[Garlic oil inhibits cyclin E expression in gastric adenocarcinoma cells].

OBJECTIVE: To explore the inhibitory effect of garlic oil on cyclin E expression in gastric adenocarcinoma cells. METHODS: Human gastric adenocarcinoma SGC7901 cells were cultured routinely and the expressions of transforming growth factor alpha (TGFalpha) and epidermal growth factor receptor (EGFR) are detected by immunofluorescent staining and flow cytometry. The SGC7901 cells were also cultured with RPMI 1640 without calf serum for 48 h, followed by further culture with RPMI 1640 in the presence of 2.5% calf serum before treatment with TGFalpha, garlic oil, or their combination, and cyclin E expression of the cells was then detected by immunofluorescent staining and flow cytometry. RESULTS: The positivity rates of TGFalpha and EGFR expressions were 46.80% and 57.78 % respectively in SGC7901 cells cultured routinely for 48 h. The positivity rate of cyclin E expression was increased by 7.06% (P<0.001) in SGC7901 cells treated with 30 microg/L TGFalpha for 5 h, decreased by 11.75% (P<0.001) following a 5-hour treatment with 10% garlic oil, and decreased further by 17.11% (Plt;0.001) after treatment with both 30 microg/L TGFalpha and 10% garlic oil for 5 h. CONCLUSIONS: The gastric adenocarcinoma SGC7901 cells express TGFalpha and EGFR and possess TGFalpha autocrine and paracrine loops to promote cell proliferation. Garlic oil inhibits cyclin E expression in routinely cultured SGC7901 cells and also in TGFalpha-treated ones, suggesting that garlic oil can inhibit the TGFalpha autocrine and paracrine loops, which can be one of the pathways of garlic oil to inhibit cancer cell proliferation.

[Effect of transforming growth factor alpha on the expression of cyclin E and cyclin D1 in gastric carcinoma cells].

OBJECTIVE: To explore the effect of transforming growth factor alpha (TGFalpha) on the expression of cyclin E and D1 in gastric carcinoma cells. METHODS: Human gastric adenocarcinoma SGC7901 cells were cultured routinely and synchronized at G(0)/G(1) phase in serum-free RPMI-1640. The percentage of the cells at G(0)/G(1) phase was detected by propidium iodide staining and flow cytometry (FCM), and the synchronized cells were cultured in RPMI-1640 supplemented with 2.5% calf serum and treated with 10, 30, and 50 microg/L TGFalpha for 5 h. The expression of cyclin E and D1 in SGC7901 cells was detected by immunofluorescent staining and FCM. RESULTS: The percentage of the cells at G(0)/G(1) phase increased from 54% in routine culture to 72% in the serum-free RPMI-1640 culture. TGFalpha treatment of the cells synchronized at G(0)/G(1) phase induced significant increment of cyclin E and D1 expressions (P<0.001), and at the dose of TGFalpha of 50 microg/L, their expressions increased by 25.18% and 27.52%, respectively (P<0.001). CONCLUSION: TGFalpha can increase the expression of cyclin E and D1 in gastric carcinoma cells to promote their cell cycle progress.

[Association of TGFalpha and cyclin E expression in gastric carcinoma and precancerous lesions].

BACKGROUND & OBJECTIVE: Previous studies have indicated that increased expression of TGFalpha and cyclin E are correlated with the oncogenesis and progress of cancer; however, their expression patterns in gastric precancerous lesions have been not clear yet. In addition, the association between expression of TGFalpha and cyclin E has not been reported. This study was designed to investigate the expression of TGFalpha and cyclin E in chronic superficial gastritis (CSG), gastric precancerous lesions, and gastric carcinoma (GCA), and analyze the association of TGFalpha and cyclin E expression, and the relationship between their expression and different development stages of oncogenesis of GCA. METHODS: The expression of TGFalpha and cyclin E in CSG, intestinal metaphases (IM), atypical hyperplasia (AH), and GCA were examined using immunohistochemical staining. The association of TGFalpha and cyclin E expression was also statistically analyzed. RESULTS: The expression rates of TGFalpha and cyclin E were 15.1%, 53.6%, 51.7%, 61.7%, and 7.5%, 28.6%, 37.9%, 42.6% in tissue samples of CSG, IM, AH and GCA, respectively. The positive expression rates of TGFalpha and cyclin E were higher in IM, AH, GCA than in CSG; the difference was significant (each P< 0.05). In addition, the positive expression rates of TGFalpha and cyclin E were higher in low differential adenocarcinoma (TGFalpha 81.0%, cyclin E 57.1%) than in mediate to high differential one (TGFalpha 41.7%, cyclin E 16.7%) (P< 0.05,for both TGFalpha and cyclin E). It is also revealed that the expression of TGFalpha and cyclin E were closely associated (P< 0.001 for the tissue of gastric chronic inflammation, and P=0.005 for gastric carcinoma, respectively). CONCLUSION: In the tissues of CSG, IM, AH, and GCA, the expression rates of TGFalpha and cyclin E are increased gradually with the severity of the pathological lesions and the malignancy of GCA. In addition, the expression of TGFalpha and cyclin E were obviously associated.

