Experimental Study On The Effect Of Brucine On Breast Cancer Line MDA-MB-231 Cell

Objective: Brucine, a main component of nux vomica, is one of indole alkaloids. In the study, we observe the effect of Brucine on proliferation inhibition and apoptosis induction on breast cancer cell MDA-MB-231. And analyze the regulation of Brucine on the expression of Bcl-2, Bax and Caspase-3 protein, thus provide an experimental basis for the clinic.Methods: The breast cancer cell MDA-MB-231 was cultured in L-15 medium. At the logarithmic growth phase, Brucine was added, the concentration of which was 50 mg/L, 100 mg/L, 200 mg/L, 400 mg/L, to stimulate the cells for 24h, 48h, 72h. MTT assay was used to measure the proliferation inhibitory effect of Brucine on the cells. The changes of cellular morphology were observed under inverted microscope. After treatment, cells were collected to prepare single cell suspension. Breast cancer cells were collected and resuspended in PBS. PI was added and the cell cycle was analyzed with flow cytometry while the apoptotic rate was calculated at the same time. PI and Annexin V-FITC were added to analyze the early apoptosis. To analyze the function of Bcl-2, Bax, Caspase-3 protein in the process of apoptosis induction, blocking assay was applied by neutralized antibody. Breast cancer cells were treated with Brucine for 48h at the concentrations of 50 mg/L, 100 mg/L, 200 mg/L, 400 mg/L. Then the cells were lysed and protein was quantified and separated with SDS-PAGE and transferred to a nitrocelular membrane with a semi-dry apparatus. The level of Bcl-2, Bax, Caspase-3 protein was detected by Western blotting assay.Results: (1) Cellular morphology: Cell adherence in control group were homogeneous and in good condition. Cell adherence in intervention group fall Off obviously , cell debris increased, and had doses and time dependent. (2)MTT: The optical density degraded significantly in every intervention group(p<0.01), and had doses and time dependent. (3)PI simple staining and flow cytometry analysis: Cell apoptotic rate increased with the increment of the concentration and treatment time, and has doses and time dependent. It had significant deviation to control group (p<0.05). S phase cells decreased while G0/G1 phase cells increased (p<0.05) and cells were blocked at G1/S phase. (4) Annexin V-FITC/PI analysis: In contrast to the control group, the early apoptosis cells increased significantly in all intervention groups (p<0.01) and had significantly differences between each intervention groups. (5) Western blotting: After treatment for 48 hours, Bcl-2 level was decreased, Bax level and Caspase-3 level were increased.Conclusion: Brucine can inhibit the proliferation of MDA-MB-231 cells effectively with doses and time dependent. Brucine can induce the apoptosis of MDA-MB-231 cells, the molecular mechanism of which is related with the activation of Bax and inhibition of Bcl-2. The decreased expression of Bcl-2 stimulates the activation of Caspase-3. The decreased expression of Bcl-2 may stimulate the release of cytochrome C, and activate Caspase-3 and Caspase-3 cascade through a key point in its downstream, then induce apoptosis.