The Effect Of Resveratrol On Expression Of Apoptosis-related Gene Pdcd5 In Bgc-823 Cell

Background and objectiveGastric carcinoma is one of the commonest alimentary carcinoma.At present, gastric cancer has become the third largest tumor in Mainland China,which serious threat people's health.Gastric carcinoma is one of the malignant tumors focus on our country.The human programmed cell death 5(PDCD5) is a new human genes which is first reported in the international by PeKing University Center for Human Disease Genimics in 1999.It is a new apoptosis-related gene cloned form human leukemia cell line TF-1(GneBank registration number AF014955).The gene of PDCD5 locate in 19q12- q13.1 and to participate in the process of apoptosis regulation.PDCD5 mRNA is not only expressed in hemapoietic system and it expressed in more than fifty human tissues,especially in the heart,testis,kidney,adrenal gland and placenta. Remarkably,the expression of PDCD5 mRNA in fetal tissues is significantly lower than that in adult tissues.Preliminary studies showed that PDCD5 had low expression in many diseases,particularly in cancer,autoimmune diseases and local injurie.Resveratrol(Res) is a naturally accruing phytoalexin polyphenolic compound found in various plants,including grapes,polygonum,and peanuts,and has been shown to exhibit a wide range of pharmacological properties and is believed to play a role in the chemoprevention and treatment of human cancer,including colon cancer, leukemia,lung cancer and breast cancer,et al.It can significantly inhibit a variety of human tumor cell growth and induced apoptosis.Researchs showed that resveratrol can play a role in cancer by anti-oxidation,inhibit cyclooxygenase(COX) and cytochrome P450 enzymes,induced tumor cells differentiation and promote tumor cells apoptosis and so on.In order to research the effect of PDCD5 protein and mRNA expression in human gastric poor differentiated adenocarcinoma cell BGC-823 induced by resveratrol and the role of resveratrol on BGC-823 cell growth inhibition,this research draws up to use the immunocytochemistry SP examination PDCD5 protein expression in BGC-823 cells;and use the in situ hybridzation examination PDCD5 mRNA expression in BGC-823 cells;and use flow cytometry and TUNEL examination proliferation and apoptosis of BGC-823 cells.Research the correlation between PDCD5 expression in BGC-823 cells and BGC-823 cells apoptosis,to provide a theories basis for clinical treatment of gastric cancer.Materials and methods1.Samples:human gastric poor differentiated adenocarcinoma cell BGC-823 purchase from Chinese Academy Science Shanghai cell biology graduate school. Through recovery incubation and cultured,the cells divided into different experiment groups and use 25μmol/L,50μmol/L,100μmol/L,200μmol/L resveratrol handled BGC-823 cells for 24h,48h,72h,respectively.And set up blank control groups(medium control group),solvent control groups(DMSO control group), cisplatin control groups(3μg/ml),combination groups(resveratrol 200μmol/L + cisplatin 3μg/ml).2.Use immunocytochemistry SP method to detect the expression of PDCD5 protein in BGC-823 cells of each groups.3.Use in situ hybridzation method to detect the expression of PDCD5 mRNA in BGC-823 cells of each groups.4.Use Flow cytometry to analyze the cell cycle of each groups for 24h,48,72h, respectively. 5.Use Flow cytometry to detect the cell apoptosis of each groups for 24h,48,72h, respectively.6.Use TUNEL to detect the cell apoptosis of each groups for 24h.7.Statistical analysis:All the data were analyzed by SPSS 11.0 statistical software package,the data were expressed by mean±standard deviation((?)±S) and analyzed using the t-test for two groups and ANVOA for more than two groups. The level of significant wasa=0.05.Results1.Immunocytochemistry results:PDCD5 protein expressed low in BGC-823 cells;the expression increased compared with blank control groups in experiment groups by resveratrol for 24h,48h,72h;with the increase of resveratrol dosage of each time period,PDCD5 protein expression higher,and show a dose-dependent manner,in 50-200μmol/L resveratrol groups,there were significant differences compared with blank control groups and solvent control groups(P＜0.05); compared between the experiment groups,there were significant differences (P＜0.05);with time extending of each dosage,the protein expression higher of PDCD5,but there was no significant difference in three time period(P＞0.05); there was no significant differences in 25μmol/L resveratrol group and solvent control group compared with blank control group(P＞0.05).Experiment groups and cisplatin group compared with combination group,have significant differences at each time periods(P＜0.05);the expression of PDCD5 protein in combination groups increased with the time extending,especially 72h,compared between three time periods,there were significant differences(P＜0.05).2.In