Overexpression of c-fos in Helicobacter pylori-induced gastric precancerosis of Mongolian gerbil.

AIM: To explore dysregulation of c-fos in several human malignancies, and to further investigate the role of c-fos in Helicobacter pylori (H. pylori)-induced gastric precancerosis. METHODS: Four-week-old male Mongolian gerbils were employed in the study. 0.5 mL 1X10(8) cfu/L suspension of H. pylori NCTC 11 637 in Brucella broth were inoculated orally into 20 Mongolian gerbils. Another 20 gerbils were inoculated with Brucella broth as controls. 10 of the infected gerbils and 10 of the non- infected control gerbils were sacrificed at 25 and 45 weeks after infection. The stomach of each gerbil was removed and opened for macroscopic observation. The expression of c-fos was analyzed by RT-PCR and immunohistochemical studies in H. pylori-induced gastric precancerosis of Mongolian gerbil. Half of each gastric antrum mucosa was dissected for RNA isolation and RT-PCR. beta-actin was used as the housekeeping gene and amplified with c-fos as contrast. PCR products of c-fos were analyzed by gel image system and the level of c-fos was reflected with the ratio of c-fos/beta-actin. The immunostaining for c-fos was conducted using monoclonal antibody of c-fos and the StreptAvidin-Biotin-enzyme Complex kit. RESULTS: H. pylori was constantly found in all infected animals in this study. After infection of H. pylori for 25 weeks, ulcers were observed in the antral and the body of stomach of 60 % infected animals (6/10). Histological examination showed that all animals developed severe inflammation, especially in the area close to ulcers, and multifocal lymphoid follicles appeared in the lamina propria and submucosa. After infection of H. pylori for 45 weeks, severe atrophic gastritis in all infected animals, intestinal metaplasia in 80 % infected animals (8/10) and dysplasia in 60 % infected animals (6/10) could be observed. C-fos mRNA levels were significantly higher after infection of H. pylori for 25 weeks (1.84+/-0.79), and for 45 weeks (1.59+/-0.37) than those in control-animals (0.74+/-0.22, P<0.01). C-fos mRNA levels were increased 2.5-fold by 25th week (P<0.01) and 2.1-fold by 45th week (P<0.01) in precancerosis induced by H. pylori, when compared with normal gastric epithelium of Mongolian gerbil. Immunohistochemical staining revealed exclusive nuclear staining of c-fos. Furthermore, there was a sequential increase in c-fos positive cells from normal epithelium to precancerosis. CONCLUSION: The study suggested that overexpression of c-fos occurs relatively early in gastric tumorigenesis in this precancerosis model induced by H. pylori.

[Effect of Helicobacter pylori infection on gastric mucosal cell proliferation in Mongolian gerbils].

OBJECTIVE: To explore the mechanism that Helicobacter pylori (H. pylori) infection can induce adenocarcinoma in Mongolian gerbil model with long-term H. pylori infection. METHODS: Mongolian gerbil model with long-term H. pylori infection was established by inoculation H. pylori NCTC 11637 strain, and immunohistochemical straining and in situ hybridization were employed to observe changes in gastric mucosal cell proliferation due to H. pylori infection. RESULTS: Mongolian gerbil model with long-term H. pylori infection was successfully established. Immunohistochemical staining of 5'-bromodeoxyuridine (BrdU) and proliferating cell nuclei antigen (PCNA) showed that H. pylori infection induced the increase in gastric mucosal cell proliferation (P<0.05), and in situ hybridization of epidermal growth factor (EGF) and epidermal growth factor receptor (EGFR) revealed elevated expressions of EGF mRNA and EGFR mRNA with time passage after H. pylori infection (P<0.05). CONCLUSION: H. pylori inoculation can induce abnormality in gastric mucosal cell proliferation, which is instrumental for the progression from chronic gastritis to glandular atrophy, intestinal metaplasis and ultimately to atypical hyperplasia. The abnormal expressions of EGF and EGFR may be the key element for abnormality of gastric mucosal cell proliferation.

Overexpression of cyclin E in Mongolian gerbil with Helicobacter pylori-induced gastric precancerosis.

