Alisol B acetate induces apoptosis of SGC7901 cells via mitochondrial and phosphatidylinositol 3-kinases/Akt signaling pathways

AIM： To examine the effect of alisol B acetate on the growth of human gastric cancer cell line SGC7901 and its possible mechanism of action.METHODS： The cytotoxic effect of alisol B acetate on SGC7901 cells was measured by 3-（4,5-dimethylthiazol- 2-yl）-2,5-diphenyltetrazolium bromide （MI-I-） assay. Phase-contrast and electron microscopy were used to observe the morphological changes. Cell cycle and mitochondrial transmembrane potential （A^Pm） were determined by flow cytometry. Western blotting was used to detect the expression of apoptosis-regulated gene Bcl-2, Bax, Apaf-1, caspase-3, caspase-9, Akt, P-Akt and phosphatidylinositol 3-kinases （PI3K）.RESULTS： Alisol B acetate inhibited the proliferation of SGC7901 cell line in a time- and dose-dependent manner. PI staining showed that alisol B acetate can change the cell cycle distribution of SGC7901, increase the proportion of cells in G0-G1 phase and decrease the proportion of S phase cells and G2-M phase cells. Alisol B acetate at a concentration of 30 pmol/L induced apoptosis after 24, 48 and 72 h incubation, with occurrence rates of apoptotic cells of 4.36%, 14.42% and 21.16%, respectively. Phase-contrast and electron microscopy revealed that the nuclear fragmentation and chromosomal condensed, cells shrank and attachment loss appeared in the SGC7901 treated with alisol B acetate. Apoptosis of SGC7901 cells was associated with cell cycle arrest, caspase-3 and caspase-9 activation, loss of mitochondrial membrane potential and up-regulation of the ratio of Bax/Bcl-2 and inhibition of the PI3K/Akt.CONCLUSION： Alisol B acetate exhibits an antiproliferative effect in SGC7901 cells by inducing apoptosis. Apoptosis of SGC7901 cells involves mitochondria-caspase and PI3K/Akt dependent pathways.

Reversal of P-glycoprotein-mediated Multidrug Resistance in SGC7901/VCR Cells by PPARγ Activation by Troglitazone

Over-expression of P-glycoprotein(P-gp),an ATP-dependent drug efflux pump,represents one of the major mechanisms that contribute to multidrug resistance(MDR) in cancer cells.This study examined the effects of troglitazone,a ligand of peroxisome proliferator-activated receptor gamma(PPARγ),on P-gp-mediated MDR in SGC7901/VCR cells(a vincristine-resistant human gastric cancer cell line).The expression of P-gp was detected by RT-PCR and Western blotting,respectively.The SGC7901/VCR cells were treated with 0.1 mg/L vincristine(VCR) alone or in combination with 1,5,10 μmol/L troglitazone for 24 h.PPARγ was measured by electrophoretic mobility shift assay(EMSA).The intracellular concentration of Rhodamine123(Rh123,a fluorescent P-gp substrate) was assayed to evaluate the activity of P-gp.The cell cycle and apoptosis were measured by flow cytometry.The results showed that the P-gp was increasingly expressed in SGC7901,BGC823 and SGC7901/VCR cells in turn,suggesting that MDR in the SGC7901/VCR cells was mediated by the increased expression of P-gp.In the SGC7901/VCR cells,the expression level of total PPARγ was increased,however,the protein level and activity of PPARγ in the nuclei of cells decreased significantly.Troglitazone elevated the PPARγ activity in SGC7901/VCR cells in a dose-dependent manner.Troglitazone decreased the P-gp expression and markedly enhanced the accumulation of Rh123 in SGC7901/VCR cells in a dose-dependent manner.We also found that troglitazone significantly increased the percentage of SGC7901/VCR cells in the G2/M phase and decreased the cell percentage in G1 and S phase in a dose-dependent manner.Troglitazone significantly increased the apoptotic rate of SGC7901/VCR cells treated by VCR or ADR in a dose-dependent manner.It was concluded that P-gp-overexpressed SGC7901/VCR cells have minor endogenous PPARγ activity.Elevation of the PPARγ activity by troglitazone can reverse P-gp-mediated MDR via down-regulating the expression and activity of P-gp in SGC7901/VCR cells.It was suggested that troglitazone can dramatically enhance the sensitivity of P-gp-mediated MDR cancer cells to chemotherapeutic agents.

