Experimental Study Of Breast Carcinomatreatment By Extract Of Centipedes

Chapter Ⅰ Assay sensibility of brest cancer MDA-MB-231Cell line to ECP in vitroObjective:To investigate sensitivity,targeted cell cycle phase and possible mechanism of the effect of centipede extract on breast cancer cell lines and provide evidences for further clinic therapy of breast cancer.Methods:Cultured breast cancer cells MDA-MB-231are intervened with different concentration of centipede extract. MTT assay and concentration-time curve are used to study the effect sensitivity of centipede extract on breast cancer cell lines; Inverted light microscope and electron microscope are used to observe cell changes in morphology and ultrastructure; flow cytometry is applicated to investigate cell cycle and apoptosis rate in centipede extract-treated group.Results:①The results of MTT assay showed that different concentrations of centipede extract all can inhibit the growth of human breast cancer cells MDA-MB-231.The inhibition rates were (71.26±2.64)%,(62.48±6.67)%,(56.26±3.17)%,(48.63±4.48)%,(42.13± 4.73)%,(34.5±2.98)%and (26.89±2.95)%respectively,while the Corresponding concentration of centipede extract were80mg/ml,40mg/ml,20mg/ml,10mg/ml,5mg/ml,2.5mg/ml and1.25mg/ml. IC50of centipede extract to MDA-MB-231was12.5mg/ml.②Drug concentration-time curve revealed that the viable cell counts of MDA-MB-231has direct relations with the concentration and administrative time of centipede extract. When treated with12.5mg/ml and25mg/ml centipede extract, viable counts of MDA-MB-231sharply decrease after24hours treatment and the difference among treatment groups is significant (P<0.01).③After48hr intervention, there were no inhibitory effect observed by light microscopy in groups with concentration less than10mg/ml; viable counts of MDA-MB-231decreased and the cells transformed to round and small when concentration were more than10mg/ml; With treated with80mg/ml, large number of necrotic cells and irregular cell debris were seemed. Meanwhile, margination and condensation of chromatin, fragmentation and condensation of nucleus and formation of apoptotic bodies were found in cells by electron microscope which had higher density④The result of flow cytometry displayed that (A)The ratio of G0/G1phase was varied by different concentrations of centipede extract:(36.7±4.2)%in control group,(56.5±4.1)%in12.5mg/ml group, and (67.3±4.4)%which was the highest in25mg/ml group.(B) Cell proliferation index (PI) of MDA-MB-231cells was significantly different as well (P<0.05):the values in control group,12.5mg/ml group and25mg/ml group were (63.3±3.2)%,(43.5±3.5)%, and(32.7±2.0)%,respectively. With the increased concentration of centipede extract, the "sub-G1peak" corresponding to apoptotic cells was gradually rising.(C) Apoptosis ratio: After48hr intervention, spontaneous apoptosis ratio in control group was (2.15±0.13)%, while in12.5mg/ml group it was (13.01±1.51)%and (21.3±1.98)%in25mg/ml group. The differences were statistically significant (P<0.05);⑤Immunohistochemistry had detected that the expression of Bcl-2gene was gradually weaken but that of Bax enhanced in MDA-MB-231cells with the increased concentration of centipede extract.Conclusion:①Extract of centipede can inhibit the growing of human breast cancer cells MDA-MB-231and the effect was concentration-time dependent;②Extract of centipede can induce MDA-MB-231cell line to apoptosis in low concentration and directly kill it in middle or high concentration;③Extract of centipede to MDA-MB-231cells were held in G1/G0phase,inhibition of its growing,and can induce apoptosis in concentration-time depedent effects.④One of the possible mechanisms of Centipede extract treatment may be owned to its ability of promoting the expression of Bax gene and inhibiting that of Bcl-2gene in MDA-MB-231. Chapter II Investigation to the effect and mechanisms of breast cancer MDA-MB-231treatment with ECP in vivoObjective:To investigate the effects and mechanisms of breast cancer MDA-MB-231treatment with ECP.Method:①To set up model of heterotopic grafting carcinoma for breast cancer for MDA-MB-231in nude mouse, and observe its tumor growth and metastasis by intragastric administration of ECP.②To detect Bcl-2,Bax,VEGF and Ang-2in tumor tissue with immunohistochemical methodsResult:①The achievement ratio for hefterotopic grafting carcinoma for breast cancer MDA-MB-231in nude mouse is100%.