br The anticancer effects of
The anticancer effects of any drug should be first evaluated by examining its cytotoxicity(A et al., 2017). The MTT assay showed that Se-β-Lg was clearly cytotoxic against breast cancer cells, in a concentration-dependent manner, but did not exhibit any cytotoxicity to normal breast cells. It is worth noting that at 24 h, the effects of Se-β-Lg on MCF-7 cells were stronger than those on MDA-MB-231 cells, whereas the viability of these two cell lines was similar at 48 h. These differences may be due to cell-type differences. The previous study also suggested that ERK1/ERK2 kinase substrate motif was identified in MDA-MB-231cells but not in MCF-7 cells (Kabir et al., 2012).
Apoptotic cells have specific features of cytoplasm, chromatin condensation, membrane depression, and apoptotic body formation, which could be observed by inverted microscopy and SEM in Se-β-Lg-treated cells in this study. In early apoptosis, Hoechst 33342 could stain cells because of changes in the membrane permeability. After induction of apoptosis, Annexin V was bound to phosphatidylserine, which translocated from the inner surface of the membrane in normal cells to the outer surface. PI could not penetrate into living and early apoptotic cells, with intact cell membranes, but could stain late apoptotic and necrotic cells. Therefore, our study could indicate that the cells progressed from early and late apoptosis to necrosis. In addition, research on A769662 arrest leading to inhibition of the growth of cancer cells is a key point and marker in the development of novel anti-cancer drugs (Azzopardi et al., 2017; Mi et al., 2018). The studies suggested that bazilein induced apoptosis by bolcking G1/G0 phase in
human vestibular schwannoma cells (Mou et al., 2019) and gnsenoside Rk1 induced cell cycle arrest in MDA-MB-231 cells (Hong and Fan, 2019). In this study, the cell cycle assay showed that Se-β-Lg induced apoptosis by blocking G0/G1 phase in MCF-7 and
MDA-MB-231 cells. Taken together, these results suggested that Se-β-Lg could induce apoptosis of MCF-7 and MDA-MB-231 cells.
There are two main pathways of caspase-dependent apoptosis: external apoptosis pathway and internal apoptosis pathway (Zhang et al., 2018). In the mitochondrial apoptotic pathway, the destruction of MMP and the production of ROS are considered as the most important signs of apoptosis (Zhong et al., 2017). Tumor cells have a higher redox status than normal cells do. An increase of the ROS production in tumor cells to the threshold level can initiate apoptosis, which can be used as a method of cancer treatment (Cui et al., 2018).
Se-β-Lg could augment the ROS production in cells, which was indicated by an increase in a proapoptotic protein and a decrease in an anti-apoptotic protein from the Bcl-2 family. Members of the Bcl-2 family are key regulators of apoptosis, affecting the release of cytochrome c, which regulates the opening and closing of the permeability transition pore in mitochondria. Se-β-Lg could induce apoptosis by loss of MMP, elevation of ROS level, increase of Bax/Bcl-2 ratio and release of cytochrome c via the mitochondrial pathway in our study. When cytochrome c forms a complex with apoptotic peptidase-activating factor 1 (APAF1), caspase-9 is activated (Zhao et al., 2017). Pro-caspase-9 was cleaved into two shear bodies of molecular weights of 37 and 39 kDa. Subsequently, cleaved-caspase-9 could active caspase-3 thereby initiating the apoptotic signaling pathway. Similarly, pro-caspase-3 was cleaved into two shear bodies of molecular weights of 17 and 19 kDa. In our study, the expression of cleaved-caspase-9 and cleaved-caspase-3 increased by increasing the dose of Se-β-Lg. Thereby, we could conclude that in human breast cancer MCF-7 and MDA-MB-231 cells, apoptosis was induced through the mitochondria-caspase-dependent apoptotic pathway after treatment with Se-β-Lg.
The novelty of this study was that NAC was used to explore the rate of apoptosis and production of ROS in Se-β-Lg-treated breast cancer cells. Further, the expression of apoptotic pathway-related proteins was comprehensively studied. This research may provide valuable information for the selection of drug candidates for the treatment of breast cancer. Based on the results of our investigation, Se-β-Lg significantly inhibited the growth of human breast cancer MCF-7 and MDA-MB-231 cells in vitro and showed no toxic effects on normal human breast cells, which suggests that Se-β-Lg can be developed as a new anticancer drug.
At the same time, additional thorough and comprehensive studies on the antitumor mechanisms of Se-β-Lg in various tumors are warranted. However, the relationship with antitumor activity of Se-β-Lg against breast cancer is still needed to further explore in vivo.