Effects And Mechanisms Of Resveratrol On The Proliferation And Apoptosis Of The Gastric Cancer Cell Lines

The effects and the mechanisms of Resveratrol on the proliferation and apoptosis of the gastric cancer linesObjectiveResveratrol(Res) is a natrually accuring phytoalexin polyphenolic compound found in various plants, including grapes, berries, 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. Resveratrol regulates multiple cellular and molecular events associated with tumor development. Resveratrol was shown to affect diverse cellular events associated with each step of carcinogenesis, i. e., tumor initiation, promotion, and progression. At the molecular level, these effects corresponded with the inhibition of free radical formation, cyclooxygenase. Resveratrol induces apoptosis in various malignant cells through multiple mechanisms, such as up-regulation of Fas and Fas-L expression, enhancement of p53 expression and activity, alteration of expression of B-cell CLL/ lymphoma 2- associated X (Bcl-2) family of proteins, up-regulation of p21 Cipl/WAF1 expression, down-regulation of survivin expression, loss of mitochondrial function, and activation of caspase. Despite these findings, however, the effect of resveratrol on gastric cancer is not well understood. In order to know the value of resveratrol in treatments of gastric cancer, this study was aimed to gain futher insight into the pathways mediating the anticancer proliferation and induction apoptosis of resveratrol. In this study ,we examinated the effects of resveratrol on the growth and apoptosis of the gastric cancer lines SGC7901, BGC823, and the change of mitchondrial potential (Δψm) and the expression of Survivin, caspase -3 and Smac/DIABLO in protein and mRNA level.Methods1. The inhibition of resveratrol on the proliferation of gastric cacer cells was analyzed by MTT assayFor MTT assay, gastric cancer cells were plated in 96-well plates the day before experiment, cells were treated with resveratrol for various time periods. After drug treatment, attached cells were incubated with MTT and subsequetly solubilized in DMSO. The absorbency at 490nm was then measured using a microplate reader.2. Cell cycle analysis was performed by Flow cytometryAfter resveratrol treatment for 24 hours, 1×10⁶ cells were collected and fixed overnight in 70% ethanol at 4℃. Samples were labeled with propidium iodide (PI) solution containing 0.1% (v/v) Triton X- 100, 0.1% (w/v) sodium citrate, and 50ug/ml PI at 4℃for 30 minutes in the dark and analyzed by flow cytometry (BD biosciences).3. Cell apoptosis assays was performed by Flow cytometryThe gastric cancer cells at the peak of their growth were incubated for 24 hours in the presence or absence of 12.5, 25, 50, 100 or 200μmol/L resveratrol. Then the cells were collected and washed twice with cold PBS and resuspended in binding buffer at a concentration of 1×10⁶cells/mL. After incubation, 100μL of solution was transferred to a 5-mL culture tube, and 5μLannexin V-FITC and 10μLPI were added. The tube was incubated for 15 minutes at room temperature in the dark. At the end of incubation, 400μL bingding buffer was added, and the cells were analyzed immediately by flow cytometry. Flow cytometric analysis was performed with a FACACaliber using the CellQuest software.4. Measurement of mitochondrial membrane potentialChanges of mitochondrial membrane potential (Δψm) were monitored by determination of the 5,5', 6,6'- tetrachloro -1,1'3,3' -tetraethyl- benzimi- dazolyl-carbocyanine iodide (JC-1) staining. After treatment, cells were loaded with JC-1 solution and incubated at 37℃for 30minutes in the dark. Cells were the harvested, washed and resuspended in PBS and analyzed immediately using cytometry with the exitation and emission wavelength of 488 and 530nm, respectively.5. Effect of CsA on cell apoptosis induced by resveratrolCyclosporin A (CsA) is a substance that blocks the opening of the mitochondrial permeability transition pore (PTP). Cells were preincubated with 10ug/mL cyclosporin A (CsA) for 30 minutes before stimulation with 100umol/L resveratrol for 24 hours and apoptosis was examined by Flow cytometry.6. Expression