Effects And Its Mechanisms Of Naringenin On Human Gastric Cancer Cell Line SGC7901

Gastric cancer(GC) is the second leading cause of cancer-related mortality worldwide and gastric cancer-related mortality accounts for ~23﹪ of all cancer-related mortalities in China. As one of the highly malignant tumors, GC is characterized by uncontrolled cell proliferation, invasion, and metastasis. Like other malignant tumors, the progression of GC is a multistep process, in which cancer cells uncontrolled growth, detach from the primary tumor, invade surrounding tissues and intravasate into blood and/or lymphatic systems. Finally, cancer cells settle and colonize at the target organs. Although chemotherapy and radiotherapy has been employed to treat GC, the prognosis of GC is generally rather poor, because most cases of GC are diagnosed in advanced stages.Traditional Chinese medicine have the characters of Convenient, inexpensive, and less toxic side effects. it has become a hot topic in medical research.Naringenin(Nar), belongs to the flavanone family, is extracted from the citrus fruits. The spectrum of biological activities of is thought to be wide, including anti-carcino-genic activity, anti-atherogenic effect, anti-inflammatory effect, anti-oxidant effect, and so on. Like other flavonoids, Nar can inhibit cancer cell growth through a series of mechanisms, such as the impairment of cell cycle progression in oral squamous cell carcinoma cell and inhibition of cell proliferation in K562 human leukemia. In addition, it has also been reported that Nar inhibits cancer cell migration, metastasis and tissue invasion. A growing number of studies provide evidence that Nar has a strong anti-cancer effect on several types of human cancer, such as bladder, hepatocellular, breast, and colorectal cancer. The cancer chemopreventive/ therapeutic effects of naringenin on an organ-specific format have been recognized and it is considered as a cancer chemopreventive/therapeutic agent. Moreover, the preliminary anti-cancer activity of Nar was also proven in GC cells. Recent studies have shown that Nar not only can inhibit beta-catenin/Tcf signaling in GC, but can up-regulate the redox status of gastric carcinomainduced rats to decrease the risk of cancer. However, the potential therapeutic effects of Nar on GC and its molecular mechanisms are not entirely elucidated yet.Based on the above information, in order to explore effects of Nar on GC, we investigated:(1) Effects of Nar on the growth and proliferation of human gastric cancer SGC7901 cells.(2) Effects of Nar on the migration and invasion of human gastric cancer SGC7901 cells.(3) Effects of Nar on apoptosis of human gastric cancer SGC7901 cells.(4) Effects of Nar on AKT signaling pathway in human gastric cancer SGC7901 cells. Finally, it was clarified that Nar could significantly inhibit the proliferation, migration and invasion of human gastric cancer SGC7901 cells, and induce cell apoptosis. These effects are related to the phosphorylation of AKT and the expression of the downstream target factors.Our study Provide experimental and theoretical basis for further clinical application. Part 1 Effects of Nar on the growth and proliferation of human gastric cancer SGC7901 cellsObjective: To investigate Effects of Nar on the growth and proliferation of human gastric cancer SGC7901 cells. clarify the inhibitory effects of Nar on the growth and proliferation of human gastric cancer SGC7901.Methods: SGC7901 cells were purchased from Cell Resource Center of Life Sciences(Shanghai, China) and grown in RPMI 1640 medium containing 10% FBS, 100 units/m L penicillin, and 100 mg/m L streptomycin. Cultures were maintained in a humidified atmosphere of 5% CO2 at 37 ℃and were passaged 2-3 times a week by treating with 0.25% trypsin containing 0.02% EDTA.Growth rate and Proliferation activity of human gastric cancer SGC7901 cells was determined by MTT. First, SGC7901 cells were incubated at a density of 1×104 cells/well in 96-well plates in six duplications for 24 h. Then, SGC7901 cells, which was treated with various concentrations(0, 5, 10, 20, 40, 80, 160 μmol/L) of Nar for 48 h or with 40μmol/L Nar for 0, 24, 48 and 72 h, were used for experiment. The growth rate of SGC7901 cells was observed.In addition, we detected the effects of Nar on the expression of PCNA protein for human gastric cancer SGC7901 cells using Western-blot. After treated with Nar(0, 20, 40, 80μmol/L) for 48 h, the SGC7901 cells were used for experiment. To observe the changes of the proliferation activity of human gastric cancer SGC7901 cells.Results:1 The results of MTT showed that the growth rate were not significantly altered in 5 μmol/L Nar