC1 was also noted in glands.Strong MUC2 expression was noted only in the goblet cells in Barrett's oesophagus.MUC3 was expressed in the superficial columnar cells of specialized intestinal metaplasia with or without globlet cells but not in gastric metaplasia of the oesophagus.In some specimens MUC3 was expressed in the vacuolus of the globlet cells and the lumen of gland.Strong staining of MUCSAC was noted in the columnar epithelium of both gastric metaplasia and specialized intestinal metaplasia in Barrett's oesophagus,as well as expressed in the cytoplasm and vacuolus of the globlet cells in some speci- mens.Expression of MUC6 protein was detected at the basement of the crypts in gastric metaplasia and spe- cialised Barrett's glands.Expression of MUC2 and MUC3 protein was found much higher in villous or irregu- lar pit pattern than that in dot or rod pit pattern(P