Epigenetic modification regulates both expression of tumor-associated genes and cell cycle progressing in human colon cancer cell lines: Colo-320 and SW1116

The aim of this study is to assess the effects of DNA methylation and histone acetylation, alone or in combination, on the expression of several tumor-associated genes and cell cycle progression in two established human colon cancer cell lines: Colo-320 and SW1116. Treatments with 5-aza-2'-deoxycytidine (5-aza-dC) and trichostatin A, alone or in combination, were applied respectively. The methylation status of the CDKN2A promoter was determined by methylation-specific PCR, and the acetylated status of the histones associated with the p21wAF1 and CDKN2A genes was examined by chromatin immunoprecipitation. The expression of the CDKN2A, p21WAF1, p53, p73, APC, c-myc, c-Ki-ras and survivin genes was detected by real-time RT-PCR and RT-PCR. The cell cycle profile was established by flow cytometry.We found that along with the demethylation of the CDKN2A gene promoter in both cell lines induced by 5-aza-dC alone or in combination with TSA, the expression of both CDKN2A and APC genes increased. The treatment of TSA or sodium butyrate up-regulated the transcription of p21 WAF1 significantly by inducing the acetylation of histones H4 and H3, but failed to alter the acetylation level of CDKN2A-associated histones. No changes in transcription of p53, p73,c-myc, c-Ki-ras and survivin genes were observed. In addition, TSA or sodium butyrate was shown to arrest cells at the G1 phase. However, 5-aza-dC was not able to affect the cell cycle progression. In conclusion, regulation by epigenetic modification of the transcription of tumor-associated genes and the cell cycle progression in both human colon cancer cell lines Colo-320 and SW1116 is gene-specific.

Inhibitory effect of oxymatrine on serum hepatitis B virus DNA in HBV transgenic mice

To study the inhibitory effect of oxymatrine on serumhepatitis B virus (HBV) DNA in HBV transgenic mice.

Expression of Dnmt1,demethylase,MeCP2 and methylation of tumor-related genes in human gastric cancer

AIM: To explore the effect of DNA methyltransferase,demethylase and methyl-CpG binding protein MeCP2 on the expressions and methylation of hMSH2 and protooncogene in human gastric cancer.METHODS: Paired samples of primary gastric cancer and corresponking para-cancerous, non-cancerous gastric mucosae were obtained from surgically resected specimens of 28 patients. TranscriplJon levels of Dnmtl, mbd2, MeCP2, p16^INK4A,hMSH2 and c-myc were detected by using real-time PCR or RT-PCR. Promoter methylation of p16^INK4A, c-myc and hMSH2 genes was assayed by methylation-specific PCR (MSP) and sequencing (mapping). Their relationships were analyzed by Fisher's exact test using the software SPSS.RESULTS: The average mRNA level of Dnmtl gene from cancerous tissue was higher and that of mbd2 gene from cancerous tissue was lower than that from non-cancerous tissue, respectively, mbd2 was lower in cancerous tissue than in non-cancerous tissue in 14 (50.0%) of patients but higher in 3 cases (10.7%) of non-cancerous gastric tissue(P<0.001). c-myc expression was up-regulated in cancer tissues (P<0.05). The up-regulation of mbd2 was found in all patients with hypomethyiated c-myc. The transcriptional levels of p16^IK4A and MeCP2 genes did not display any differenoe between gastric cancerous and matched non-cancerous tissues. There were down-regulation and hypermethylation of hMSH2 in cancer tissues, and the hypermethylation of hMSH2 coexisted with down-regulated transcription.However, the transcription level of the above genes was not associated with biological behaviours of gastric cancers.CONCLUSION: The up-regulation of proto-oncogene may be the consequence of epigenetic control of gene expression by demethylase, and mbd2 is involved in the regulation of hMSH2 expression in human gastric cancer.