of Survivin, caspase-3, Smac protein were detected by Western-blot assay7. Reverse transcription-PCR was used to detect transcriptional regulation of Survivin, caspase-3 and Smac8. Caspase-3 activity assayThis assay was done using an colorimetric caspase assay kit. Cells (1×10⁶) were plated into 6 cm dished and subjected to various treatments. Cells were then collected and lysed in lysis buffer provided in the kit. Caspase-3 activity was measured at a wavelength of 405nm.9. Statistical AnalysisData were presented as means±SD. Student's t test was used for analyzing differences between groups, a P value＜0.05 was considered signifiant.Results1. Effects of resveratrol on the proliferation of gastric cancer cell lines. Resveratrol of 25μmol/L inhibited the proliferation of SGC7901 and BGC823 cells after treating cells for 48 hours and 72 hours (P＜0.05). After exposed to 50-300μmol/L resveratrol for 24 hours, the growth of two gastric cancer cell lines were significantly inhibited in dose-dependent manner (P＜0.01). When cells were incubated with resveratrol for from 24 hours to 72 hours, cell viability was decreased in a time-dependent manner.2. Effect of resveratrol on the cell cycle status of SGC7901, BGC823 cellsThe percentage of cells in the G₁, G₂ and S phases of the cell cycle in untreated SGC7901 cells were 81.63%±1.38% , 13.55%±0.87% and 4.81%±1.50%. After exposed to 12.5,25,50,100μmol/L resveratrol for 24 hours, the rates of cells in the G₁ phase were 77.04%±2.95%, 73.19%±7.60%, 66.66%±2.31% (P＜0.01), 61.39%±1.62% (P＜0.01), respectively, the rates of cells in the G₂ phase were 4.31%±2.63%, 5.25%±2.55%, 4.65%±3.33%, 0.05%±0.07%(P＜0.01), respectively, the rates of cells in the S phase were 21.98%±9.37%, 21.58%±5.52%, 28.70%±3.25% (P＜0.01), 38.77%±1.41% (P＜0.01), respectively. The percentage of cells in the G₁, G₂ and S phases of the cell cycle in untreated BGC823 cell line were 67.29%±5.83%, 5.77%±1.69% and 27.04%±4.45%. After exposed to 12.5,25,50,100μmol/L resveratrol for 24 hours, the rates of cells in the G₁ phase were 63.70%±1.60%, 65.76%±3.54%, 59.21%±6.29%, 34.60%±2.79% (P＜0.01), respectively, the rates of cells in the G₂ phase were 3.30%±2.53%, 3.92%±3.25%, 0.70%±0.53% (P＜0.01), 0.74%±1.05% (P＜0.05), respectively, the rates of cells in the S phase were 32.67%±3.81%, 30.17%±4.88%, 40.09%±6.79% (P＜0.05), 64.66%±1.89% (P＜0.01), respectively. Resveratrol in the concentration of 50, 100μmol/L induced the accumulation of gastric cancer cells in the S phase of the cell cycle.3. Effect ofresveratrol on apoptosis of SGC7901, BGC823 cellsThe percentage of cells undergoing apoptotic cell death and the percentage of cells undergoing necrotic cell death in control SGC7901 cells were 2.74%±0.32% and 5.10%±0.75%, respectively. Compared with the control group, after exposed to 12.5,25,50,100,200μmol/Lresveratrol for 24 hours, the percentage of SGC7901 cells undergoing apoptotic cell death gradually increased, the percentage of apoptotic cells were 5.55%±2.15%, 12.67%±4.68%(P＜0.05),20.60%±4.54%(P＜0.01), 52.94%±8.36% (P＜0.01), 69.60%±11.20% (P＜0.01), respectively, the percentage of cells undergoing necrotic cell death were 8.54%±1.66%, 10.99%±3.88%, 9.39%±1.46%, 17.13%±6.26% (P＜0.05), 22.37%±9.17% (P＜0.05), respectively. The percentage of cells undergoing apoptotic cell death and the percentage of cells undergoing necrotic cell death in control BGC823 cells were 3.36%±0.41% and 5.43%±1.49%. Compared with the control group, after exposed to 12.5,25,50,100,200μmol/L resveratrol for 24 hours, the percentage of BGC823 cells undergoing apoptotic cell death gradually increased, the percentage of apoptotic cells were 4.57%±0.79%, 5.81%±1.91%, 18.59%±6.29% (P＜0.05), 35.35%±9.01% (P＜0.01), 56.12%±22.86% (P＜0.01), respectively, the percentage of cells undergoing necrotic cell death were 5.74%±0.96%, 6.88%±1.55%, 9.22%±1.82% (P＜0.01), 14.64%±5.01% (P＜0.05), 19.51%±6.12% (P＜0.01), respectively. Results showed that resveratrol induced a dose-dependent apoptosis in both cell lines.4. Effect of resveratrol on mitochondrial membrane potential (Δψm)The loss ofΔψm was presented by the decreased percentage of red light by flow