Susceptibility for NSAIDs-induced apoptosis correlates to p53 gene status in gastric cancer cells.

The anti-tumor effect of non-steroidal anti-inflammatory drugs (NSAIDs) remains unclear. Here, we found that the susceptibility for NSAIDs-induced apoptosis might correlate with the status of the p53 gene in gastric cancer cells. Apoptosis in gastric cancer cells expressing wild-type p53 is induced through up-regulation of bax and down-regulation of bcl-2 and that regulation of the bax-bcl-2 heterodimer may be a major target of NSAIDs. As to gastric cancer cells expressing mutant-type p53, other key factors may exist in the NSAIDs' growth inhibition action.

[The relationship of mTOR signaling pathway and histone acetylation in human gastric cancer cell lines].

OBJECTIVE: To evaluate the relationship between mammalian target of rapamycin (mTOR) signaling pathway and histone acetylation in cell survival, cell cycle, gene expression and protein level on human gastric cancer cells. METHODS: Human gastric cancer cell lines, MKN45 and SGC7901 were treated with trichostatin A, rapamycin and/or LY294002, a PI3K inhibitor. Cell viability was analyzed by methylthiazolyl tetrazolium. Cell cycle distribution was evaluated by flow cytometry. The transcription level of p21(WAF1) gene was detected by using real-time polymerase chain reaction. Proteins were detected by Western blotting. RESULTS: Cell viability remarkably reduced after treatment by more than two drugs (P< 0.01). Through flow cytometry assessment, MKN45 cells were arrested in G2 phase (P< 0.05), while SGC7901 cells were in G2 or G1 phase (P< 0.05) whether treated with single or more than two drugs. The expression of p21(WAF1) mRNA was remarkably increased in the gastric cancer cells treated with conjoined drugs (P< 0.01). Phosphorylation of Akt, p70S6K and 4E-BP1 was significantly reduced in cells treated with conjoined drugs (P< 0.01). And histone acetylation of H4/H3 was also increased in cells treated with conjoined drugs (P< 0.01). CONCLUSION: mTOR singnaling pathway has an important relationship with histone acetylation in gastric cancer cell lines. There is a co-effect of mTOR inhibitor and histone deacetylase inhibitor on gastric cancer cells.

[Synergistic effect of rapamycin (RPM) and PD98059 on cell cycle and mTOR signal transduction in human colorectal cancer cells].

OBJECTIVE: To investigate the synergistic effect of rapamycin (RPM) and PD98059 on human colorectal cancer cells and its potential mechanisms. METHODS: Three human colorectal cancer cell lines SW480, HCT116 and HT29 were treated with RPM 10 nmol/L, PD98059 (10 micromol/L, 20 micromol/L, 40 micromol/L, 50 micromol/L), or RPM plus PD98059, respectively, and the sensitivity was analyzed by MTT assay. The cell cycle progression was evaluated by flow cytometry. Western blotting analysis was performed to examine the total and phosphorylated levels of mammalian target of rapamycin (mTOR) and its downstream translational signaling intermediates, 70 kDa ribosomal protein S6 kinase (p70s6k) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1). RESULTS: Both RPM and PD98059 could inhibit viability of the three cell lines. The anti-proliferative effect of PD98059 exhibited a time/dose dependent manner and was strengthen by RPM. All the treatment with RPM, PD98059, and RPM + PD98059 induced arrest of cell cycle, although the arrest was confined at different cell cycle phases. In addition to their effect on proliferation and cell cycle, both inhibitors also reduced phosphorylation levels of mTOR, p70s6k, and 4E-BP1, as well as total 4E-BP1 levels in SW480 and HCT116 cells. That effect was reinforced when cells were treated with RPM plus PD98059 simultaneously, whereas total protein levels of mTOR and p70s6k remained unchanged. CONCLUSION: RPM and PD98059 inhibit proliferation of colorectal cancer cells synergistically, and induce cell cycle arrest. The modulation of mammalian target of rapamycin signaling pathway is involved in its potential mechanisms.

