br group were stained uniformly blue by
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. 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
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.
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 Endothelin 1 swine arrest
To investigate the effects of Se-β-Lg on the cell cycle of breast cancer cells, PI staining 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
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
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
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.