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 breastcancer, 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 (MCF7/TAX, MDA-MB-231/PT, MDA-MB-231and MCF-7), and as well as the non-tumor cell lines MCF-10A, in variousdrug concentrations (from 0.004 to 1 μM). Western Blotting and Real-Time PCR assay were used to detect therelative protein and gene expression level after treatment with BOC26P in MCF-7/TAX. The effect of BOC26Pon Specific fluorescent P-gp substrate accumulation in MCF-7/TAX was analyzed by flow cytometry; Moleculardocking was used to analyze the binding capacity between BOC26P, Cyclosporine A, and Verapamil. FCM assaystaining with Annexin V-FITC/PI and Propidium iodide was used to measure the apoptosis and the cell cycleafter treatment with BOC26P in MCF-7/TAX, MDA-MB-231/PT, MDA-MB-231, and MCF-7; Detection ofmitochondrial membrane potential after treatment with BOC26P inMCF-7/TAX, MDA-MB-231/PT, MDA-MB231and MCF-7; Western Blotting and Real-Time PCR assay was used to detect the apoptosis relative proteinand gene expression level after treatment with BOC26P in MDA-MB-231, MCF-7, MDA-MB-231/PT, and MCF7/ADR.Results Cytotoxicity assay showed that BOC26P could effectively suppress 4 breast cancer cell lines (MCF7/TAX, MDA-MB-231/PT, MDA-MB-231, and MCF-7) with an IC50 value of under 0.5 μM. The IC50 value ofBOC26P on non-tumor cells MCF-10A was 32.29 μM. The binding ability of BOC26P to P-gp in breast cancercells was weak. There was no significant effect on the intracellular accumulation of Rhodamin 123(Rh123), Pgp binding specific fluorescence substrate, and multi-drug resistance protein P-gp expression in MCF-7/ADRand MDA-MB-231/PT tumor cells; BOC26P induced MCF-7/TAX, MDA-MB-231/PT, MDA-MB-231 and MCF-7cells cycle arrest at G2/M phase and lead to cell apoptosis. BOC26P induced significant activation of p53protein in MCF-7/ADR and MAD-MB-231/TAX cells. Under the same conditions, BOC26P promoted Baxexpression while inhibited Bcl-2 expression, and could significantly cause activation of Cleveland PARP andClevead Caspase3. The results demonstrated that BOC26P may induce apoptosis through the death receptorapoptosis pathway.Conclusion It is known that BOC26P has a significant proliferation inhibitory effect on breast cancer cellswithout serious side effects. BOC26P has the Potential to be developed into a clinical substitute drug for triple-

Development of a LC-MS/MS Method for a New Ortho-aryl Chalcone Compound of OC26 and its Application to Bile

Background OC26 and its pro-drug BOC26P, both ortho-aryl chalcone compounds, showed a well-definedantitumor activity in various cancer cells especially in drug-resistant tumor cell lines.Aim The purpose of this study was to investigate the bile excretion characteristics of OC26 after OC26 and BOC26Padministered in rats respectively.Method An ultra-performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) method wasdeveloped and validated for OC26 in rat bile. Liquid-liquid extraction with ethyl acetate method was use to pretreatthe bile samples. After that, a gradient mobile phase at a flow rate 0.5mL/min of acetonitrile and 2mM CH3COONH4with 0.1% aqueous ammonia solution (v/v) and the positive ion alternate mode separated and quantified OC26.Bile samples were collected from rats after intravenous injection (i.v) 12.5mg/kg of OC26 and BOC26P, respectively.Results For method validation, the method showed high extraction recovery. The assay showed a good linearitywith correlation coefficient >0.99 at the concentration ranges of 20-2000ng/mL. All data were within the requiredlimits. The bile excretion results showed that the excretion amount of OC26 was gradually stabilized after 2h. Theaccumulative excretion percentage of OC26 after i.v 12.5mg/kg BOC26P was significantly higher than that of OC26after i.v 12.5mg/kg OC26. Significant gender differences were also observed in bile excretion of OC26.Conclusion This method was selective, sensitive and reliable and successfully applied to the bile excretion of OC26.This study provided theoretical basis for OC26 further research.

Developing an UPLC-MSMS Method to Quantify a Novel Anticancer Chalcone BOC26P for Its Pharmacokinetic Study In vivo

Objective BOC26P is a potent anticancer candidate which inhibits microtubule polymerization and shows strongcytotoxic activity against numerous cancer cell lines and drug resistant cell lines. To support the pharmacokineticstudy of BOC26P, a rapid, selective and reproducible UPLC-MS/MS method was developed.Method Dexamethasone sodium phosphate (DSP) was used as an internal standard (IS). Following proteinprecipitation by using methanol-acetonitrile solution (1:1, v/v) with an internal standard DSP, the processedsamples were chromatographed on an UPLC X Bridge 71 TM C8 column (4.6 mm × 100 mm, 3.5 μm) with a mobilephase that consisted of acetonitrile and 2mmol/L ammonium acetate aqueous solution (containing 0.25%ammonia) with a gradient elution pumped at a flow rate of 0.4 mL/min. Mass spectrometric detection wasperformed in the positive electrospray ionization mode by multiple reaction monitoring (m/z 428.84→198.92 and472.90→434.93 for BOC26P and DSP, respectively). The quantification of BOC26P in rat plasma was fully verified.Results The linearity was established in the range of 50 to 2000 ng/mL(r2≥0.99). The recovery of BOC26P fromspiked plasma were ranged from 96.7% to 110.5%. This method showed acceptable accuracy (3.7% to 6.3%) andprecision (1.5% to 3.1%) both of intra- and inter-day.Conclusion The developed method was successfully applied for three intravenous dose (2, 5, 12.5 mg/kg BOC26P)pharmacokinetics in male and female rats