Folate levels in mucosal tissue but not methylenetetrahydrofolate reductase polymorphisms are associated with gastric carcinogenesis.

AIM: To evaluate whether folate levels in mucosal tissue and some common methylenetetrahydrofolate reductase (MTHFR) variants are associated with the risk of gastric cancer through DNA methylation. METHODS: Real-time PCR was used to study the expression of tumor related genes in 76 mucosal tissue samples from 38 patients with gastric cancer. Samples from the gastroscopic biopsy tissues of 34 patients with chronic superficial gastritis (CSG) were used as controls. Folate concentrations in these tissues were detected by the FOL ACS:180 automated chemiluminescence system. MTHFR polymorphisms were analyzed by PCR-RFLP, and the promoter methylation of tumor-related genes was determined by methylation-specific PCR (MSP). RESULTS: Folate concentrations were significantly higher in CSG than in cancerous tissues. Decreased expression and methylation of c-myc accompanied higher folate concentrations. Promoter hypermethylation and loss of p16(INK4A) in samples with MTHFR 677CC were more frequent than in samples with the 677TT or 677CT genotype. And the promoter hypermethylation and loss of p21(WAF1) in samples with MTHFR 677CT were more frequent than when 677CC or 677TT was present. The 677CT genotype showed a non-significant higher risk for gastric cancer as compared with the 677CC genotype. CONCLUSION: Lower folate levels in gastric mucosal tissue may confer a higher risk of gastric carcinogenesis through hypomethylation and overexpression of c-myc.

Regulation of hMSH2 and hMLH1 expression in the human colon cancer cell line SW1116 by DNA methyltransferase 1.

Aberrant DNA methylation is now recognized as an important epigenetic alteration occurring early in human cancer. To directly study the role of DNA methyltransferase 1 (DNMT1) in the regulation of expression of tumor-related genes in human colon cancer cells, we stably transfected expression constructs containing sense or antisense DNMT1 into the human colon cancer cell line, SW1116. The expression level of mismatch repair genes (MMR), human mut-L homologue 1 (hMLH1) and human Mut S homologue 2 (hMSH2), was monitored by real-time RT-PCR. The methylation status of hMLH1 and hMSH2 promoters was determined by bisulfite modification and methylation-specific PCR (MSP). The protein levels of DNMT1, hMSH2 and hMLH1 were determined by Western analysis. The results show that DNMT1 protein expression was increased or decreased in transfected cell lines containing sense or antisense DNMT1 constructs, respectively. In cells expressing the sense DNMT1 construct, the expression of hMLH1 and hMSH2 was down-regulated through hypermethylation of their respective promoters. Furthermore, antisense DNMT1 expression induced promoter demethylation and up-regulated transcription of hMSH2 (P<0.05) and hMLH1 (P=0.064) in SW1116 cells.

Clinical relevance of iceA and babA2 genotypes of Helicobacter pylori in a Shanghai population.

OBJECTIVE: To determine the distribution of the iceA and babA2 genotypes of Helicobacter pylori in patients with various gastroduodenal diseases in Shanghai, and to explore the association between genotype and the clinical outcome of infection. METHODS: One hundred and forty-one strains of H. pylori were isolated from gastric biopsies of 43 patients with chronic gastritis, 47 with duodenal ulcer (DU), 30 with gastric ulcer (GU) and 21 with non-cardia gastric carcinoma. The iceA, babA2, cagA and vacA genotypes were determined by polymerase chain reaction (PCR). RESULTS: The iceA1, iceA2 and babA2 genotypes were detected in 74.5% (105/141), 15.6% (22/141) and 63.8% (90/141), respectively, of the H. pylori strains studied. Two H. pylori isolates (1.4%) were positive for both iceA alleles and 16 (11.3%) were negative for both. The prevalence of babA2 and its combination with cagA (cagA(+)/babA2(+)) in DU patients was significantly higher than that in GU patients (74.5%vs 50.0% for babA2, P = 0.028; 70.2%vs 46.7% for cagA(+)/babA2(+), P = 0.039). There was no significant difference in the prevalence of babA2 among the other disease groups, and no significant association of the iceA genotypes with the different clinical diseases (P > 0.05). CONCLUSIONS: The most predominant genotype of the H. pylori strains isolated from patients in Shanghai are iceA1(+)/babA2(+), and the babA2 genotype may play a different role in the pathogenesis of DU and GU. An association between iceA status and clinical outcome of H. pylori infection could not be confirmed in the present study.

