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  • Cell apoptosis rate br To further evaluate the rates

    2020-08-12

    3.3. Cell apoptosis rate
    To further evaluate the rates of apoptosis induced by Se-β-Lg in breast cancer cells, the cells were detected by Annexin V-FITC/PI staining. As shown in Fig.1D, E, with an increase
    in Se-β-Lg concentration, the proportion of apoptotic cells (Annexin VFITC+/PI+) increased in a concentration-dependent manner compared with that in the control group. The apoptotic rates
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    MDA-MB-231 cells were detected by flow cytometry after Annexin VFITC/PI staining. *p values < 5% (p < 0.05) was considered statistically
    significant, compared with control group.
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    Fig.2. (A, E) Morphological changes were observed by an inverted light microscope (×20). (B, F) Ultra-structure alterations were observed by a SEM
    detected by an inverted fluorescence microscope after Hoechst 33258 staining (×20). (D, H) Apoptosis cells were observed by a confocal laser
    3.4. Se-β-Lg-induced A-769662 arrest
    To investigate the effects of Se-β-Lg on the cell cycle of breast cancer cells, PI staining A-769662 was performed. As shown in Fig. 3A, B, the proportion of apoptotic cells increased, reflecting the activation of the sub-G1 phase. The cell cycle data (Fig. 3C, D) showed that the G0/G1 phase of MCF-7 cells increased from 52.23% to 90.76% (p < 0.05) at a dose of 20 μg/mL to
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    Fig.3. (A, B) Effects of Se-β-Lg on cell cycle distribution were detected by flow cytometry in breast cancer cells. (C, D) Columns show the
    percentages of the corresponding cell cycle phase in MCF-7 and MDA-MB-231 cells, respectively. *p values < 5% (p < 0.05) was considered
    statistically significant, compared with control group.
    3.5. Se-β-Lg-induced disruption of MMP
    We observed the changes in MMP, the main marker of early apoptosis, in breast cancer cells treated with Se-β-Lg. As shown in Fig. 4C, D, the MMP values of MCF-7 and MDA-MB-231 cells decreased in a concentration-dependent manner after treatment with Se-β-Lg. In other words, with an increase in concentration, the proportions of rhodamine 123-positive cells (Fig. 5A) declined compared with those in the control groups. These results suggested that Se-β-Lg might induce apoptosis through mitochondrial-mediated internal apoptosis pathway. 3.6. Se-β-Lg-induced intracellular ROS production
    The use of DCFH-DA as a fluorescent probe to detect ROS production (Fig. 4E, F) showed that Se-β-Lg could induce ROS generation in MCF-7 and MDA-MB-231 cells compared
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    Fig.4. (A, B) Effects of NAC pretreatment on the rates of cell apoptosis in cultures of MCF-7 and MDA-MB-231 cells treated with 80 µg/mL
    Se-β-Lg for 24 h. (C, D) The loss of MMP was induced by Se-β-Lg in breast cancer cells. (E, F) The levels of ROS were detected by flow cytometry
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    3.7. Se-β-Lg-induced expression of apoptosis-related proteins
    To further demonstrate whether treatment with Se-β-Lg could cause changes in the mitochondrial function of breast cancer cells, the levels of related proteins (Bax, Bcl-2, and cytochrome c) were detected by western blotting. As shown in Fig. 5C, E, the expression of Bax was significantly upregulated, whereas that of Bcl-2 was downregulated in Se-β-Lg-treated MCF-7 and MDA-MB-231 cells compared with their expression in the control groups. In addition, the levels of cytochrome c in the cytosol of Se-β-Lg-treated cells increased in a concentration-dependent manner. The results (Fig. 5D, F) also showed that the fractions of cleaved caspase-9/3 increased in Se-β-Lg-treated MCF-7 and MDA-MB-231 cells, suggesting the involvement of the mitochondrial caspase-dependent apoptotic pathway.
    Fig.5. (A) Columns represent MMP damage in MCF-7 and MDA-MB-231 cells. (B) Columns represent the production of ROS in MCF-7 and
    apoptosis-related proteins were measured by western blotting. (C, D, E, F) Western blot analysis of Bax, Bcl-2, cytochrome c, pro-caspase-9/3 and
    cleaved-caspase-9/3.
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    4. Discussion
    Although several therapeutic options exist for the treatment of cancer, many are not successful because of the adverse effects. One of the important considerations for new anticancer drugs is that they should have a lower toxicity profile within the therapeutic range. It has been reported that Se, an essential trace element, has many beneficial properties, such as the ability to maintain various physiological functions, improve the immunity, and protect against cancers (Cheng et al. 2018). Similarly, many studies have shown that β-Lg has a number of prominent functions, such as lowering blood pressure and cholesterol levels, as well as antioxidant and anticancer activities (Ma et al., 2018). Hence, our research team has been working on evaluating the mechanism of action of these two compounds. In this study, we evaluated the effects of treatment with Se-β-Lg on human breast cancer MCF-7 and MDA-MB-231 cells and further elucidated the molecular mechanisms of the antitumor activities of this compound, focusing on the mitochondrial-dependent apoptosis pathway through the production of ROS.