treated SGC7901 cells. However, Nar(10, 20, 40, 80 and 160 μmol/L) caused progressive reduction on growth rate of SGC7901 cells with a dose-dependent manner(P<0.05). After SGC7901 cells were treated with 40 μmol/L Nar for 0, 24, 48 or 72 h, the proliferation of SGC7901 cells were decreased with a time-dependent manner(P<0.05).2 The results of Western-blot showed that Nar(0, 20, 40, 80μmol/L) inhibited the protein expression levels of PCNA of SGC7901 cells with a dose-dependent manner(P<0.05).Conclusions: Nar could inhibit SGC7901 cells growth and proliferation with a time- and dose-dependent manner. Part 2 Effects of Nar on the migration and invasion of human gastriccancer SGC7901 cellObjective: To investigate the effects of Nar on on the migration and invasion of human gastric cancer SGC7901 cells. and to explore the role of Nar in the migration and invasion of human gastric cancer SGC7901 cells and its possible mechanism.Methods: The cell culture method is the same as the first part.The effects of Nar on the migration of human gastric cancer SGC7901 cells was detected by the wound-healing assay. SGC7901 cells were seeded(5×104cells/well) into 12-well plates and grown to 80~90% confluence for the experiment. Monolayer cells were wounded by scratching with 200-μL sterile pipette tips and washed twice with 1×PBS. And then, cells were treated with Nar(0, 20, 40, 80μmol/L) for 48 h or with 40μmol/L Nar for 24, 48 and 72 h. The cell migration activity was expressed as the number of cells migrating into the wound.Cell invasion ability of human gastric cancer SGC7901 cells was detected in transwell invasion assay. After treated with Nar(20, 40, 80μmol/L) for 48 h or with 40μmol/L Nar for 24, 48 and 72 h. SGC7901 cells were used for experiment.In addition, we detected the effects of Nar on the expression of MMP2, MMP9, TIMP1, TIMP2 m RNA and protein for human gastric cancer SGC7901 cells using Western-blot or real-time quantitative RT-PCR assays. After treated with Nar(0, 20, 40, 80μmol/L) for 48 h,the SGC7901 cells were used for experiment.Results:1 The results of wound-healing assay showed that Nar(0, 20, 40, 80μmol/L) caused progressive reduction on migration of SGC7901 cells with a dose-dependent manner(P<0.05). After SGC7901 cells were treated with 40 μmol/L Nar for 24, 48 or 72 h, the migration of SGC7901 cells were decreased significantly compared with the control groups(P<0.05).2 The results of transwell invasion assay showed that Nar(0, 20, 40, 80μmol/L) caused progressive reduction on invasion of SGC7901 cells with a dose-dependent manner(P<0.01). After SGC7901 cells were treated with 40 μmol/L Nar for 24, 48 or 72 h, the invasion of SGC7901 cells were decreased significantly compared with the control groups(P<0.01).3 The results of Western-blot or real-time quantitative RT-PCR assays showed that Nar(0, 20, 40, 80μmol/L) inhibited the m RNA and protein expression levels of MMP2 and MMP9 of SGC7901 cells with a dose-dependent manner(P<0.05), while there were no significant changes for TIMP1 and TIMP2 expression.Conclusions: Nar could inhibit the migration and invasion of human gastric cancer SGC7901 cells, and reduce the expression of MMP2 and MMP9. These results suggested that Nar may play an inhibitory regulatory role in the migration and invasion of human gastric cancer SGC7901 cells by decreasing the expression of MMP2 and MMP9. Part 3 Effects of Nar on apoptosis of human gastric cancer SGC7901 cellsObjective: To investigate the effects of Nar on apoptosis of human gastric cancer SGC7901 cells, and to explore the possible mechanism of Nar on the apoptosis of human gastric cancer SGC7901 cells.Methods: Flow cytometry was used to detect the apoptosis rate of human gastric cancer SGC7901 cells. After treated with Nar(0, 20, 40, 80μmol/L) for 48 h or with 40μmol/L Nar for 24, 48 and 72 h, SGC7901 cells were incubated with Annexin V-FITC and were used for experiment.In addition, we detected the effects of Nar on the m RNA and protein expression of Bax,Bcl-2,Survivin and Cleaved-Caspase-3 for human gastric cancer SGC7901 cells using Western-blot or real-time quantitative RT-PCR assays. After treated with Nar(20, 40, 80μmol/L) for 48 h,the SGC7901 cells were used for experiment.Results:1 The results of flow cytometry showed that Nar(0, 20, 40, 80μmol/L) increased apoptosis rate of SGC-7901 cells in a dose-dependent manner(P<0.05). After SGC7901 cells were treated with 40 μmol/L Nar for 24, 48 or 72 h, the apoptosis rate of SGC7901 cells were significantly increased compared with the control groups(P<0.05).2 The results of Western-blot or real-time quantitative RT-PCR assay showed that Nar(20, 40, 80μmol/L) up-regulated the m RNA and