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 proto-oncogene in human gastric cancer. METHODS: Paired samples of primary gastric cancer and corresponding para-cancerous, non-cancerous gastric mucosae were obtained from surgically resected specimens of 28 patients. Transcription levels of Dnmt1, 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 Dnmt1 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 hypomethylated c-myc. The transcriptional levels of p16(INK4A) and MeCP2 genes did not display any difference 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.

Histone acetylation regulates p21WAF1 expression in human colon cancer cell lines.

AIM: To investigate the effect of histone acetylation on regulation of p21WAF1 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 p21WAF1 mRNA and protein were detected by real-time RT-PCR and Western blotting, respectively. Acetylation of two regions of p21WAF1 gene-associated histones and total cellular histones were examined by chromatin immunoprecipitation assay and Western blotting. RESULTS: Trichostatin or sodium butyrate re-activated p21WAF1 transcription resulted in up-regulated p21WAF1 protein level in colon cancer cell lines. Those effects were accompanied by an accumulation of acetylated histones in total cellular chromatin and p21WAF1 gene-associated region of chromatin. CONCLUSION: Histone acetylation regulates p21WAF1 expression in human colon cancer cell lines, Colo-320 and SW1116.

[Effect of eukaryotic expression plasmid DNA methyltransferase gene on methylation status and transcription level of DNA mismatch repair genes in human colon cancer cell line].

OBJECTIVE: To analyze the effect of eukaryotic plasmids containing sense or antisense DNA methyltransferase (Dnmt1) genes on the methylation status and transcription level of DNA mismatch repair (MMR) genes and microsatellite instability (MSI) in human colon cancer cell line. METHODS: Human colorectal cells of the SW1116 line were cultured. Recombinant plasmids containing sense Dnmt1 (HMT) or antisense Dnmt1 (THM) gene, pCMV-HMT and pCMV-THM, were constructed. Then pCMV-HMT, pCMV-THM, and pcMV blank plasmid were transfected into SW1116 cells respectively by using lipofectAMINE. The expression of Dnmt1 protein was examined by Western blotting. The transcription levels of hMLH1 and hMSH2 genes were detected by using real-time (RT-PCR). The status of methylation in promoters of hMLH1 and hMSH2 genes were examined with methylation specific PCR (MSP). The MSI of DNA in SW1116 cells was evaluated by silver-stained polyacrylamide gel electrophoresis. RESULTS: Both the expressions of the hMLH1 and hMSH2 gene mRNAs were remarkably decreased in the SW1116-HMT cells in comparison with those in the untransfected cells. The expression of hMSH2 gene mRNA in the SW1116-THM cells was remarkably increased in comparison with that in the untransfected cells. No significant difference in the expressions of the hMLH1 and hMSH2 gene mRNAs was found between the SW1116 cells transfected with blank pCMV and the untransfected SW1116 cells. MSP showed that the methylation level in the regions of hMLH1 and hMSH2 promoters was remarkably increased in the SW1116 cells transfected with sense Dnmtl plasmid. However, in the SW1116 cells the hMSH2 promoter region was changed from partially-methylated into de-methylated, and the hMLH1 promoter region remained non-methylated. MST test showed that extra bands indicating MSI were seen only in the D2S123 groups. CONCLUSION: Dnmt1 regulates the expression and methylation status of MMR genes and affects MSI in human colon cancer cell line SW1116.

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 p21WAF1 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.