Effects of bile reflux on gastric mucosal lesions in patients with dyspepsia or chronic gastritis

AIM: To investigate the influences of bile reflux on profiles of gastric mucosal lesions in patients with dyspepsia or chronic gastritis.METHODS: A total of 49 patients diagnosed with dyspepsia and chronic gastritis underwent 24-h ambulatory andsimultaneous monitoring of intragastric bilirubin absorbance and pH values, and then they were divided into bile refluxpositive group and bile reflux negative group. Severity of pathological changes in gastric mucosa including activeinflammation, chronic inflammation, intestinal metaplasia, atrophy and dysplasia as well as Helicobacter pylori (H pylori) infection at the corpus, incisura and antrum were determined respectively according to update Sydney system criteria. The profiles of gastric mucosal lesions in the two groups were compared, and correlations between time-percentage of gastric bilirubin absorbance ＞0.14 and severity of gastric mucosal lesions as well as time-percentage of gastric pH ＞4 were analyzed respectively. RESULTS: Thirty-eight patients (21 men and 17 women, mean age 44.2 years, range 25-61 years) were found existing with bile reflux (gastric bilirubin absorbance ＞0.14) and 11 patients (7 men and 4 women, mean age 46.2 years,range 29-54 years) were bile reflux negative. In dyspepsia patients with bile reflux, the mucosal lesions such as active inflammation, chronic inflammation, intestinal metaplasia, atrophy or H pylori infection in the whole stomach, especially in the corpus and incisura, were significantly more severe than those in dyspepsia patients without bile reflux. Moreover, the bile reflux time was well correlated with the severity of pathological changes of gastric mucosa as well as H pylori colonization in the near-end stomach, especially in the corpus region. No relevance was found between the time of bile reflux and pH ＞4 in gastric cavity. CONCLUSION: Bile reflux contributes a lot to mucosal lesions in the whole stomach, may facilitate H pylori colonization in the corpus region, and has no influence on acid-exposing status of gastric mucosa in patients with dyspepsia or chronic gastritis.

Effects of histone acetylation and DNA methylation on p21^(WAF1)regulation

Cell cycle progression is regulated by interactions betweencyclins and cyclin-dependent kinases (CDKs). p21wAF1 is oneof the CIP/KIP family which inhibits CDKs activity. Increasedexpression of p21WAF1 may play an important role in thegrowth arrest induced in transformed calls. Although thestability of the p21wAF1 mRNA could be altered by differentsignals, cell differentiation and numerous influencingfactors. However, recent studies suggest that two knownmechanisms of epigenesis, i. e. gene inactivation bymethylation in promoter region and changes to an inactivechromatin by histone deacetylation, seem to be the bestcandidate mechanisms for inactivation of p21WAF1. To date,almost no coding region p21wAF1 mutations have been foundin tumor cells, despite extensive screening of hundreds ofvarious tumors. Hypermethylation of the p21WAF1 promoterregion may represent an alternative mechanism by which thep21WAF1/ClPl gene can be inactivated. The reduction of cellularDNMT protein levels also induces a corresponding rapidincrease in the cell cycle regulator p21wAF1 proteindemonstrating a regulatory link between DNMT and p21WAF1which is independent of methylation of DNA. Both histonehyperacetylation and hypoacetylation appear to be importantin the carcinoma process, and induction of the p21WAF1 geneby histone hyperacetylation may be a mechanism by whichdietary fiber prevents carcinogenesis. Here, we review theinfluence of histone acetylation and DNA methylation onp21WAF1 transcription, and affection of pathways or factorsassociated such as p53, E2A, Sp1 as well as several histonedeacetylation inhibitors.

Capsule endoscopy in diagnosis of small bowel Crohn's disease

AIM: To evaluate the effectiveness of wireless capsule endoscopy in patients with suspected Crohn's disease (CD) of the small bowel undetected by conventional modalities,and to determine the diagnostic yield of M2A Given Capsule.METHODS: From May 2002 to April 2003, we prospectively examined 20 patients with suspected CD by capsule endoscopy. The patients had the following features:abdominal pain, weight loss, positive fecal occult blood test, iron deficiency anaemia, diarrhoea and fever. All the patients had normal results in small bowel series (SBS) and in upper and lower gastrointestinal endoscopy beforethey were examined. Mean duration of symptoms before diagnosis was 6.5 years.RESULTS: Of the 20 patients, 13 (65%) were diagnosed as CD of the small bowel according to the findings of M2A Given Capsule. The findings detected by the capsule were mucosal erosions (2 patients), aphthas (5 patients),nodularity (1 patient), large ulcers (2 patients), and ulcerated stenosis (3 patients). The distribution of the lesions was mainly in the distal part of the small bowel,and the mild degree of lesions was 54%.CONCLUSION: Wireless capsule endoscopy is effective in diagnosing patients with suspected CD undetected by conventional diagnostic methods. It can be used to detect early lesions in the small bowel of patients with CD.

