br addition consistent with the above results that
addition, consistent with the above results that APS alone failed to in-hibited cell proliferation, no significant diﬀerence of Fulvestrant (ICI 182,780) phase distribution was identified among APS groups (Fig. S1).
The formation of apoptotic bodies is a typical characteristic of cells undergoing apoptosis. After treatment with CM for 48 h, DAPI staining indicated that CM led to a strong chromatin condensation and nuclear fragmentation with the increasing concentration of APS in CM, while the cells presented normal nuclear morphology and slight blue stain in nuclei in the negative control group (Fig. 6a), and also in those groups after treatment with APS at the diﬀerent concentrations (Fig. S2). In-creased nuclear fragmentation and dot-like apoptotic bodies were identified in the presence of 5-FU compared with that in CM (1000 μg/ mL APS) group (Fig. 6a).
MCF-7 cells were labeled by AO/EB and observed with a fluorescent microscope after exposure to CM for 48 h. No significant apoptosis was detected in the negative control group. As is presented in Fig. 6b, we found that the number of apoptotic cells, especially the late-stage proportion, gradually increased with growing concentrations. More-over, it was notable that the overall number of cells tended to decrease in a dose-dependent manner. Basically, all cells were apoptotic at dif-ferent stages and appeared to disintegrate in 5-FU group.
Fig. 5. DNA cell cycle analyses of MCF-7 cells after treatment with CM. (a) Cell cycle distribution by flow cytometry of MCF-7 cells in the presence of CM mediated by various APS and 5-FU for 48 h. (b) Cell cycle profile of MCF-7 cells after exposure to CM for 48 h. The fraction of cells from apoptosis, G1, S and G2 phases analyzed from PI-A vs. cell accounts of each phase were shown in as percentages. (c) Proliferation index of MCF-7 cells. Data expressed as mean ± SD of three independent experiments. * P < 0.05, ** P < 0.01 vs. negative control group (untreated cells).
To further evaluate the role of APS in the apoptosis of MCF-7, we used flow cytometry to quantitatively analyze the apoptotic cells. As is shown in Fig. 6c and d, the apoptotic rates of MCF-7 cells were gra-dually increased in a dose-dependent manner. Significant elevation was noticed in the experimental group with the concentration of 1000 μg/ mL APS compared with that of untreated cells (P < 0.001). However, the apoptotic rate (29.7%) was markedly inferior as compared to the rate of 57.1% in 5-FU group (P < 0.001).
SEM examination revealed that MCF-7 cells are polymorphic and triangular in shape with elongated pseudopods and microvilli on cell surface. No significant change of cell morphology was noticed after treatment with APS at various concentrations, as shown in Fig. S3.
Instead, CM incubation led to cell deformation with decreased cell size, and loss of microvilli and pseudopods with smooth surface in the ma-jority of cells. Meanwhile, CM treatment resulted in apoptotic body formation with diﬀerent sizes (Fig. 6e). Cell morphological changes with apoptotic bodies were also identified after exposure to 5-FU. Here, the reduction in microvilli in CM group may potentially inhibit MCF-7 cell attachment and invasion.
3.3.4. CM down-regulated Bcl-2 and upregulated Bax expression
To further explore the apoptotic mechanism of APS mediated CM on MCF-7 cells, the expression levels of apoptosis-related genes and pro-teins (Bcl-2 and Bax) were examined. It was found that CM inhibited the
Fig. 6. APS mediated CM induced the apoptosis in MCF-7 cells. (a) Nuclear condensation of MCF-7 cells was visualized under fluorescent microscope after exposure to CM and 5-FU. Condensed nuclei and the apoptotic bodies are indicated by arrows. (b) Apoptosis cells were detected by AO/EB staining in the presence of APS mediated CM and 5-FU. Green nuclei of living cells, green–yellow nuclei of cells at early stages of apoptosis with diﬀerent forms of condensed, bright orange nuclei of cells at the late stage of apoptosis with condensed chromatin, and uneven orange-red or bright red nuclei of necrotic cells. Scale bar = 50 μm. (c) Flow cytometry analysis of apoptosis induced by CM in MCF-7 cells using Annexin V-FITC staining. (d) The percentage of apoptotic MCF-7 with the intervention of CM. Significant
diﬀerences between the CM-treated groups and negative control group were indicated as *** (P < 0.001), while significant diﬀerence between APS-mediated CM (1000 μg/mL) and 5-FU was indicated as ###(P < 0.001). (e) SEM observation of MCF-7 apoptosis after exposure to CM and 5-FU. Magnification of the images was
3000× and 10,000× (green border) on electron microscope. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 7. Bcl-2 and Bax expression in MCF-7 cells in diﬀerent groups. (a) Immunofluorescent pictures of FITC-Bcl-2 and DAPI staining and their merge of MCF-7cells after exposure to APS mediated CM and 5-FU, respectively. (b) Immunofluorescent pictures of Bax protein after treatment with APS mediated CM and 5-FU, respectively. Scale bar = 30 μm. (c) Bcl-2 and Bax protein expression by flow cytometry intracellular staining. (d) The ratio of Bax and Bcl-2 protein expression. * P < 0.05, ** P < 0.01, vs. negative control group (untreated cells). # P < 0.05, compared between 5-FU and APS mediated CM at the concentration of 1000 μg/mL.