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  • Measurement of mitochondrial membrane potential MMP br Based

    2020-08-12

    2.7.Measurement of mitochondrial membrane potential (MMP)
    Based on the manufacturer’s instructions, the alteration of MMP was assayed by rhodamine 123 staining. All cells incubated with different concentrations of Se-β-Lg for 24 h were collected, stained with rhodamine 123, and detected by flow cytometry. 2.8. Measurement of intracellular ROS generation
    The generation of ROS was analyzed by flow cytometry using the ROS detection kit. The collected cells were washed with PBS two times and incubated with 10 μM 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) at 37 °C in the dark. Simultaneously, a control group was incubated with N-acetyl-L-cysteine (NAC), a type of ROS inhibitor. The cells were co-cultured with 10 mM NAC for 1 h, and then, NAC was discarded and Se-β-Lg (80 µg/mL) was added. Later, the stained cells were observed to detect the apoptosis rate using Annexin V-FITC/PI kits and ROS production using flow cytometry. 2.9. Analysis of western blot
    Western blot analysis was used to evaluate the expression levels of apoptotic proteins. After incubation with Se-β-Lg at diverse concentrations for 24 h, the cells were digested with trypsin, centrifuged, and collected. Radio immunoprecipitation assay buffer with phenylmethylsulfonyl fluoride (100:1) was added to cells to extract proteins after placing on
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    ice for 30 min. The concentration of the extracted protein was quantified by the Bradford protein assay kit. Later, the protein samples were mixed with 2× protein buffer and boiled for 5 min. The lysates of treated and control cell samples were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrotransfered onto a polyvinylidene difluoride membrane. The membranes were blocked with 5% bovine serum albumin in Tris-buffered saline-Tween-20 (TBST) and then incubated with the primary Pronase E at 4 °C overnight. Subsequently, the membranes that were washed three times with TBST for about 15 min each time were further incubated with the corresponding secondary antibodies for 1.5 h. The protein bands were examined using the ECL reagent according to instructions and visualized by the Quantity One software. 2.10. Statistical analysis
    Each experiment was repeated at least three times, and data were analyzed using the SPSS software. Analysis of variance (ANOVA) was used to determine significant differences, and a p-value of <0.05 was considered statistically significant. The results are presented as mean ± standard deviation. 3. Results
    3.1. Effects of Pronase E Se-β-Lg on the viability of breast cancer cells
    To determine the cytotoxic effects of Se-β-Lg on human breast cancer cell lines, MCF-7, MDA-MB-231, and MCF-10A cells were treated with Se-β-Lg for 24 and 48 h, and cell viability was evaluated using the MTT assay. The results (Fig. 1A, B) showed that Se-β-Lg significantly inhibited the growth of MCF-7 and MDA-MB-231 cells in a concentration-dependent manner. At lower Se-β-Lg concentrations, cells treated for 24 h showed a higher cell viability
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    than did those treated for 48 h. However, the effects of time were less prominent with an increase in the Se-β-Lg concentration. As shown in Fig. 1C, Se-β-Lg had no significant effects on MCF-10A cells, indicating that the compound was less cytotoxic to normal breast cells. The half-maximal inhibitory concentrations of Se-β-Lg were 40.84 µg/mL for MCF-7 cells and 46.04 µg/mL for MDA-MB-231 cells at 24 h (Table.1). Therefore, the Se-β-Lg concentrations of 20, 40, and 80 µg/mL were selected for the follow-up experiments.
    Breast cancer cells IC50 values of Se-β-Lg (μg/mL)
    Table.1 IC50 values of Se-β-Lg on breast cancer cells. IC50 values were calculated by SPSS (mean ± S.D.).
    3.2. Changes in cell morphology
    Inverted microscope observation (Fig. 2A, E) showed that the untreated MCF-7 and MDA-MB-231 cells had a normal shape, and compact structure, and were filled with cytoplasm. However, with an increase in the Se-β-Lg concentration, the cell morphology significantly changed including reduction of cell volume, collapse of the structure, and formation of apoptotic bodies in the treated cells. The cellular ultrastructure (Fig. 2B, F) showed that the untreated cells had a smooth surface and maintained the integrity of the plasma membrane, whereas the Se-β-Lg-treated cells displayed a cell shrinkage, even collapse, and eversion of the cytoplasmic membrane.
    Morphological alterations of apoptotic nucleus of breast cancer cells were observed by an inverted fluorescence microscopy. The results (Fig. 2C, G) showed that cells in the control
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    group were stained uniformly blue by Hoechst 33258, whereas the nuclei gradually disintegrated and chromatin fibers aggregated in the treated groups, appearing as bright fluorescence singals. The morphological changes of apoptotic cells (Fig. 2D, H) were viewed by a confocal laser scanning microscope. Most of the treated cells were stained bright blue with Hoechst 33342 in the early stage of apoptosis. With increasing concentration, the cells in the late stage of apoptosis appeared pale purple upon Hoechst 33342/PI double staining. Taken together, these results indicated that Se-β-Lg could induce the morphological changes of MCF-7 and MDA-MB-231 cells.