| 1 Background Gastric cancer is one of malignant cancers which threaten the health of human. It ranks the first in all cancers in china. Furthermore, its early detection rate is about 10%. The therapeutic efficacy is unsatisfacty to severe patients. The survival rate of five years is 20%-30%. The etiological factors of gastric cancer are not completely clear. It is an accepted opinion that its natural process is above 15 years, most of which belong to latent period without obvious clinical symptoms. So preventing measurements are important to reduce its incidence rate before the clinical symptoms occur.Shandong Academy of Medical Science first found in the world that garlic could prevent the human gastric cancer by the comparative study of the high incidence area—Qixia county and the low incidence area—Changshan county of gastric cancer in Shandong province, and verified it by numerous epidemiological studies and experiment studies. A project of an intervention trial of gastric cancer using garlic allitrum with selenium was founded by the Ministry of Chinese health in 1988 (code:1988baldadbl). This study was performed in about 5000 high-risk population of gastric cancer aged 35 -74 years in seven towns of Quxia county. We collected and analyzed the data of the incidence, mortality, and all death cause during from 1992 to 2001 year. Furthermore, we performed two experiments to explore the suitable does, optimalcombinations of garlic ingredient and other plant ingredients, and their molecular mechanisms.2 Objectives To observe the long-term effect of the combination of allimin with Selenium preventing gastric cancer; To find out the optimal combinations of garlic oil and other drugs to inhibit gastric cancer cells by the experiment; To explore the inducing apoptotic effect and the molecular mechanism of the combinations to gastric cancer cells in vitro.3 Materials and MethodsTechniques of the analysis epidemiology, the cell biology and the molecular biology are applied in this study. This study includes two parts.3.1 Part one: the long-term effect of allimin and selenium preventing gastric cancerThe analysis epidemiological methods were applied. Compared the accumulative incidence rate and the accumulative mortality rate between the intervention group and the control group. Analyzed the relative risk and 95% confidence interval by the single or multi factors logistic regression mode.3.2 Part two: The inhibitory effect of the combination of garlic oil and other plant ingredients to gastric cancer cell and its mechanism 3.2.1 The experiment of the optimal combinations The proliferation inhibitory rate of the different gradient concentration of garlic oil and other drugs to two gastric cancer cell lines were detected by enzyme-linked immunosorbent assay, then calculated their 50% inhibitory concentration (ICJ according to their inhibitory rate. Found out the suitable does each drugs. Detected the inhibitory effect of two gastric cancer cell lines in vitro by using the garlic oil respectively combined with other drugs, and confirmed the optimal combinations of garlic oil and other drugs by their interaction effect analysis.3.2.2 Detection of the apoptosis effect to gastric cancer cells Detected the inducing apoptosis effect of the combinations of garlic oil to gastric cancer MGC-803 cells by the methods as following: The morphology changes were observed by Giemsa staining; Analyzed the DNA electrophoregram by gel alectrophoresis; observed the change of DNA content in cell by confocal microscopy; the annexin v content about early apoptosis cells were detected by flow cytometry.3. 2. 3. The study of the molecular mechanism The cell cycle and the rate of Fas positive antigen of each group cells were detected by flow cytometry. Analyzed the change of reduced glutathione hormone(GSH) content of each groups by the confocal microscopy. Detected the changes of bcl-2, bax, hTERT, c-myc and Mad gene mRNA transcription level by RT-PCR method.4 Results4.1 The analysis results on the long-term effect of allimin and seleniumpreventing gastric cancer4. 1. 1 The cumulative incidence and mortality rate of gastric cancer in two groups begun to separate after the second year of end intervention. The distance of separation got to maximum after five years of end intervention, then began to deflate.4.1.2 Relative risk(RR) and 95 percent confidence interval (95%CI) in theintervention group compared to the control group respectively were 0.43and 0.19-0.98. After adjusted the confound factors, the RR and the 95%CI respectively were 0.41 and (0.18-0.93).4. 2 The results on the experiment of the optimal combinants4.2. ] The IC50 of garlic oil to two gastric cancer cell lines of MGC-803and SGC-7901 were 74 u g/ml and 90 \x g/ml in vitro. The IC50 of sodiumselenite were 93 umol/L and 105fimol/L. The IC50 of curcumine were 16umol/L and 22 umol/L. The IC50 of isoflavone were 105ymol/L and 133umol/L. The lCm of resveratrol were 99 u g/ml and 120yg/ml.4. 2. 2 The evident inhibition effect could be observed when the garlic oil at 1/3 I Cm does and/or other drugs at 1/4 IC50 does to two gastric cancer cell lines for 12, 24, and 48h, furthermore the time-effect relationship could be observed. The combinations of garlic oil and sodium selenite to gastric cancer cell displayed the weak antagonistic effect by the analysis of interaction effect. The combinations of garlic oil and curcumine or isoflavone displayed the additional effect. The combinations of garlic oil and resveratrol displayed the synergistic effect. Resveratrol was the optimal combination of garlic oil.4.3 The results on garlic oil and resveratrol inducing the apoptosis effect of gastric cancer cells4.3.1 Garlic oil and/or resveratrol to gastric cancer cell for 24h, apoptosis cells could be observed by Giemsa staining. Furthermore, the numbers of apoptosis cells of the combination administration group were more than those of the solo administration group.4. 