The Mechanism pf Weichang'an in Inducing Apoptosis of Gastric Cancer SGC7901 Cells

Objective:To investigate the effect of Chinese medicine Compound Weichang'an(胃肠安)for invig-orating the spleen on apoptosis of gastric cancer SGC7901 cells and its possible mechanism.Methods:The gas-trie cancer SGC-7901 cells were divided into different mass concentration groups(0 mg·L^(-1),500 mg·L^(-1)1000 mg·L^(-1),1500 mg·L^(-1),2000 mg·L^(-1)).CCK8 and monoclonal test were applied to detect prolifera-tion ability;comet assay was used to detect DNA damage.After DCFH-DA fluorescent labeling,the level of ROS activity was detected by flow cytometer;after AnnexinV-FTC/PI double labeling,the proportion of apoptotic ellls was detected by flow cytometer;after JC-1 staining,the mi tochondri almembrane potential was detected by flow cytometer;after FTTC-DEVD-FMK staining,the ratio of Caspase activity was detected by flow cytometer.Results:Weichang an inhibited cell proliferation and reduced cell colony formation in a time-dose-dependent manner;the results of comet electrophoresis showed that Weichang'an could induce DNA damage in gastric cancer cells;com-pared with control group.the ratio of Weichang'an's intervention with the apoptosis of gastric cancer cells in-creased(P<0.05),the mitochondrial membrane potential decreased(P<0.05),the activity of Caspase3 and Caspase9 increased(P<0.05),and the intracellular ROS level increased(P<0.05).Among them,the effect of Weichang'an treatment group(1000 mg·L^(-1))was the most significant.Conclusion:Weichang'an has an inhibi-tory effect on the proliferation of gastric cancer SGC7901 cells and can induce cell apoptosis.Its mechanism may be related with the ROS-mediated pathway of mitochondrial apoptosis and DNA damage.

Study on biological characters of SGC7901 gastric cancer cell-dendritic cell fusion vaccines

AIM： To detect the biological characters of the SGC7901 gastric cancer cell-dendritic cell fusion vaccines.METHODS： The suspending living SGC7901 gastric cancer cells and dendritic cells were induced to be fusioned by polyethylene glycol. Pure fusion cells were obtained by selective culture with the HAT/HT culture systems. The fusion cells were counted at different time points of culture and their growth curves were drawn to reflect their proliferative activities. The fusion cells were also cultured in culture medium to investigate whether they could grow into cell clones. MTT method was used to test the stimulating abilities of the fusion cells on T lymphocytes＇ proliferations. Moreover, the fusion cells were planted into nude mice to observe whether they could grow into new planted tumors in this kind of immunodeficiency animals.RESULTS： The fusion cells had weaker proliferative activity and clone abilities than their parental cells. When they were cultured, the counts of cells did not increase remarkably, nor could they grow into cell clones in culture medium. The fusion cells could not grow into new planted tumors after planted into nude mice. The stimulating abilities of the fusion cells on T lymphocytes＇ proliferations were remarkably increased than their parental dendritic cells. CONCLUSION： The SGC7901 gastric cancer cell-dendritic cell fusion vaccines have much weaker proliferative abilities than their parental cells, but they keep strong abilities to irritate the T lymphocytes and have no abilities to grow into new planted tumors in immunodeficiency animals. These are the biological basis for their antitumor biotherapies.

Effect of low-dose X-ray radiation on adaptive response in gastric cancer cell

Objective： We aimed to study the effect and mechanism of low-dose radiation （LDR） on adaptive response of gastric cancer cell. Methods： SGC7901 cells were cultured in vitro, and divided into 4 groups： control group （DO group）, low-dose radiation group （D1 group, 75 mGy）, high-dose radiation group （D2 group, 2 Gy）, low-dose plus high-dose radiation group （D1 ＋ D2 group, 75 mGy ＋ 2 Gy, the interval of low and high-close radiation being 8 h）. Cell inhibition rate was detected by cytometry and CCK8 method; the proportion of cell cycle at different times after irradiation was determined by using a flow cytometry. The ATM mRNA levels were detected by using quantitative real-time reverse transcription polymerase chain reaction （RT-PCR）. Results： There was no significant different between groups DO and D1, groups D2 and D1 ＋ D2 cell inhibition rate （P 〉 0.05）. There was a significant increase G2/M arrest in groups D2 and D1 ＋ D2 than groups DO and D1 after 6 h of radiation and did not recover at 48 h （P 〈 0.05）. The ATM mRNA expression of group D2 and D1 ＋ D2 increased highly than that of group DO and D1 （P 〈 0.05）. However, differences between group D2 and D1 ＋ D2, group DO and D1 were not statistical significant （P 〉 0.05）. Conclusion： LDR cannot induce adaptive response in SGC7901 cells in vitro, which may be associated the regulation of cell cycle, and its ATM mRNA expression cannot be affected by 75 mGy X-ray radiation.