Histone acetylation regulates p21^(WAF1) expression in human colon cancer cell lines

AIM: To investigate the effect of histone acetylation on regulation of p21^WAF1 gene expression in human colon cancer cell lines.METHODS: Two cell lines, Colo-320 and SW1116 were treated with either trichostatin or sodium butyrate.Expressions of p21^WAF1 mRNA and protein were detected by real-time RT-PCR and Western blotting, respectively.Acetylation of two regions of p21^WAF1 gene-associated histones and total cellular histones were examined by chromatin immunoprecipitation assay and Western blotting.RESULTS: Trichostatin or sodium butyrate re-activated p21^WAF1 transcription resulted in up-regulated p21^WAF1 protein level in colon cancer cell lines. Those effects were accompanied by an accumulation of acetylated histones in total cellular chromatin and p21^WAF1 gene-associated region of chromatin.CONCLUSION: Histone acetylation regulates p21^WAF1 expression in human colon cancer cell lines, Colo-320 and SW1116.

Capsule oxymatrine in treatment of hepatic fibrosis due to chronic viral hepatitis:A randomized,double blind,placebo-controlled,multicenter clinical study

AIM: To evaluate the efficacy and safety of oxymatrine capsule in treatment of hepatic fibrosis in patients with chronic viral hepatitis. METHODS: It was a randomized, double blind, placebocontrolled, multicenter clinical study. One hundred and fortyfour patients were divided into oxymatrine capsule group (group A) and placebo group (group B). The course was 52wk. Patients were visited once every 12wk and the last visit was at 12wk after cessation of the treatment. All patients had liver biopsy before treatment, part of them had a second biopsy at the end of therapy. Clinical symptoms, liver function test, serum markers of hepatic fibrosis were tested. Ultrasound evaluation was performed before, during and at the end of therapy. RESULTS: One hundred and forty-four patients enrolled in the study. Of them 132 patients completed the study according to the protocol, 49 patients had liver biopsy twice (25 patients in group A and 24 in group 13). At the end of therapy, significant improvements in hepatic fibrosis and inflammatory activity based on Semi-quantitative scoring system (SSS) were achieved in group A. The total effective rate of the treatment was 48.00%, much higher than that of 4.17% in group B (P<0.05). Significant improvement in serum markers of hepatic fibrosis such as hyaluronic acid (HA) and type Ⅲ procollagenic peptide (PⅢ P) in group A was seen (P<0.05). The total effective rate of serum markers at the end of therapy in group A was 68.19%, much higher than that of 34.85% in group B (P<0.05). The total effective rate of noninvasive markers at the end of therapy in group A was 66.67%, much higher than that of 30.30% in group B (P<0.05). The rate of adverse events was similar in two groups. CONCLUSION: Oxymatrine capsule is effective and safe in treatment of hepatic fibrosis due to chronic viral hepatitis.

Oxymatrine therapy for chronic hepatitis B:A randomized doubleblind and placebo-controlled multi-center trial

AIM: To evaluate the efficacy and safety of capsule oxymatrine in the treatment of chronic hepatitis B.METHODS: A randomised double-blind and placebocontrolled multicenter trial was conducted. Injection of oxymatrine was used as positive-control drug. A total of 216 patients with chronic hepatitis B entered the study for 24 weeks, of them 108 received capsule oxymatrine, 36 received injection of oxymatrine, and 72 received placebo.After and before the treatment, clinical symptoms, liver function, serum hepatitis B virus markers, and adverse drug reaction were observed.RESULTS: Among the 216 patients, six were dropped off,and 11 inconsistent with the standard were excluded.Therefore, the efficacy and safety of oxymatrine in patients were analysed. In the capsule treated patients, 76.47 % became normal in ALT level, 38.61% and 31.91% became negative both in HBV DNA and in HBeAg. In the injection treated patients, 83.33 % became normal in ALT level,43.33 % and 39.29 % became negative both in HBV DNA and in HBeAg. In the placebo treated patients, 40.00 % became normal in ALT level, 7.46 % and 6.45 % became negative both in HBV DNA and in HBeAg. The rates of complete response and partial response were 24.51% and 57.84 % in the capsule treated patients, and 33.33 % and 50.00 % in the injection treated patients, and 2.99 % and 41.79 % in the placebo treated patients, respectively.There was no significance between the two groups of patients, but both were significantly higher than the placebo. The adverse drug reaction rates of the capsule,injection and placebo were 7.77 %, 6.67 % and 8.82 %,respectively. There was no statistically significant difference among them.CONCLUSION: Oxymatrine is an effective and safe agent for the treatment of chronic hepatitis B.

