Study On The Effects Of Compoud Cremastra Appendiculata Ethanol Etraction On Proliferation Inhibition And Apoptotic Induction In Human Breast Cancer MDA-MB-231 Cells In Vitro And In Vivo

Objective:To investigate the influences of compound cremastra appendiculata ethanol extraction（CCAEE）on the proliferation and apoptosis in human breast cancer MDA-MB-231 cells in vitro.To investigate the anti-tumor effect of CCAEE in human breast cancer MDA-MB-231 cells xenograft nude mice in vivo.Methods:1.Human breast cancer MDA-MB-231 cells were exposed to different concentrations of compound cremastra appendiculata ethanol extraction for 24h,48h and 72h,the cell viabilities were examined by CCK-8 assay,the morphological changes of cells were observed under microscope.The apoptosis morphological changes of MDA-MB-231 cells were observed under microscope by Hoeschst 33342 apoptotic cells staining,the apoptosis rates and mitochondrial membrane potential changes of MDA-MB-231 cells were examined by Annexin V-APC/7-AAD double staining flow cytometric assay and JC-1 staining respectively.In addition,the expressions of cell proteins related to mitochondrial apoptosis including Bax,Bcl-2,Cyt-C and Caspase-3 were detected by Western-blot assay.2.The subcutaneous xenograft tumor model bearing MDA-MB-231 cells in BALB/c nude mice was establish and then mice were divided into 4 groups:model group,CCAEE 0.9g/kg（crude drug）and 1.8g/kg（crude drug）group and arubicin hydrochloride group（n=5 per group）,another 5 normal nude mice were as normal control group.The model and normal group were administered with equal volume of CMC-Na.The positive control group was introperitoneally injected arubicin hydrochloride at a dosage of 1.25 mg/kg every other day.The mice of CCAEE groups were orally administered at a dosage of 0.9g/kg（crude drug）and 1.8g/kg（crude drug）respectively every day.All the drugs were given for 21 days.During the administration,the mice weight and tumor volume were measured every other day.At the end of the treatment,the nude mice were executed and the tumor were extracted and weighted for calculating tumor inhibition rate.The pathological changes of tumor cells were observed under microscope after HE staining.The protein expressions of Bax,Bcl-2,Cyt-C and Caspase-3 in tumor were examined by Western blot assay.Results:1.The results of CCK-8 showed that CCAEE could dose-dependently inhibited the proliferation of human breast MDA-MB-231 cells and induced the apoptosis-like morphological changes.The IC₅₀ of MDA-MB-231 cells was 0.51mg/m L（crude drug）for 24h,0.26 mg/m L（crude drug）for 48h and 0.10mg/m L（crude drug）for 72h.The Hoechst 33342 staining results revealed that the treatment of 0.25,0.5,1.0 mg/ml（crude drug）of CCAEE for 24h dose-dependently induced the apoptosis-like morphological changes.Annexin V-APC/7-AAD double staining streaming results showed that,compared with the control group,CCAEE increased the apoptosis rate of MDA-MB-231 cells（p<0.05 or p<0.01）in a dose-dependent manner.JC-1results showed that,compared with the control group,CCAEE significantly decreased the red/green fluorescence ratio in a dose-dependent manner（p<0.05or p<0.01）,indicating that CCAEE could dose-dependently reduced mitochondrial membrane potential.In addition,Western blot results showed 0.5mg/m L（crude drug）CCAEE for 24h significantly up-regulated the cell protein expression levels of Bax,Cyt-C and Caspase-3（p<0.05 or p<0.01）,while down-regulated the expression level of Bcl-2（p<0.05 or p<0.01）.2.The nude mice xenograft tumor model bearing human breast MDA-MB-231cells was successfully established.Compared with the normal group,the body weight of the model group and the treatment groups were significantly reduced（p<0.05 or p<0.01）.Compared with the model group,arubicin hydrochloride and CCAEE 0.9g/kg（crude drug）group and 1.8g/kg（crude drug）group significantly reduced the tumor volume and weight with the inhibitory rate of 89.67%,67.39%and 43.48%respectively（p<0.05 or p<0.01）.HE staining results showed that compared with the model group,CCAEE0.9g/kg（crude drug）group and 1.8g/kg（crude drug）group could inhibit the growth of human breast tumor cells:with the number of cells decreasing and the necrosis morphological changing.Western-blot results showed that CCAEE groups could up-regulated the protein expression levels of Bax,Cyt-C and Caspase-3（p<0.05 or p<0.01）,down-regulated the expression level of Bcl-2（p<0.05 or p<0.01）.Conclusion:CCAEE inhibits the proliferation of human breast cancer MDA-MB-231 cells in vitro and in vivo,an effect that may be related to its induction on mitochondrial apoptosis.