[Toosendanin induces apoptosis of human gastric cancer MGC-803 cells and its mechanism].

Objective: To explore the apoptosis of human gastric cancer MGC-803 cells induced by toosendanin(TSN) and its mechanism. Methods: The human gastric cancer MGC-803 cells were divided into 5 groups, each group was set with 3 replicate. Fluorouracil (5-FU) and 0 nmol/L toosendanin (TSN) were used as positive control and negative control, respectively. The other three groups were treated with toosendanin at the final concentrations of 30, 50, and 70 nmol/L, respectively. After 48 h of treatment with toosendanin, the morphology of the cells were observed under laser confocal microscopy. Flow cytometry was used to detect the mitochondrial membrane potential, and enzyme-labeled assays were used to detect the activities of Caspase-3 and Caspase-9. The mRNA and protein levels of Bcl-2, Bax, Cyt c and APAF-1 were measured by qRT-PCR and Western blot. Results: Compared with the 0 nmol/L TSN group, after the human gastric cancer MGC-803 cells were treated with toosendanin at the concentrations of 30, 50, and 70 nmol/L for 48 h, the cell volume shrinkage, nucleus cleavage and chromatin morphological changes were observed under the microscope. The activities of Caspase-3 and Caspase-9 were increased significantly (P＜0.05), while the mitochondrial membrane potential was decreased significantly (P＜0.05). In addition, the mRNA and protein expression levels of Bax, Cyt c and APAF-1 were increased significantly (P＜0.05), while the mRNA and protein expression levels of Bcl-2 were decreased significantly (P＜0.05). Conclusion: Toosendanin up-regulates the expressions of Bax, Cyt c and APAF-1, down-regulates the expression of Bcl-2 gene, enhances the activities of caspase-3 and caspase-9, and induces the apoptosis of human gastric cancer MGC-803 cells.

[Effects of hedyotis diffusa on mitochondrial membrane potential and expressions of apoptosis-related genes in human gastric cancer cell line MNK-45].

Objective: To investigate the effects of hedyotis diffusa (injection) on mitochondrial membrane potential and expressions of apoptosis-related genes in human gastric cancer cell line MNK-45 cells. Methods: The human gastric cancer MNK-45 cells were divided into 4 groups, each group was set with 3 replicates. The control group was MNK-45 cells without added hedyotis diffusa; the 3 groups of experimental groups were treated with hedyotis diffusa at final concentrations of 20 , 30, 40 µg / ml respectively; each group was incubated for 48 h in a 5% carbon dioxide incubator, and the morphological changes of the cells were observed under a laser confocal microscope. Mitochondrial membrane potential was detected by flow cytometry. The expressions of Cytochrome C (Cyt c), caspase3 and caspase9 genes and proteins were detected by qRT-PCR and Western blot respectively. Results: Compared with the control group, the mitochondrial membrane potentials of MNK-45 cells were significantly reduced in the hedyotis diffusa treated groups at final concentrations of 20, 30, and 40 µg / ml (P＜0. 01). The gene expressions of Cyt c, caspase3, and caspase9 were significantly up-regulated (P＜0. 01) and their protein expressions were also significantly increased (P＜0. 05 or P＜0. 01). The 40 µg / ml hedyotis diffusa treatment group performed best. Conclusion: In the final concentration range of 20 ~ 40 µg / ml, hedyotis diffusa can reduce human gastric cancer MNK-45 cells mitochondrial membrane potential, induce apoptosis and up-regulate Cyt c, caspase3 and caspase9 gene expressions.

[Effects of Toosendanin on the formation of CTPS cytoophidium in human gastric cancer cell MKN-45 and its mechanism].

Objective: To investigate the relationship between toosendanin(TSN) and CTP synthase(CTPS) cytoophidium formation in gastric cancer MKN-45 cells. Methods: In this study, the experimental material is MKN-45 human gastric cancer cells. It contains 7 treatment groups of 0, 20, 40, 60, 80, 100, and 120 nmol/L TSN. Each group was treated in triplex privately for 24、48 and 72 hours. Cell counting kit-8 (CCK8) was used to detect the inhibitory effect of TSN on the proliferation of MKN-45 cells. After immunofluorescence detection, the morphology of CTPS cells was observed by a laser confocal microscope. The effect of TSN on MYC gene expression was detected by qRT-PCR. In addition, it contains 2 treatment groups of 1 mmol/L DON and 1 mmol/L MPA, each group was treated in triplex privately for 6 hours and then the cytoophidium morphology was detected by immunofluorescence. Results: The results of immunofluorescence showed that CTPS formed a filamentous cytoophidium structure after treating MKN-45 cells with 1 mmol/L DON and 1 mmol/L MPA, which means that the cells have the ability to form CTPS cytoophidium; The cell proliferation rate of TSN treatment group was significantly lower than that of 0 nmol / L TSN group (P＜0.01); Immunofluorescence results showed that CTPS cytoophidium was the most abundant in MKN-45 cells after treated with 80 nmol/L TSN for 72 h. The results of qRT-PCR showed that MYC expression in cells was significantly decreased after treated with 80 nmol/L TSN for 24 h (P＜0.05), and MYC expression was significantly increased after 48 h (P＜0.01), and then decreased. Conclusion: Toosendanin may affect intracellular cytoophidium assembling by regulating the expression of MYC.

Reversal of MDR1 gene-dependent multidrug resistance in HL60/HT9 cells using short hairpin RNA expression vectors.

Multidrug resistance (MDR) is a serious obstacle to cancer chemotherapy. Overexpression of P-glycoprotein (P-gp), the MDR1 gene product, confers MDR to tumor cells. This study explored the possibility of reducing drug resistance by targeting the mdr1 gene using short hairpin RNA (shRNA). Two different shRNAs were designed and constructed in a pSilencer 2.1-U6 neo plasmid. The shRNA recombinant plasmids were transfected into HT9 leukemia cells. Real-time polymerase chain reaction and Western blotting were used to characterize the inhibited expression of MDR1 mRNA and P-gp, and the drug sensitivity of the transfected cells was assessed using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. The results indicated that the inhibition of P-gp expression by small interfering RNA selectively restored sensitivity to the drugs transported by P-gp. Evaluation of chemosensitivity showed 52.58% reversal by p2.1-shRNA1 and 73.07% reversal by p2.1-shRNA2 in drug resistance for harringtonine, and 84.87% reversal by p2.1-shRNA1 and 94.23% reversal by p2.1-shRNA2 in drug resistance for curcumin in the transfected cells. The results demonstrated the efficacy and selectivity of shRNA in reversing MDR in drug-resistant HT9 cells in vitro.