protein expression of Bax and Cleaved-Caspase-3(P<0.05), while the m RNA and protein expression of Bcl-2 and Survivin were significantly decreased(P<0.05).Conclusions: Nar could promote the apoptosis of human gastric cancer SGC7901 cells. It was suggested that Nar could promote apoptosis of SGC7901 cells by increasing the SGC7901 cells apoptosis factor Bax and Cleaved-Caspase-3 m RNA and protein expression, inhibitting apoptosis factor Bcl-2 and Survivin m RNA and protein expression. Part 4 Effects of Nar on AKT signaling pathway in human gastriccancer SGC7901 cellsObjective: To investigate the effects of Nar on the signal transductions of AKT and confirm whether Nar inhibited the proliferation, migration and invasion of human gastric cancer SGC7901 cells, as well as increased apoptosis through down-regulating AKT signaling pathway.Methods: The cell culture method is the same as the first part.First, SGC7901 cells were treated with Nar(0, 20, 40, 80μmol/L) for 24, 48 or 72 h, and then total protein lysates of each sample was collected and then subjected to western blotting with AKT and p-AKT(phosphorylation of AKT) antibodies.Second, SGC7901 cells was divided into four groups as follows: 1 group(Nar-, LY294002-); 2 group(Nar-, LY294002+); 3 group(Nar+, LY294002-); 4 group(Nar+, LY294002+). SGC7901 cells were pretreated with 50 μmol/L AKT inhibitor LY294002 for 2h and incubated with Nar(40 μmol/L) for 48 h. Cells were collected and subjected to Western-blot assay to detected the protein expression of PCNA, Bax, Bcl-2, MMP2, MMP9, TIMP1, TIMP2, AKT and p-AKT. Cell growth rate was detected by MTT. Cell migration and invasion activities were detected by wound healing and transwell assay. Cell apoptosis was detected by flow cytometry. The activities related factors of AKT signaling pathway, PCNA, Bax, Bcl-2, MMP2, MMP9, TIMP1, TIMP2, AKT and p-AKT were detected by Western-blot assayResults:1 The effects of Nar on the expression of AKT and its phosphorylation of human gastric cancer SGC7901 cells.The results of Western-blot showed that Nar inhibited phosphorylation of AKT in a dose- and time-dependent manner, whereas Nar had no obvious effects on the AKT protein levels.2 The effects of Nar and AKT inhibitor LY294002 on the growth of human gastric cancer SGC7901 cells.The results of MTT showed that the growth rate of SGC7901 cells was decreased by the single treatment with Nar or LY294002(P<0.01), but markedly decreased by the combination of treatment(P<0.01).3 The effects of Nar and AKT inhibitor LY294002 on the migration and invasion of human gastric cancer SGC7901 cells.The results of wound-healing assay showed that the migration of SGC7901 cells was decreased by the single treatment with Nar or LY294002(P<0.01), but markedly decreased by the combination of treatment(P<0.01).The results of transwell invasion assay showed that the invasion of SGC7901 cells was decreased by the single treatment with Nar or LY294002(P<0.01), but markedly decreased by the combination of treatment(P<0.01).4 The effects of Nar and AKT inhibitor LY294002 on the apoptosis of human gastric cancer SGC7901 cells.The results of flow cytometry showed that the apoptosis of SGC7901 cells was increased by the single treatment with Nar or LY294002(P<0.01), but markedly increased by the combination of treatment(P<0.01).5 The effects of Nar and AKT inhibitor LY294002 on the related factors of human gastric cancer SGC7901 cells proliferation, migration, invasion and apoptosis as well as the expression of AKT and its phosphorylationTo further determine the mechanisms of synergistic effect induced by the combination treatment of Nar and LY294002 on SGC-7901 cells, the protein expression of PCNA, Bax, Bcl-2, MMP2, MMP9, TIMP1, TIMP2, AKT and p-AKT were examined by Western-blot.The results of Western-blot showed that the protein expression of Bax partly increased by the single treatments with Nar or LY294002(P<0.05), but markedly increased by the combination of treatment(P<0.05). The protein expression of PCNA, Bcl-2, MMP2, MMP9, and phosphorylation of AKT partly decreased by the single treatments with Nar or LY294002(P<0.05), but mildly decreased by the combination of treatment(P<0.05). However, total AKT protein level was not changed under the same treatment conditions. Also there were no significant changes for TIMP1 and TIMP2 levelsConclusions: Nar could inhibited phosphorylation of AKT in human gastric cancer SGC7901 cells. combination of LY294002 and Nar could synergistically inhibited phosphorylation of AKT. Nar Inhibited SGC7901 cells proliferation, migration, invasion and apoptosis may through inhibiting phosphorylation of AKT and down-regulating AKT signaling pathway.