3. 2 The evident DNA ladder was showed in the combination group, the DNA degraded band could be seen in each solo administration group, the strip band showed in the control group by gel electrophoresis. 4. 3. 3 The strongest fluorescence could be observed in the control group, the second in each solo group, and the weakest in the combination group. 4.3.4 By flow cytometry, the annexin v positive rate in the combination group was (35. 83 + 0. 72)%, (25.73 + 0.45)% in garlic oil group, and (4. 84 ± 0.12)% in resveratrol group, the significant difference could be observed compared to the control group. Furthermore, the obvious synergistic effect was detected in the combination group by the interaction analysis.4.4 The results on the study of molecular mechanism4. 4. ] Go/Gi stages of gastric cancer cells were arrested in garlic oil group, then S stages cells were arrested in resveratrol group, but GJGl stageswere blocked in the combination group by flow cytometry way. 4. 4. 2. By flow cytometry, Fas positive expression rate was (5. 27 + 0. 23)% in the control group, (8.23 + 0.56)% was observed in resveratrol group, and (10.59 + 0.41)% in garlic oil group, the Fas positive rate in the combination group was (14.16 + 0.74)%. The significant difference could be observed compared to the control group. Furthermore, the obvious synergistic effect in the combination group was detected.4.4.3 Scanned six cells each groups by confocal microscopy, garlic oil and resveratrol could decrease the GSH contents of gastric cancer cells, the more evident effect could be observed in the combination group.4.4.4 Garlic oil and resveratrol could decrease the expression of bcl-2 gene mRNA of gastric cancer cell, and promoted the expression of bax gene mRNA by RT-PCR method. This effect in their combination group was stronger than that in their solo administration group.4. 4. 5 Detected the expression of c-myc, Mad and hTERT gene mRNA by RT-PCR way. Garlic oil or resveratrol could decrease the expression of c-myc gene, but the significant difference could not be observed. The combination group could evidently decrease the expression of c-myc gene, and the synergistic effect was obvious too. Garlic oil and resveratrol either solo or their combination using could enhance the expression of Mad gene, but the synergistic effect was not evident. Garlic oil and resveratrol either solo or their combination using could decreased the expression of hTERT gene, furthermore the synergistic effect was evident.5 Conclusions5. 1 The combination of allicin and sodium selenite has the protective effect of five years to gastric cancer.5.2 The combination of garlic oil and curcumine or isoflavone to gastric cancer cells showed the additional inhibitory effect on the proliferation. The combination of garlic oil and sodium selenite showed the weakantagonistic effect. The combination of garlic oil and resveratrol showedthe synergistic effect.5.3 The combination of garlic oil and resveratrol could induce the cellapoptosis of gastric cancer cell MGC-803 line in vitro, and thesignificant synergistic effect could be observed.5. 4 Go/G, stages of gastric cancer cells were arrested in garlic oil group.5 stages were arrested in resveratrol group. G0/Gi stages were arrested in the combination application group.5.5 Garlic oil and/or resveratrol could enhance the expression of Fas antigenic and decrease the GSH content in the gastric cancer cell. The significant synergistic effect could be observed in their combination application group.5. 6 Garlic oil and/or resveratrol could enhance the expression of bax gene mRNA in gastric cancer MGC-803 cell line, and at the same time decreased the expression of bcl-2 gene mRNA. The more obvious effect could be observed in their combination application group.5.7 Garlic oil or resveratrol to gastric cancer cell could decrease the expression of c-myc gene mRNA, but the statistic significance was not observed. The statistic significance could be observed in their combination application group. Either solo or combination application of garlic oil and resveratrol could obviously enhance the expression of Mad gene mRNA, but the statistic significance of synergistic effect could not be observed in their combination application. Both solo and their combination application could decrease the expression of hTERT gene, furthermore the synergistic effect had a statistic significance in their combination application group.6 Meaning and creativityDiscovered that the preventing effect of allimin and selenium to gastric cancer might maintain within five years end administration bylong-term following up the high-risk population in the intervention study. Performed the trials of the suitable combinations of garlic oil and other plant ingredients to gastric cancer cells. Found out that the combination application of garlic oil and resveratrol was optimal. Explored the apoptosis effect of the combinations inducing gastric cancer cells and the molecular mechanism by the molecular biology technique. Revealed that the mechanisms were relative to decrease the GSH content, the expression of bcl-2, c-myc, hTERT gene mRNA, and increase the expression the Fas antigen, bax, Mad gene mRNA. Provided important science fundament for preventing gastric cancer in population.7 Shorting and SuggestionsThe shorting in this study is lacking the animal experiment of the combinations of garlic oil and resveratrol to gastric cancer, and lacking the studies of the relative protein expression. We would perform the studies of pharmacodynamic action, pharm-metabolism and acut- or chronicity- toxicology, and the studies of the relative protein expression in future. |
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