Expression of nuclear factor-kappa B and target genes in gastric precancerous lesions and adenocarcinoma:Association with Helicobactor pylori cagA (+) infection

AIM:To examine the expression of nuclear factor kappaB (NF-κB) and its target genes in intestinal metaplasia (IM),dysplasia (DYS) and gastric carcinoma (GC) infected with Helicobacter pylori (H pylori) and to investigate the mechanism underlying Hpyloricytotoxin associated gene A(cag A) infection leading to gastric adenocarcinoma.METHODS: Expressions of NF-κB/p65 and its target genes:c-myc, cyclinD1 and bcl-xl were immunohistochemically examined in 289 cases of gastric biopsy and resection specimens from patients with IM, DYS and GC infected with H pylori. H pylori in the above mentioned tissues was detected by Warthin-Starry stain and rapid urease tests.IgG antibody to cagA in sera of the patients was measured by ELISA.RESULTS:The positive rates of NF-κB/p65 were significantly higher in groups with cagA of IMI-Ⅱ(28/33), IM III(48/52),DYSI(27/31), DY5 Ⅱ-Ⅲ(28/32), GC(35/40) than in groups without cagA of IMI-Ⅱ(4/17), IMⅢ(3/20), DYSI(3/20),DYSⅡ-Ⅲ(6/21), GC(10/23). The expressions of c-myc,cyclinD1, and bcl-xl were significantly higher in groups with cagA of IM Ⅲ(47/52, 49/52, 46/52), DYSⅡ-Ⅲ(29/32, 26/32,25/32) than in groups without cagA of IM Ⅲ(8/20, 7/20,5/20), DYSⅡ-Ⅲ(10/21, 8/21, 3/21), which were in conformity with the expression of NF-κB in IM Ⅲ, and DYSⅡ-Ⅲ. Asignificantly higher expression level of NF-κB/p65, c-myc,cyclinD1 and bcl-xl was detected in intestinal type GC(27/28,18/28, 22/28, 24/28) than in diffuse type GC(8/12, 3/12,3/12, 6/12), respectively.CONCLUSION: There may be two different molecular mechanisms in the occurrence of intestinal and diffuse type gastric carcinomas. Intestinal type gastric carcinoma is strongly associated with high expression of c-myc, cyclinD1 and bcl-xl through NF-κB/p65 activated by Hpylori cagA.Inhibiting the activity of NF-κB is an effective and promising way to prevent intestinal type gastric carcinoma.

Association of tumor necrosis factor genetic polymorphism with chronic atrophic gastritis and gastric adenocarcinoma in Chinese Han population

AIM:To investigate the association of TNF polymorphisms with chronic atrophic gastritis (CAG) and gastric adenocarcin-oma in Chinese Han patients.METHODS:The TNFa-e 5 microsatellites and 3 RFLP sites were typed using PCR technique,followed by high-voltage denaturing PAGE with silver staining and restriction enzyme digestion respectively in specimens from 53 patients with CAG and 56 patients with agstric daenocarcinoma and 164 healthy controls.The PCR products were cloned and sequenced.RESULTS:The frequency of TNF-β Ncol*1/2 genotype was higher in patients with chronic atrophic gastritis than in healthy controls,but no significant difference was observed(60.38% vs 46.34%,p=0.076).The frequency of TNa10 allele was significantly higher in patients with chronic atrophic gastritis than in healthy controls(19.81% vs 11.89%,p=0.04).However,it did not relate to age,gender,atrophic degree or intestinal metaplasin in patients with chronic atrophic gastritis,The frequency of TNF-β Ncol*1/2 and d2/d6 genotypes were significantly higher in patients with gastric adenocarcinoma than in healthy indiveduals(p>0.05).However,TNF-β Ncol*1/2 and d2/d6 genotypes did not relate to age,gender,grade of differentiation and clinicopathologic state in patients with gastric adenocarcinoma.The frequency of TNFa6b5c1 hapolotype homoaygote was significantly lower in patients with gastric adenocarcinoma than in healthy controls (1.79% vs 15.85%,p=0.006).CONCLUSION:TNFa10 allele may be a risk factor for chronic atrophic gastritis ,TNF-β Ncol*1/2 and d2/d6 genotypes are associated with the suscepotibility to gastric adenocarcinoma,whereas TNFa6b5c1 haplotype homozygote may contribute to the resistance against gastric adenocarcinoma.

