br mitochondrial anti apoptotic protein Bcl in a dose depend
mitochondrial anti-apoptotic protein Bcl-2 in a dose-dependent manner (Fig. 7a). Compared with the experimental group, the protein level of Bcl-2 was significantly down-regulated after the exposure to 5-FU. In addition, the protein level of Bax was gradually up-regulated with the intervention of CM. Following 5-FU treatment, the protein level of Bax was markedly enhanced in the MCF-7 13(S)-HODE (Fig. 7b). The results of flow cytometry intracellular staining showed that Bcl-2 protein levels were markedly decreased and Bax protein levels were significantly increased in MCF-7 cells after treatment with CM and 5-FU (Fig. 7c). Significant up-regulation of Bax/Bcl-2 ratio was identified in CM group (200–1000 μg/mL) compared with that of negative control group (Fig. 7d). Notably, the Bax/Bcl-2 ratio increased from 0.227 in negative control to 3.01 in the CM group (1000 μg/mL).
3.4. CM inhibited MCF-7 cells migration and invasion
The previous SEM results indicated that CM led to the loss of mi-crovilli and pseudopods of MCF-7 cells which may contribute to the inhibition of cell migration and invasion. On this basis, we examined the eﬀect of CM on the migration and invasion activities of MCF-7 cells by Transwell migration assays. As is shown in Fig. 8a, cells migrated to the lower chamber by shrink motion, and penetrated Matrigel by se-creting hydrolases and further invaded to the lower chamber. The Transwell assays indicated that CM depressed the migration and inva-sion of MCF-7 cells in a dose-dependent manner (Fig. 8b). The selected random fields of cells number suggested that the migration of MCF-7 cells were significantly decreased when exposed to CM mediated by APS with the concentration of 200–1000 μg/mL (Fig. 8c) as compared to that of untreated cells (P < 0.001). Similarly, APS mediated CM made the invasive potential of MCF-7 cells markedly decreased in comparison with that of negative control group (Fig. 8d). Expectedly, APS had no inhibitory eﬀect either on cell migration or invasion. No significant diﬀerence was observed of the migrated or invasive numbers of MCF-7 cells after treatment with various concentrations of APS (Fig.
S4–S5). Here, Transwell assay further verified that CM possessed highly strong inhibitory eﬃcacy on cell migration and invasion, and the un-derlying mechanism still needs to be determined in our subsequent research.
Nowadays, traditional herbal medicine and natural products are becoming increasingly popular among cancer patients responsible for their enhanced immunity potential and quality of life. Particularly, numerous polysaccharides isolated from microorganisms, plants and animals have attracted growing interests in biomedical field and appear to be a favorable source for oncotherapy, as they possess anti-tumor activities or augment the eﬃcacy of chemotherapy agents [25–27]. Among these polysaccharides, APS has been considered as an ideal candidate for oncotherapy, particularly being used in combination with chemotherapy and radiotherapy . As an adjuvant in cancer treat-ment, APS can enhance therapeutic eﬀects and reduce adverse eﬀects, contributing to the improvement of host immune response. However, few reports are available concerning whether APS can activate the immune potential in vitro. In the present study, we initially examined the eﬀect of APS on MCF-7 cells viability and received unsatisfactory outcome. Considering the antitumor mechanism of APS in vivo, the macrophage-like RAW264.7 cells was chosen as model in vitro to evaluate whether APS was able to stimulate immune cells and further inhibited cancer cells growth. We found the obtained APS could acti-vate RAW264.7 cells accompanied by up-regulation of NO and TNF-α production, which may act as cell apoptosis inducer to inhibit MCF-7 cells viability. Based on the encouraging observation, APS may provide an adjuvant strategy for the treatment of breast cancer. The corre-sponding anti-breast cancer eﬀect of APS and its mechanism in vivo will be further elucidated.
The immune regulation and antitumor activity of polysaccharides are closely associated with their structure variability which presents a
Fig. 8. APS mediated CM suppressed MCF-7 cells migration and invasion. (a) Representative SEM images of cells that passed through the Transwell membrane with/ without Matrigel at 1000× and 2000× magnification. Green arrows indicated that the secreted hydrolases digested Matrigel, and blue arrows indicated that cells migrated to low chamber. (b) Representative images of migrating or invading MCF-7 cells after treatment with APS mediated CM, respectively. Scale bar = 100 μm. (c–d) MCF-7 cell numbers that passed through the Transwell chambers without Matrigel (motile cells/field) or with Matrigel (invasive cells/field). ** P < 0.01, ***