Investigation Of Annonaceous Acetogenins Inhibit Breast Cancer Cells And Its Mechanisms

Background:With the continuous development of living standards, the incidence of breast cancer keeps rising. The paper shows that advanced breast cancer 5-year survival rate is only between 10% and 15%.There is a close relationship between MAPK signal transduction system and breast cancer, so the research with related signal transduction pathway are expected to find a new target for curing breast cancer. As an important component of comprehensive treatment, Chinese medicine has a certain effect and unique advantage. Based on molecular biology methods and Chinese medicine, preventing and treating breast cancer have always being an extraordinary topic on breast cancer in China. Recently, there are some progresses on Annona anti-cancer, which is known as the "Star of Tomorrow Anti-cancer". Annona lactone compound, followed by the taxol, is considered as another promising and natural source of anti-tumor compounds. Bullatacin is the strongest anti-cancer activity lactone isolated from Annona lactone compound and its activity in mouse vivo is 400 to 600 times stronger than taxol. This project was to explain inhibition and mechanism of inhibition in human breast cancer cells of Bullatacin and to provide theoretical and experimental basis for the anti-cancer drug development of Chinese medicine. Furthermore, it established a theoretical basis for Chinese and Western combined treatment of breast cancer.Purpose:1. Clarifying the toxicity of cancer is the key factor, standardized and individualized, unity on the contradiction between treatment of diseases and treatment of dialectical in the process of development of breast cancer and provide a theoretical basis for orientation and thinking of Chinese and Western combined treatment of breast cancer.2. Selecting a stronger anti-cancer activity Annona lactone monomer, called Bullatacin, from Alcohol extract of Annona seed. The main purpose was to study on inhibition and mechanism of inhibition in human breast cancer cells of Bullatacin and to provide theoretical and experimental basis for the anti-cancer drug development of Chinese medicineMethods: The method was combination of literature and modern experimental research:1. literature research:1.1 Accessed to ancient and modern Chinese medical literature on breast cancer in order to find its occurrence and development factors, and then explained.1.2 According to evidence-based Medicine, analysing theory and clinical practice on breast cancer, and exploring a point about Chinese and Western combined treatment of breast cancer.2. Experimental study:2.1 Extraction Annona lactone monomer Bullatacin: Crushed seeds of Annona, extracted 3 times with 95% ethanol, successively extracted with petroleum ether, chloroform saturated with water, methanol, Annona lactone compound was isolated from the chloroform part according to Silica gel column chromatography and recrystallizationh, Annona total lactones parts and Bullatacin monomer compound was obtained by the same way.2.2 Observed the inhibition effect of Bullatacin on proliferation of ER (+)MCF-7 and ER (-) MDA-MB-231 cells with MTT method in vitro. Examined if Bullacatin had inhibiting or not and effect potency with ER(+)MCF-7 and ER(-)MDA-MB-231 cells, in addition selected their own effective dose and time.2.3 Observed the effects of Bullatacin on cell morphology of MDA-MB-231: Observed the effect of 20ug/ml Bullatacin to MDA-MB-231 cells on morphology by inverted microscope and transmission electron microscopy.2.4 The study on cell cycle of MDA-MB-231 treated with Bullacatin by flow cytometry: According to analysis of FCM, the effects on cell cycle of MDA-MB-23 at time 0h,12h,24h,48h after 20ug/ml Bullatacin treatment.2.5 Observed the effects of Bullatacin on apoptosis of MDA-MB-231 with Annexin V-FITC staining: The apoptosis was measured by FCM, tested the apoptosis of MDA-MB-231 treated with 20ug/ml Bullatacin. After adding to MAPK pathway agonists and the inhibitor of main pathway respectively, testing the effects of apoptosis2.6 Western Blotting detected on Bullacatin regulated MAPK pathway with MDA-MB-231 With treating 20ug/mlBullatacin,MAPKpathway agonists and the inhibitor of main pathway on MDA-MB-231 respectively, tested the expression of different experimental groups phosphorylation and the total protein as well as regulation of MAPK pathway according to western blotting.Results:1. Caner poison breakthrough the occurrence and development of breast carcinoma, Evidence-Based Medicine request standardized and individualized on the Treatment of Diseases and on the treatment of Syndrome should go to contradictory unity;2. We get Annona lactones compound from chloroform part of Annona Seeds,by silicagel column chromatography and recrystallization,we get the compound, According to the physical constants and spectral data to identify the structure, learn to Bullatacin> 95% purity.3.Samples of bullacatin have the inhibition on both MCF7 cells of ER(+)and M-DA-MB-231 ce 11-s of ER(-) and a dose and time dependence.Compared with taxol group, there is the no diffe-rence on the 20 ug/ml of Bullacatin roled in the MDA-MB-231 cell inhibition rate for 48h4.20ug/ml of Bullacatin role in cells, cell ratio of G1 period reducecell ratio of S-period ad-d a nd the cell cycle stay on the S- period obviously. Alongwith the ti-me of the Bullacatin roles, t he cell ratio of G1 period reduces and the cell ratio of S-period a-dds. The bullacation influe nces the cell cycle with the time dependence.5. Bullacatin 20ug/ml act on MDA-MB-231 cell for 48hours:5.1 be detected by optical microscope:cell density and proliferation rate is lower in than that in control group,the cells in model group become larger and more round gradually.the space of cells is looser,cytoplasma granula is increasing,blebbing, dropped, so chips around cells is increasing.5.2 be detected by electric mirror membrane microvilli is almost lost, cytoplasm of organelle show marked dilation.mitochondria is the worst one.they have apparent vacuole to form,endoplasmatic enlargement and organelle has exosmose,but typical apoptosis and apoptotic body are noshown.Conclusions:1. Breast cancer required the standardization of modern medicine and individualized therapy. Traditional Chinese medicine theory advocated by the same disease with different treatment, different diseases. To a certain extent, cancer by targeting therapy and the theory of caner poison.2. Bullatacin on human breast cancer cells MDA-MB-231 has strong inhibitory effect.It can make enabling cell cycle arrest in S- phase. The inhibition has a dose and time dependent;3. Bullatacin induced programmed cell death in the form of paraptosis, the mechanism may be upstream of MAPK pathway activation.