5-Aza-2'-deoxycitydine induces demethylation and up-regulates transcription of p16INK4A gene in human gastric cancer cell lines.

BACKGROUND: To investigate the effects of DNA methylation on the expression of tumor-associated genes and the cell cycle in human gastric cancer cells. METHODS: Two gastric cancer cell lines (MKN-45 and HGC-27) were treated with DNA methyltransferase (DNMT) inhibitor, 5-aza-2'-deoxycytidine (5-aza-dC). The expressions of p16INK4A, p21WAF1, p53, p73, c-Ha-ras and c-myc genes mRNA were detected by using reverse transcription PCR (RT-PCR). DNA methylation status of p16INK4A gene promoter was assayed by bisulfite modification and sequencing. The cell cycle was analyzed by using flow cytometry (FCM). RESULTS: 5-aza-dC induced the demethylation of p16INK4A gene promoter. The expression of p16INK4A mRNA was obviously up-regulated by treatment with 10 micro mol/L (MKN-45 cells) or 5 micro mol/L (HGC-27 cells) of 5-aza-dC for 24 hours. However, 5-aza-dC treatment failed to regulate the expressions of p21WAF1, p53, p73, c-Ha-ras and c-myc genes in MKN-45 and HGC-27 cells. Furthermore, 5-aza-dC induced the cell cycle arrest in G1 phase in HGC-27 cell, but not in MKN-45 cell. CONCLUSIONS: DNA methylation regulates the transcription of p16INK4A but not p21WAF1 and proto-oncogenes in human gastric cancer cell lines MKN-45 and HGC-27.

[Prophylactic and therapeutic effect of oxymatrine on D-galactosamine-induced rat liver fibrosis].

OBJECTIVE: To investigate the prophylactic and therapeutic effect of oxymatrine on experimental liver fibrosis and to reveal its mechanism. METHODS: By establishing D-galactosamine-induced rat liver fibrosis model, we observed the effect of oxymatrine on serum and tissue biochemical indexes, content of liver hydroxyline, expression of TGF?1 mRNA and changes of tissue pathology. RESULTS: There was a decline of liver hydroxyline and serum AST and ALT in oxymatrine group compared to those of the D-GalN group. The hydroxyline content in oxymatrine pretreatment group was (0.50 0.11)mug/mg compared with (0.99 0.14)mug/mg in D-GalN group (t=8.366, P<0.01). The content in oxymatrine treatment group was (0.44 0.04)mug/mg compared with 0.70 0.06 in D-GalN group (t=9.839, P<0.01). The SOD activity was (149.81 15.28) NU/mg in oxymatrine pretreatment group and (95.22 16.33) NU/mg in the model group (t=7.309, P<0.01); (157.68 19.54) NU/mg in the treatment group compared with (119.88 14.94) NU/mg in the model group (t=4.348, P<0.01). MDA in the pretreatment group was (2.06 0.17) nmol/mg, lower than (4.57 0.37) nmol/mg in the model group (t=17.529, P<0.01). In the treatment group, it was (1.76 0.24)nmol/mg, lower than (3.10 0.17) nmol/mg in the model group (t=12.697, P<0.01). TGF?1 mRNA reduced in the pretreatment and treatment groups as compared with that in the model group (0.21 0.01 vs 0.50 0.01, t=48.665, P<0.01; 0.18 0.02 vs 0.38 0.01, t=22.464, P<0.01). Electron microscopy showed that oxymatrine group had milder hepatocyte degeneration and less fibrosis accumulation than did the model group. Microscopy revealed wide septa expansion from the portal area to the central venous, piecemeal and confluent necrosis and pseudo-nodular formation in part of the lobular in the model group. While in oxymatrine group these lesions were much improved. CONCLUSIONS: Oxymatrine shows prophylactic and therapeutic effect in D-galactosamine induced rat liver fibrosis. This is partly by protecting hepatocyte and suppressing fibrosis accumulation through anti-lipoperoxidation.