②It has distinctive inhibitive effect for ECP to hefterotopic grafting carcinoma for breast cancer MDA-MB-231in nude mouse. At the25th day postgraft of nude mouse,the average grafting carcinoma volume of the contrast is (1011.52±212.68)mm3, but for the treatment group is (238.83±65.45) mm3;The average grafting carcinoma weight of the contrast is (983±97.3)mg, but for the treatment group is (556±98.1)mg, The inhibitive ratio reaches to44.2%. there are significant difference for them (P<0.05).③The staining cells population, staining intensity and the optical density (OD) of Bcl-2,VEGF and Ang-2for the contrast group are higher than the treatment group, but they are lower for Bax. there are significant difference for them (P＜0.01)Conclusion:ECP can inhibit hefterotopic grafting carcinoma for breast cancer MDA-MB-231in nude mouse, the mechanisms relate to inhibit tumor Angiogenesis and to promote cells to apoptosis.. Chapter Ⅲ:Application of the techniques and methods of Proteomics into separating differential expression proteins in breast cancer cells treatment with ECPObjective:To screen the relevant differential expression proteins after treatment with ECP, and to investigate the molecule mechanism of ECP for breast cancer MDA-MB-231.Method:we use proteomic Techniques and methods to analysis the mechanism of treatment with ECP for breast cancer MDA-MB-231. Firstly, comparative two-dimensional gel electrophoresis (2-DE) technology was applied to separate the total protein of MDA-MB-231 treatment group by ECP and its control group respectively. The well-resolved, reproducible2-DE patterns of treatment group of ECP and control group were established. Then, PDQuest software was used to analyze2-DE images, and the differential expression proteins between the two groups were identified by both peptide mass fingerprint (PMF) and peptide sequence tag (PST) based on MALDI-TOF-MS (Matrix-assisted laser desorption/ionization time of flight mass spectrometry).Result:We select the differential expression proteins for between treatment group and control group which spots variance is over double to analyse with MALDI-TOF-MS after spots enzymolyed,and found out76protein spots at difference level of expression (p<0.05), including41spots decreasing in treatment group, and35spots increasing in control groups.12of the total pieces of PMF were be matched searching in SWISS-PROT/TREMBL database by Mascot software. Among the identified12protein spots, the expression level of proteins which up-regulates in treatment group is5in total including:S100A9, CALML5,CK-19,Gal-7,Alpha-enolase. But6proteins in treatment group is down-regulation, which include GSTO1-1,PGAMA,HSP90-β, AKP-P,IMPDH and GAPDH.Conclusion:①Through analysis of two-dimensional gel electrophoresis (2-DE),76protein spots at difference level of expression (p<0.05) have been found out, including41spots decreasing in treatment group, and35spots increasing in control groups.②Among the identified11of the total of protein spots, the expression level of proteins which up-regulates in treatment group is5in total including:S100A9, CALML5,CK-19,Gal-7,Alpha-enolase and which down-regulates in treatment group is6proteins in total, including:GSTO1-1,PGAMA, HSP90-β,AKP-P,IMPDH and GAPDH.. It hint that ECP can inhibit MDA-MB-231cell line to growth by inducing it to apoptosis or directly to death in multi-channels. Chapter Ⅳ:Verification of the differential expression levels of the partial proteins by western-blotting analysis and its function studyObjective:To verify the differential expression levels of the partial proteins S100A9,CK19and Gal-7which identified with compared proteomic techniques.Method:To verify the differential expression levels of S100A9, CK19and Gal-7again by Western-blot methods, which express in MDA-MB-231treatment with ECP group and control group ever identified with comparative proteomic techniques.Result:The proteins of S100A9,CK19and Gal-7express up-regulation in treatment group with ECP, and the results were identical with the proteome analysis.Conclusion:The differential expression proteins of S100A9,CK19and Gal-7screened by proteome analysis are indeed differential expression in treatment group and control group of MDA-MB-231cell.