Relationship between clinical and pathologic findings in patients with chronic liver diseases

AIM: To explore the relationship between clinical findings of patients with chronic liver diseases and the pathologic grading and staging of liver tissues.METHODS: The inflammatory activity and fibrosis of consecutive liver biopsies from 200 patients were determined according to the diagnosis criteria of chronic hepatitis in China established in 1995. A comparative analysis was carried out for 200 patients with chronic liver diseases by comparing their clinical manifestations, serum biochemical markers with the grading and staging of liver tissues.RESULTS: It was revealed that age, index of clinical symptoms and physical signs were obviously relevant to the pathologic grading and staging of liver tissues (P＜0.05). Blood platelet, red blood cells, aspartate aminotransferase (AST),N-terminal procollagen Ⅲ (PⅢ NP) were apparently correlated with the degree of inflammation. PGA (prothrombin time,GGT, apoprotein A1) index, PGAA (PGA+△2-macroglobublin)index, albumin and albumin/globulin were relevant to both inflammation and fibrosis. Hyaluronic acid (HA) was an accurate variable for the severity of hepatic inflammation and fibrosis. The combination of serum markers for fibrosis could increase the diagnostic accuracy. It was notable that viral replication markers were not relevant to the degree of inflammation and fibrosis.CONCLUSION: There is a good correlation between clinical findings and the pathologic grading and staging of liver tissues, which may give aid to the noninvasive diagnosis of liver fibrosis.

Grading and staging of hepatic fibrosis,and its relationship with noninvasive diagnostic parameters

AIM: To explore the grade and stage of pathology and the relationship between grading and staging of hepatic fibrosis and noninvasive diagnostic parameters.METHODS: Inflammatory activity and fibrosis of consecutive liver biopsies from 200 patients with chronic liver disease were determined according to the Diagnostic Criteria of Chronic Hepatitis in China, 1995. A comparative analysis was made in these patients comparing serum markers,Doppler ultrasonography, CT and/or MR imaging with the findings of liver biopsy.RESULTS: With increase of inflammatory activity, the degree of fibrosis also rose. There was a close correlation between liver fibrosis and inflammatory activity. AST, GGT, albumin,albumin/globulin, ALP, AFP, hyaluronic acid, N-terminal procollagen Ⅲ(P Ⅲ NP), collagen type Ⅳ(Col Ⅳ), tissue inhibitors of metalloproteinases-1(TIMP-1), alpha-2-macroglobulin, natural killer cells(NK), some parameters of Doppler ultrasonography, CT and/or MR imaging were all related to the degree of inflammatory activity. GGT, albumin,albumin/globulin, ALP, AFP, hyaluronic acid, Col Ⅳ, TIMP1, alpha-2- macroglobulin, transforming growth factor-beta 1(TGFβ1), NK, some parameters of Doppler ultrasonography,CT and/or MR imaging were all related to the staging of fibrosis. By regression analysis, the parameters used in combination to differentiate the presence or absence of fibrosis were age, GGT, the parameter of blood flow of portal vein per minute, the maximum oblique diameter of right liver by B ultrasound, the wavy hepatic surface contour by CT and/or MR. The sensitivity, specificity and accuracy of the above parameters were 80.36 %, 86.67 %, and 81.10 %,respectively.CONCLUSION: There is close correlation between liver fibrosis and inflammatory activity. The grading and staging of liver fibrosis are related to serum markers, Doppler ultrasonography, CT and/or MR imaging. The combinationof the above mentioned noninvasive parameters are quite sensitive and specific in the diagnosis of hepatic fibrosis.

High Prevalence of cagA-positive Helicobacter pylori Strains in Chinese Patients with Peptic Ulcer Disease, Chronic Gastritis and Gastric Carcinoma

The object in this study is to investigate the cagA stares of H. pylori isolates from Chinese patients with duodenal ulcer (DU), chronic gastritis (CG) and gastric carcinoma (GC). We examined 66 H. pylori isolates from patients, including 30 patients with DU, 30 patients with CG, and 6 patients with GC. The cagA gene was amplified by polymerase chain reaction (PCR), the PCR product is about 324bp. IgG antibodies to H. pylori were present in the sera of all patients, which were detected by ELISA.