Studies on the Anti-Proliferative Effects and Molecular Mechanism of a Novel Small Molecular BOC26P in Breast Cancer

Background To explore the pharmacodynamic evaluation and mechanism research of BOC26P against breast cancer, and to provide a basis for the treatment of breast cancer. Method MTT assay was used to detect the cytotoxicity of BOC26P against 4 breast cancer cell lines (MCF-7/TAX, MDA-MB-231/PT, MDA-MB-231and MCF-7), and as well as the non-tumor cell lines MCF-10A, in various drug concentrations (from 0.004 to 1uM). Western Blotting and Real-Time PCR assay were used to detect the relative protein and gene expression level after treatment with BOC26P in MCF-7/TAX. The effect of BOC26P on Specific fluorescent P-gp substrate accumulation in MCF-7/TAX was analyzed by flow cytometry; Molecular docking was used to analyze the binding capacity between BOC26P, Cyclosporine A, and Verapamil. FCM assay staining with Annexin V-FITC/PI and Propidium iodide was used to measure the apoptosis and the cell cycle after treatment with BOC26P in MCF-7/TAX, MDA-MB-231/PT, MDA-MB-231, and MCF-7; Detection of mitochondrial membrane potential after treatment with BOC26P inMCF-7/TAX, MDA-MB-231/PT, MDA-MB-231and MCF-7; Western Blotting and Real-Time PCR assay was used to detect the apoptosis relative protein and gene expression level after treatment with BOC26P in MDA-MB-231, MCF-7, MDA-MB-231/PT, and MCF-7/ADR. Results Cytotoxicity assay showed that BOC26P could effectively suppress 4 breast cancer cell lines (MCF-7/TAX, MDA-MB-231/PT, MDA-MB-231, and MCF-7) with an IC50 value of under 0.5 ?M. The IC50 value of BOC26P on non-tumor cells MCF-10A was 32.29 uM. The binding ability of BOC26P to P-gp in breast cancer cells was weak. There was no significant effect on the intracellular accumulation of Rhodamin 123(Rh123), P-gp binding specific fluorescence substrate, and multi-drug resistance protein P-gp expression in MCF-7/ADR and MDA-MB-231/PT tumor cells; BOC26P induced MCF-7/TAX, MDA-MB-231/PT, MDA-MB-231 and MCF-7 cells cycle arrest at G2/M phase and lead to cell apoptosis. BOC26P induced significant activation of p53 protein in MCF-7/ADR and MAD-MB-231/TAX cells. Under the same conditions, BOC26P promoted Bax expression while inhibited Bcl-2 expression, and could significantly cause activation of Cleveland PARP and Clevead Caspase3. The results demonstrated that BOC26P may induce apoptosis through the death receptor apoptosis pathway. Conclusion It is known that BOC26P has a significant proliferation inhibitory effect on breast cancer cells without serious side effects. BOC26P has the Potential to be developed into a clinical substitute drug for triple-negative breast cancer and drug- resistance breast cancer. BOC26P, a cytotoxic antitumor candidate compound but not a P-gp substrate, does not interfere with P-gp expression and efflux function in the P-gp-positive drug-resistant breast cancer cells, that is, it can avoid P-gp mediated multidrug resistance pathway in breast cancer. BOC26P activates the membrane stability protein Bcl-2 family in MDA-MB-231/PT, MCF-7/ADR and MA-MB-231 mitochondrial pathways of drug-resistant human breast cancer cells and their maternally sensitive cells, causing mitochondrial rupture, releasing P53, and activating the caspase-mediated apoptosis pathway, thus causing apoptosis of breast cancer cells. The significant inhibitory activity of BOC26P on breast cancer resistant cells may be related to P53, Activation of P53 and other tumor suppressor genes alters the expression of Bcl-2 and Bax, further activates the Caspase cascade initiates apoptosis pathway, and cause apoptosis eventually.