situ hybridration results:PDCD5 mRNA expressed low in BGC-823 cells; the expression increased compared with control groups in experiment groups by resveratrol for 24h,48h,72h;with the increase of resveratrol dosage of each time period,PDCD5 mRNA expression higher,above 50μmol/L resveratrol groups, there were significant differences compared with blank control groups and solvent control groups(P＜0.05),compared between the experiment groups,there were significant differences(P＜0.05);with time extending of each dosage,the mRNA expression higher of PDCD5;but there was no significant difference in three time period(P＞0.05).There was no significant difference in 25μmol/L group and solvent control group compared with blank control group(P＞0.05).Experiment groups and cisplatin group compared with combination group,have significant differences at each time periods(P＜0.05);the expression of PDCD5 mRNA in combination groups increased with the time extending,especially 72h,compared between three time periods,there were significant differences(P＜0.05).3.Flow cytometry results of the cell cycle:The rates of G₁ phase and G₂ phase decline and S phase increase in experiment groups compared with blank control group by reveratrol for 24h,48h,72h;with the increase of resveratrol dosage of each time period,the rates of cells in G₁ phase and G₂ phase were declined and the rate of cells in S phase was increased,50～200μmol/L resveratrol groups compared with blank control groups and solvent control groups there have significant difference(P＜0.05);compared between the experiment groups the rate of S phase increased have a significant difference(P＜0.05).With the time extending of each dosage,the rate of cells in S phase was increasing and the rates of cells in G₁ phase and G₂ phase were declining,but there were no significant difference in three time period(P＞0.05).There was no significant cycle difference in 25μmol/L group and solvent control group compared with blank control group(P＞0.05).Experiment groups and cisplatin group compared with combination group,the cell cycle have significant differences at each time periods(P＜0.05).4.How cytometry results of the cell apoptosis:The blank control groups were almost all live cells;with the increase of resveratrol dosage of each time period,the percentage of cells undergoing apoptotic cell death and the percentage of cells undergoing necrotic cell death were gradually increased,and the percentage of cells undergoing apoptotic cell death was significant increased,above 50μmol/L resveratrol groups,there were significant differences compared with blank control group and solvent control group(P＜0.05);compared between the experiment groups,the percentage of cells undergoing apoptotic cell death has a significant difference(P＜0.05);with the time extending of each dosage the percentage of cells undergoing apoptotic cell death and the percentage of cells undergoing necrotic cell death were gradually increased,but there was no significant difference(P＞0.05). There was no significant difference in 25μmol/L group and solvent control group compared with blank control group(P＞0.05).Experiment groups and cisplatin group compared with combination group,the percentage of cells undergoing apoptotic cell death have significant differences at each time periods(P＜0.05),and the percentage of cells undergoing necrotic cell death have no significant differences at each time periods(P＞0.05).5.TUNEL results:The apoptotic cells showed nuclei brown,chromatin distribute unevenly and dyeing depth varies detected by TUNEL method.Blank control groups almost did not see apoptotic cells and in experiment groups the number of apoptotic cells increased with the dosage of resveratrol increased,above 50μmol/L experiment groups,cisplatin group and combination group compared with blank control group and solvent control group there were significant difference(P＜0.05); compared between the experiment groups,there were significant differences (P＜0.05);200μmol/L resveratrol group compared with cisplatin group,there was no significant differences(P＞0.05),the other experiment groups compared with the cisplatin group there were significant differences(P＜0.05);experiment groups and cisplatin group compared with combination group,have significant differences(P＜0.05).Conclusions1.Resveratrol can up-regulated PDCD5 protein and mRNA in BGC-823cells and induce BGC-823 cells stagnation in the S phase of the cell cycle and induce apoptosis in a dose-dependent manner.2.The BGC-823 cells appear series of morphological change of apoptosis by resveratrol,while the expression of PDCD5 protein and mRNA in BGC-823 cells were up- regulated,indicating that PDCD5 involved in the gastric cancer cell apoptosis and resveratrol can induce gastric carcinoma cell apoptosis by upregulated the expression of PDCD5.3.Resveratrol can enhance the effect of cisplatin induce apoptosis.