AIM: To explore dysregulation of cyclin E in malignancies, and to further investigate the role of cyclin E in Helicobacter pylori (H. pylori)-induced gastric precancerosis. METHODS: Four-week-old specific pathogen-free male Mongolian gerbils were employed in the study. 0.5 mL 1 x 10(8) cfu x L(-1) suspension of H.pylori NTCC11637 in Brucella broth was inoculated orally into each of 20 Mongolian gerbils, and a further 20 gerbils were inoculated with Brucella broth as controls. 10 of the infected gerbils and 10 of the non-infected control gerbils were sacrificed at 25, 45 wk after infection. The expression of cyclin E was analyzed by RT-PCR and immunohistochemical studies with monoclonal antibody to cyclin E in Mongolian gerbil of H. pylori-induced gastric precancerosis. RESULTS: H. pylori was constantly detected in all infected animals throughout the study. At 25 wk after infection of H. pylori, ulcers were observed in the antral and body of stomach (n=6). Histological examination showed that all animals developed severe inflammation and multifocal lymphoid follicles appeared in the lamina propria and submucosa of gastric antrum. At 45 wk after infection of H. pylori, severe atrophic gastritis (n=10), intestinal metaplasia (n=8) and dysplasia (n=6) could be observed. Cyclin E mRNA levels were significantly more at 25 wk after infection of H. pylori (1.27+/-0.26), and at 45 wk after infection of H. pylori (1.82+/-0.39) than control-animals (0.59+/-0.20, P<0.01) cyclin E mRNA levels were evaluated by 2.2-fold at 25 wk (P<0.01) and 3.1-fold at 45 wk (P<0.01) precancerosis induced by H. pylori, when compared with control gastric epithelium of Mongolian gerbil. Immunohistochemical staining revealed exclusive nuclear staining of cyclin E. Furthermore, there was a sequential increase in cyclin E positive cells from normal epithelium to precancerosis. CONCLUSION: Overexpression of cyclin E occurs relatively early in gastric tumorigenesis in this model.

Tumor growth inhibition effect of hIL-6 on colon cancer cells transfected with the target gene by retroviral vector.

AIM:To observe the tumor inhibitory effects by transfecting IL-6 cDNA into colon cancer cell line HT-29 with retroviral vector pZIP cDNA.METHODS:Human IL-6 gene was reconstructed in retrovirus vector and transfected into incasing cells PA317 by lipofectamine mediated method, the clones of the cells transferred with hIL-6 were selected by G418,and targeted HT-29 cells were infected with the virus granules secreted from PA317 and also selected by G418.Test gene transcription and expression level by hybridization, ELISA and MTT assay,etc. Analyze tumor inhibitory effects according to the cell growth curve, plating forming rate and tumorigenicity in nude mice.RESULT:Successfully constructed and transfected recombinant expressing vectors pZIPIL-6 cDNA and got positive transfected cell lines. The colon cancer cell line (HT-29 IL-6) transfected with the hIL-6 gene by retroviral vector was estab-lished. The log proliferation period and the doubling time of this cell line was between 4 to 7 days and 2.5 days according to the direct cell count, the cell proliferation was obviously inhibited with MTT assay, the plating inhibitory rate was 50% by plating efficiency test. When HT-29 IL-6 cells were inoculated into the nude mice subcutaneously, carcinogenic activity of the solid tumor was found superior to the control group and the size of tumor was not significantly enlarged. Injection of combination virus fluid containing IL-6 gene into transplantation tumors could inhibit the growth and development of the tumor.CONCLUSION:IL-6 could inhibit the growth and proliferation of colon cancer cells by retroviral vector-mediated transduction.

Induction of apoptosis of lymphocytes in rat mucosal immune system.

AIM:To undergo apoptosis during negative and positive selection processes in rat mucosal immune system which are implicated in the pathogenesis of various mucosal diseases.METHODS: Female Sprague-Dawley rats were given protein synthesis inhibitor, cycloheximide, intravenously or intraperitoneally, an apoptosis was recognized by morphological hallmark under light and electronmicroscopy, and the expression of proliferating cell nuclear antigen was visualized immunohisto-chemically.RESULTS: The apoptosis of mucosal lymphocytes in the digestive tract, as well as in trachea, uterus and lacrimal gland was induced by cycloheximide (>1.0mg·kg(-1) body weight), which were located mainly in lamina propria and germinal centers of lymphoid nodules. At the same time, a portion of crypt epithelial cells of proliferating zone in small and large intestine, and the epithelial cells in genital tract were also found to undergo apoptosis. Immunostainings showed that apoptotic cells expressed proliferating cell nuclearantigen.CONCLUSION: Apo-ptosis of lymphocytes in mucosal immune system can be induced by cycloheximide. This model will facilitate the understanding of normal mucosal immune system and its role in the pathogenesis of related diseases such as inflammatory bowel diseases.