| Clinical and experimental data more and more shows, breast cancer is one of the common malignant tumors in China, is also a focus on the prevention and treatment of malignant tumors in china. The second leading cause of death in malignant tumors, only second to gastric cancer and esophageal cancer. Surgical operation is the only effective method for the treatment of early breast cancer, but most patients has reached later in the course of operation, curative effect is limited, so finding new treatments are particularly important. With the in-depth study of the mechanism of carcinogenesis, explore the method of inducing apoptosis and cell cycle of tumor cell block becomes the new ideas to improve clinical curative effect. At present, the clinical treatment of tumor appeared resistance, multidrug resistance (MDR) refers to tumor cells to1kind of antitumor drug, drug resistance at the same time, different to the other structure, targeting different antitumor drugs have drug resistance. With the wide application of chemotherapy, tumor drug resistance problem is becoming more and more serious, which has become one of the major barriers to effective treatment of tumor. The resistant mechanisms of tumor is started from the MDR gene product expression, to explore the mechanism of drug resistance caused by such products. The main P-glycoprotein (P-gP), intracellular glutathione (GSH) and glutathione transferase (GST), DNA topoisomerase Ⅰ, Ⅱ (TOPO Ⅰ, Ⅱ), multidrug resistance associated protein (MRP) and lung resistance protein (LRP).The research of a new compound ophiobolin (ophoiobolin) on MCF-7/ADR breast cancer resistant cells, ophiobolin S is screened out12from the Ocean University of China provided microbial extract of favorable antitumor activity compounds, which belongs to the ophiobolin two sesquiterpene compounds, its molecular weight was434.39. Domestic and foreign literatures have not reported antitumor activity of this compound. This paper selects the resistance to adriamycin in human breast cancer cell line MCF-7/ADR as the experimental object, to study the effect of Ophiobolin O on the proliferation, cycle and apoptosis and its mechanism of action.Objective:To study the effect of Ophiobolin O on MCF-7/ADR cell proliferation, cycle and apoptosis and its mechanism of action.Methods:MTT assay was used to detect the effect of Ophiobolin O on human breast cancer eell line MCF-7human breast cancer cell line MCf-7/ADR proliferation; flow cytometry was used to Propidium Iodide (PI) single standard effect method for the detection of Ophiobolin O in MCF-7/AD cell cycle progression; change detection based on Real-time PCR for resistance of mRNA expression by flow cytometry; Annexin V-FTTC/PI double labeling and PI single labeled sub-Gl (cell hypodiploid apoptotic peak) was used to detect apoptosis rate; changes in cell morphology was observed by Ophiobolin O and ADM after the application of Hoechst33342and DAPI fluorescence staining; the expression level of Western protein and blotting detection of apoptosis by flow cytometry cycle; JC-1staining was used to detect the mitochondrial membrane potential (△ψ m) level; the active oxygen fluorescent probe for detecting DCFH-DA marker flow cytometry intracellular (reactive oxygen species, ROS); the expression of Western protein in apoptosis pathway in detection of blotting; NAC and ROS inhibitor JC-1and ROS levels, animal experiment in vivo detection of Ophiobolin O and ADM in the tumor inhibition rate; transfection experiment detection of intracellular MDR1gene promoter leve.Results:Ophiobolin O could inhibit the proliferation of MCF-7/ADR cells, and its effect is concentration dependent, in the role of MCF-7/ADR cells after48h, ADMIC50.6.67±0.16μ M0.1μ M of MCF-7/ADR cells after Ophiobolin O flow cytometry cell cycle progression and sub-Gl results show that, compared with the control group, with the concentration increased, the percentage of cells in G2/M phase gradually increased (p<0.05). and the percentage of cells in S phase decreased (p<0.05), the percentage of cells in GO/Gl phase remained basically unchanged, showed that Ophiobolin O+ADM cells were arrested in G2/M phase, suggesting that Ophiobolin O has induced the apoptosis of MCF-7/ADR cells. Results the rate of apoptosis was detected by flow cytometry and Annexin V VPI were also showed with the drug action time prolonged, early apoptosis cells (Annexin V+VPI-) to total cell number is increasing (p<0.05), further proved that Ophiobolin O could induce apoptosis of MCF-7/ADR for. The morphological changes of Hoechst33342and DAPI fluorescent dye detection cells showed typical apoptotic morphology visible change Ophiobolin O role of MCF-7/ADR cells after48h, the performance of some cells shrink, cytoplasm condensed, nuclear chromosome condensed highly, marginalization. nuclear fragmentation was fragmented, visible body crescent apoptosis. and normal control groups of intact nucleus. Induction of Ophiobolin () apoptosis in MCF-7cells associated with the following a series of intracellular events:(1) decreased mitochondrial membrane potential;(2) temporarily increased intracellular reactive oxygen species level;(3) factors from mitochondria to the nucleus Translocation Induced apoptosis.Conclusion:Ophiobolin O can inhibit the proliferation of MCF-7/ADR cells, Ophiobolin O can induce apoptosis in tumor cells, and can make the cell cycle change. The combined effect of animal experiment in vivo, the tumor inhibition rate increased. |
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