cytometry assay. The numbers of cells with loss of or reducedΔψm were 3.53%±0.92% and 4.20%±0.43% in untreated SGC7901 and BGC823 cell lines. After exposed to 100μmol/Lresveratrol in various time point, the number of SGC7901 and BGC823 cells with loss ofΔψm were 13.93%±2.53% and 13.60%±2.11%(P＜0.01) at 6h post-treatment, 20.52%±2.41% and 18.89%±3.32% (P＜0.01) at 12h posttreatment, 38.87%±4.86% and 38.2%±7.87%(P＜0.01) at 18 h post-treatment, 52.2%±9.21% and 47.87%±8.54% (P＜0.01) at 24h post-treatment. This suggested that the loss ofΔψm of the gastric cancer lines began after treated with resveratrol for 6 hours, and further increased for 24 hours. After exposed to 12.5,25,50,100μmol/L resveratrol for 24 hours, the numbers of SGC7901 cells and BGC823 cells with loss ofΔψm were 6.62%±1.37% and 5.79%±0.52%, 12.02%±2.92% and 10.76%±2.50%, 22.82%±7.96% and 22.48%±4.82%, 52.2%±9.21% and 47.87%±8.54%, and were significantly higher than those of control group (3.53±0.92% and 4.20±0.43%, P＜0.01).5. Effect of CsA on SGC7901 and BGC823 cell apoptosis induced by resveratrolCells were preincubated with 10ug/mL CsA for 30 minutes before stimulation with 100umol/L resveratrol for 24 hours and apoptosis was examined by Flow cytometry. Compared with the resveratrol treated cells (52.94%±8.36%å’Œ35.35%±9.01%), the rates of apoptotic cell in CsA+ Res groups significantly decreased both in SGC7901 and BGC823 cells (28.61%±4.27%, and 19.08%±5.40%, P＜0.05). But the percentage of apoptotic cells still were higher than those of untreated cells (2.74%±0.32% and 3.36%±0.41%, P＜0.01).6. Effect of resveratrol on the expression of SurvivinTo confirm the modulation of Survivin expression by Resveratrol at the mRNA levels and protein levels, semi- quantitative RT-PCR and Western blotting at 24 hours after the treatment of Resveratrol were performed. We found that there was a marked decrease in both the mRNA and protein expression of Survivin within 24 hours in a dose- dependent manner.7. Effect of resveratrol on the expression of SmacWithin 24 after exposure to 12.5, 25, 50, 100μmol/L resveratrol, the levels of Smac protein expression in cell cytosol in treated cells became gradually increased. The expression of Smac mRNA in BGC 823 cell lines became gradually increased in a dose- dependent manner, but the expression of Smac mRNA in SGC 7901 cell lines was not significantly different between groups.8. Effect of resveratrol on the expression of caspase-3After exposure to 12.5, 25, 50, 100μmol/L resveratrol, the levels of caspase-3 mRNA and protein expression were increased in a dose- dependent manner.9. Effect of resveratrol on the expression of the activity of caspase-3After exposed to 12.5,25,50,100μmol/L resveratrol for 24 hours, caspasc-3 activity in SGC 7901 cells were 0.073±0.011, 0.112±0.008, 0.195±0.011 and 0.245±0.006, respectively, and compared with control group, increased by 1.16- fold, 1.78-fold, 3.01-fold and 3.89 -fold. After exposed to 12.5,25,50,100μmol/L resveratrol for 24 hours, caspase-3 activity in BGC 823 cells were 0.074±0.010, 0.109±0.015, 0.188±0.015 and 0.243±0.010, respectively, and compared with control group, increased by 1.12- fold, 1.65- fold, 2.85- fold and 3.69- fold.Conclusions1. Resveratrol inhibited the proliferation of gastric cancer SGC7901 and BGC823 cell lines in dose- and time- dependent maimers.2. Resveratrol induced the accumulation of SGC7901 and BGC823 cells in the S phase of the cell cycle and induced apoptosis in a dose-dependent manner.3. Resveratrol decreased theΔψm in SGC7901,BGC823 cells in dose-dependent manner and time-dependent manner.4. The Cyclosporin A (CsA), a substance that blocks the opening of the PTP, could in partly block cell apoptosis induced by resveratrol in SGC7901,BGC823 cells. Resveratrol induced apoptosis via a mitochondria pathway.5. Resveratrol down-regulated Survivin protein and mRNA levels in SGC7901 and BGC823 cells.6. Resveratrol increased the expression of Smac protein in plasm in SGC7901 and BGC823 cells. 7. Resveratrol up-regulated the expression of caspase-3, and activated the activities of caspase-3 in cellular and induced apoptosis in SGC7901 and BGC823 cells.