HLA-DRB1 allele polymorphisms in genetic susceptibility to esophageal carcinoma

AIM: To probe into the genetic susceptibility of HLA-DRB1 alleles to esophageal carcinoma in Han Chinese in Hubei Province.METHODS: HLA-DRB1 allele polymorphisms were typed by polymerase chain reaction with sequence-specific primers (PCR-SSP) in 42 unrelated patients with esophageal cancer and 136 unrelated normal control subjects and the associated HLA-DRB1 allele was measured by nucleotide sequence analysis with PCR.SAS software was used in statistics.RESULTS: Allele frequency (AF) of HLA-DRB1·0901 was significantly higher in esophageal carcinoma patients than that in the normal controls (0.2500 vs0.1397, P=0.028, the odds ratio 2.053, etiologic fraction 0.1282). After analyzed the allele nucleotide sequence of HLA-DRB1·0901 which approachs to the corresponded exon 2 sequence of the allele in genebank. There was no association between patients and controls in the rested HLA-DRB1 alleles.CONCLUSION: HLA-DRB1·0901 allele is more common in the patients with esophageal carcinoma than in the healthy controls, which is positively associated with the patients of Hubei Han Chinese. Individuals carrying HLA-DRB1·0901may be susceptible to esophageal carcinoma.

Synergistic antitumor effect of TRAIL and doxorubicin on colon cancer cell line SW480

AIM: TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) has been reported to specifically induce apoptosis of cancer cells although only a small percentage of cell lines were sensitive to it. Cell lines not responding to TRAIL in vitro were said to be more prone to apoptosis when TRAIL was combined with another anticancer agent.Generally, factors affecting drug-sensitivity involve many apoptosis-related proteins, including p53. The expression of wild-type p53 gene was proposed as an important premise for tumor cells responding to chemotherapy. The present study was to investigate the cell killing action of TRAIL on colon cancer cell line SW480, its synergistic effect with doxorubicin, and the possible mechanisms.METHODS: SW480 cells were cultured in the regular condition and incubated with different levels of agents.Morphologic changes in these cells after treatment were observed under phase-contrast microscope and cytotoxicity by TRAIL alone and in combination with doxorubicin was quantified by a 1-day microculture tetrazolium dye (MTT) assay. In addition, flow cytometry assay (FCM) and transmission electron microscopy were used to detect apoptosis among these cells. Variation of p53 protein level among different groups according to concentrations of agents was measured by Western blot assay.RESULTS: (1) SW480 cells were not sensitive to TRAIL,with IC50>l mg·L^1 and dose-independent cytotoxicity. (2)SW480 cells were sensitive to doxorubicin at a certain degree,with dose-dependent cytotoxicity and IC50=65.25±3.48μmol·L^-1. (3) TRAIL could synergize with doxorubicin to kill SW480 cells effectively, which was represented by the boosted killing effect of doxorubicin on theses cells. IC50 of doxorubicin against SW480 cells sharply reduced when it was combined with TRAIL. (4) Subtoxic TRAIL (100 μg·L^-1),combined with subtoxic doxorubicin (0.86 μmol·L^-1), could kill SW480 cells sufficiently. Cytotoxicity by MTT assay arrived at 80.12±2.67 %, which was significantly higher than that by TRAIL or doxorubicin alone, with P=0.006 and 0.003 respectively. This killing effect was partly due to apoptosis. It was proved by large amounts of apoptotic cells under phase-contrast microscopy, cell apoptosis rate of 76.82±1.93 % by FCM assay and typical apoptotic morphology observed through transmission electron microscopy. Increase of apoptosis after combined treatment had no relation with protein level of p53 (p>0.05).CONCLUSION: SW480 cells are not sensitive to TRAIL, but TRAIL can synergize with lower concentra^on of doxorubidn to induce apoptosis effectively. The status of p53 protein is not involved in the mechanism of synergistic apoptosis. It suggests the potential therapeutic applicability of the combination of TRAIL with doxorubidn against colon cancers.