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  • br ACCEPTED MANUSCRIPT br of ROS

    2020-08-12


    ACCEPTED MANUSCRIPT
    of ROS on the mitochondrial apoptosis pathway is mediated through the opening of osmotic transition pores, which activate the pore-destabilizing B-cell lymphoma 2 (Bcl-2)-associated X (Bax) protein and inhibit the pore-stabilizing Bcl-2 protein from the Bcl-2 family. The internal mitochondrial pathway initiates apoptosis by modulating the release of cytochrome c with the help of Bcl-2 and activation of members of the caspase family, which are involved in cell differentiation and important regulation of cell apoptosis (Jose et al., 2018). This study investigated the growth inhibitory effects and revealed the molecular mechanism of Se-β-Lg-induced apoptosis in PR-positive (MCF-7) and triple-negative (MDA-MB-231) cancer cells.
    2. Materials and methods
    2.1. Reagents and chemicals
    As described previously, β-Lg, purified from fresh milk, and seleninic 9036-06-0 were used as raw materials to synthesize Se-β-Lg at a low temperature under vacuum. (Zheng et al., 2016). Fetal bovine serum (FBS) was obtained from Thermo Scientific Hyclone (Shanghai, China). Dimethyl sulfoxide (DMSO) was purchased from Sigma Chemical Co. (St. Louis, MO, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), propidium iodide (PI), Rnase, Hoechst 33258 and Hoechst33342/PI double-staining kits were provided by Solarbio Science &Technology Co. Ltd. (Beijing, China). ROS assay kits, rhodamine 123, antibodies against Bax, Bcl-2, cytochrome c, caspase-3, caspase-9, and β-actin, as well as an enhanced chemiluminescence (ECL) kit were obtained from Beyotime Institute of Biotechnology (Shanghai, China). A fluorescein isothiocyanate (FITC)-conjugated Annexin V/PI (Annexin V-FITC/PI) apoptosis detection kit was provided 9036-06-0 by Keygen Biotech (Nanjing, China). All
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    other chemical reagents were of analytical grade.
    2.2. Cell culture and conditions
    Human breast carcinoma MCF-7 and MDA-MB-231 cells, as well as normal breast MCF-10A cells, were purchased from the Chinese Academy of Medical Sciences & Peking Union Medical College(Beijing, China). All the cells were cultured in RPMI-1640 medium supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin in a humidified incubator with 5% CO2 at 37 °C. Cells in the exponential growth phase were used for experiments.
    2.3. Determination of cell viability
    Growth inhibition of MCF-7, MDA-MB-231, and MCF-10A cells by Se-β-Lg was assessed using the MTT assay. After cells attached to plate wells, different concentrations of Se-β-Lg (0, 10, 30, 90, 120, 200, and 400 µg/mL) were added, and the plate was incubated for 24 h and 48 h. Then, 20 µL of the MTT reagent (5 mg/mL) was added to each well, and incubation continued for an additional 4 h. After removing the supernatants, 150 µL of DMSO was added, and the plate was shaken at room temperature for approximately 10 min to dissolve the formazan crystals formed. Cell viability was measured using an ELISA reader at 570 nm (Model 680; BioRad, Hercules, CA, USA). 2.4. Observation of cell morphology
    2.4.1. Observation under an inverted microscope
    The morphology of cells treated with Se-β-Lg at different concentrations (0, 20, 40, and 80 µg/mL) for 24 h was observed under an inverted microscope. (Nikon,Tokyo, Japan). 2.4.2. Observation under a scanning electron microscope (SEM)
    MCF-7 and MDA-MB-231 cells were washed three times with phosphate-buffered saline
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    Morphological changes in the apoptotic cell nucleus were observed using Hoechst 33258 staining. Treated cells after 24 h were collected by centrifugation and fixed with polyformaldehyde for 10 min. Then, the cells were stained with 2.5 µg/mL Hoechst 33258 and observed under an inverted fluorescence microscope (Nikon,Tokyo, Japan). 2.4.4. Hoechst 33342/PI double staining assay
    Hoechst 33342/PI double staining assay was used to examine the morphological changes of the apoptotic cells. Cells treated with Se-β-Lg for 24 h were fixed with 4% polyformaldehyde, then stained with 10 µM Hoechst 33342 and PI and viewed under a confocal laser scanning microscope (Nikon,Tokyo, Japan). 2.5. Determination of apoptosis rate
    Breast cancer MCF-7 and MDA-MB-231 cells grown to the exponential phase were exposed to various concentrations of Se-β-Lg for 24 h. According to the manufacturer's protocol, the cells were centrifuged at 300 g for 7.5 min after adding 1 mL of binding buffer. Next, the cells were incubated with 5 µL of Annexin V and PI in the dark. The apoptosis rate was detected by flow cytometry (BD FACSCalibur, San Jose, CA, USA).
    2.6.Analysis of the cell cycle
    ACCEPTED MANUSCRIPT
    The cells were treated with Se-β-Lg for 24 h. Ethanol (70%) was slowly added to the collected cells, followed by incubation at 4 °C for 18 h. After the cells were washed with PBS, RNase was added, followed by incubation at 37 °C. The subsequent changes in the cell cycle after adding PI were estimated by flow cytometry and analyzed using the ModFit LT software.