| Objectives:1. To observe the effect of piperine on gastric cancer cell’s proliferation viability in vitro.2. To observe the effect of piperine on gastric cancer cell’s apoptosis, as well as discussing its molecular mechanism.3. To observe piperine’s effect on cell cycle of gastric cancer cells and discussing its molecular mechanism。Method:1. To incubate gastric cancer cell lines HGC-27, SGC-7901, and BGC-823in RPMI1640culture medium with10%fetal serum. Piperine of different concentration (0μmol/L～200μmol/L) are added to the medium, and allowed to stand for24hours,36hours and48hours. CCK-8is applied to examine piperine’s effect on human gastric cancer cells’ proliferation in vitro.2. HGC-27cell line is incubated in medium with10%veal serum. After different level of piperine’s acting on the medium for24hours, Hoechst33258staining is applied to examine piperine’s effect on enhancing HGC-27’s apoptosis. Also, Annexin V and PI double staining is applied with flow cytometry to examine piperine’s effect on HGC-27’s apoptotic capacity. Western Blot is also applied in order to check pierine’s effect on apoptotic protease (caspase), anti-apoptotic protein (Bcl-2), and apoptosis enhancing protein (Bax) level.3. HGC-27cells are planted in6-well plate, and cell synchronization is achieved by serum starvation. Piperine of assorted concentrations are to act on the cells24hours after starvation, and flow cytometer is used to inspect piperine’s effect on cell cycle progression. Meanwhile, Western Blotting is applied for cell cycle proteins including Cyclin A, Cyclin Bl and cell cycle inhibitor p21level change.4. Western Blotting is applied for assessment on piperine’s effect on Akt level and its phosphorylation level in HGC-27cell line.Results:1. CCK-8analysis suggests that piperine inhibited the proliferation of HGC-27, BGC-823, and SGC-7901cell obviously and the effects are both dose-and time-dependent. The IC50of piperine to HGC-27cells at24and48hours are96.73μmol/L and41.34μmol/L, respectively. To BGC-823cells, piperine’s IC50at24 and48hours are97.01μmol/L and15.68μmol/L. For SGC-7901cells, IC50at24and48hours are89.98μmol/L and19.60μmol/L, respectively.2. Annexin V and PI double staining suggests that piperine is effective in inducing apoptosis in human gastric cancer cells. In comparison to blank control,24hours after treatment, HCG-27cells’ apoptotic rate increased to17.09%(p<0.05).3. Hoechst3325staining analysis shows that in piperine groups, apoptotic bodies containing nuclear fragments were generated in apoptosis cells. While in control group, there was little distinct morphological features of apoptotic cells.4.Western blot analysis shows that piperine may up-regulate apoptosis promoting cleaved Caspase-3(p<0.05), while having no obvious inference on Bcl-2and Bax expression in HGC-27cells.5.Cell cycle examination results suggest that piperine may cause HGC-27cells arrest in G2/M phase. In contrast to blank control, distribution ratio of cells at G2/M phase increased to21.98%±6.89(p<0.05).6. Western Blot suggest that piperine may lower cyclin A and cyclin B1expression in human gastric cancer, while have no obvious effect on cyclin-dependent kinase inhibitor p21.Conclusion:1.Piperin has the capacity of inhibiting HGC-27, BGC-823and SGC-7901proliferation in vitro2. Piperine may active apoptosis protein kinase caspase-3and induce HGC-27cell apoptosis in vitro.3. Piperin may arrest HGC-27cell cycle progression, which may be related to down-regulation of cyclin A, cycline B. Purpose:1. Observe the synergic effect of piperine and cisplatin on proliferation of human gastric cancer cell line.2. Analyze the synergic effect of piperine and cisplatin on apoptosis of human gastric cancer cell line (BGC-823)3. To explore the synergic effect of piperine combined with cisplatin on PI3K/Akt signal transduation pathway in human gastric cancer cell line (BGC-823)Method:1. The human gastric cancer cells were cultured with RPMI.1640medium containing10%fetal bovine serum. Piperine were added at dose of75μM,5μM cisplatin, or piperine in combination with cisplatin. After24h treatment, human gastric cancer cell proliferation in each group then was detected with CCK-8assay.2. The human gastric cancer cell line BGC-823cells were cultured in medium containing10%fetal bovine serum RPML1640. Cells were treated with Piperine75μM, cisplatin5μM or piperine75μM in combination with cisplatin5μM for24hours, Hoechst33258staining was used to detect the role of synergic effect of piperine and cisplatin on apoptosis of gastric cancer cells, and Annexin V/PI double labeling apoptosis detection analysis was used to detected apoptosis rate of cells in each group.3. Further study of synergic effect of piperine and cisplatin was detected by Western blot, including: Caspas-3, Cleaved-Caspas-3, Bax, Bcl-2and pAkt/Akt were detected for analysis.Results:1. CCK-8analysis showed that piperine could significantly inhibit proliferation of human gastric HGC-27cells; When human gastric HGC-27cells were treated with piperine combined with cisplatin, a significant synergic effect was found in inhibition of proliferation.2. Annexin V/PI double labeling apoptosis detection analysis showed that both of monotherapy group and the combination showed apoptotic effect in human gastric cancer BGC-823cells. Compared with piperine and cisplatin monotherapy group, piperine combined with cisplatin showed a significant synergic effect on apoptosis (p<0.05). 3. Western blot analysis showed that pipeline could increase pro-apoptotic protein Bax (p<0.05) and apoptosis protease caspase-3(p<0.05), and downregulated anti-apoptotic protein Bcl-2gastric (p<0.05) in cancer BGC-823cells;4. Compared to control group, cisplatin showed no significant difference in impact of on phosphorylation of Akt, while piperine combined with cisplatin significantly inhibit phosphorylation of Akt, indicating that pretreatment with piperine could inhibit activation of Akt.Conclusion:1. Piperine enhances proliferation inhibitory activity of cisplatin on human gastric cancer cells.2. Piperine increased cytotoxicity of cisplatin, which may be related to up-regulating pro-apoptotic protein Bax and down-regulating anti-apoptotic Bcl-2proteins and promoting apoptosis of gastric cancer cell.3. Piperine combined with cisplatin may reduce Akt phosphorylation levels in BGC-823cells, which may result in up-regulating the pro-apoptotic proteins, thereby, inhibiting cell proliferation. |
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