Anti-proliferative and pro-apoptotic effects of cinobufagin on human breast cancer MCF-7 cells and its molecular mechanism

Abstract Cinobufagin (CBF) is an active ingredient isolated from Venenum Bufonis extracted and dried from the secretory glands of Bufo gargarizans Cantor. The purpose of the study was to investigate the effects and underlying mechanisms of CBF on human breast cancer MCF-7 cells in vitro. Our results showed that CBF exhibited obvious cytotoxicity on MCF-7 cells in a dose- and time-dependent manner, as indicated by CCK-8 assays. Also, Hoechst 33258 staining and flow cytometry assays showed that CBF strongly induced MCF-7 cell apoptosis and G1 phase arrest. In addition, further molecular mechanistic investigation demonstrated that cinobufagin significantly increased Bax expression, decreased Bcl-2 expression level and up-regulated the ratio of the pro-apoptosis/anti-apoptosis protein Bax/Bcl-2, which were demonstrated by RT-qPCR and western blot assays. Taken together, our data confirm that CBF inhibits growth and triggers apoptosis of MCF-7 cells by affecting the expression of Bax and Bcl-2 in vitro.


Introduction
Breast cancer, with its high incidence rate, is presently the most common malignant neoplasm among women . Traditional methods for the treatment of breast cancer, such as chemotherapy and radiotherapy, even when used in conjunction with surgery or endocrine therapy, may have many side effects (Overgaard et al. 1997). Thus, novel and effective treatments or drugs with lower toxicities specifically targeting breast cancer are urgently needed. Recent studies (Daniel et al. 2006;Jo et al. 2010) have shown that a large number of original components derived and extracted from natural products may have some anti-cancer potential. In this regard, bufadienolides have been attracting attention for the potential effectiveness of their antitumour properties (Cheng et al. 2014). Cinobufagin (CBF) (Figure 1), a major bioactive ingredient of the traditional Chinese medicine Venenum Bufonis (He et al. 2012), which was extracted and dried from white exudates of the skin and secretory glands of Bufo gargarizans Cantor, belongs to the bufadienolide group (Ye & Guo 2008). CBF has been proved to have obvious cytotoxicity towards several types of cancer cells, including prostate cancer cells (Yeh et al. 2003) and human osteosarcoma U2OS cells (Ma et al. 2016). However, its anti-cancer mechanisms are not completely understood, and research on the relationship between CBF and breast cancer is lacking. For this reason, the efficiency of CBF on breast cancer MCF-7 cells and the underlying mechanisms were investigated in our study.

CBF inhibited growth in MCF-7 cells
Exploring novel and promising anti-cancer agents extracted and separated from nature products has become a significant strategy in cancer therapy. Our CCK-8 assay showed that CBF strongly inhibited the growth of MCF-7 cell lines in a time-and dose-dependent manner. IC50 values at 24, 48 and 72 h post-treatment were 0.94 ± 0.08, 0.44 ± 0.12 and 0.22 ± 0.03 μM, respectively ( Figure S1). Taxol, a proven anti-cancer drug, has been reported to induce MCF-7 cell apoptosis. As it was used as one of the first-line therapies for breast carcinoma in clinical settings (Zhang et al. 2014), we chose to use it as the positive control medicine in our cell viability analysis. As shown in Figure S1, the IC50 value in taxol-treated MCF-7 cells at 48 h was 0.08 ± 0.02 μM. Although MCF-7 cells seemed to be much more sensitive to taxol, with a lower IC50 value than CBF, the anti-proliferative effect of CBF on MCF-7 was still clear.

CBF induced apoptosis in MCF-7 cells
Apoptosis, a programmed cell death, is typically characterised by evident morphological change with pyknosis (Elmore 2007). The process has been targeted as a promising drug target for cancer therapy, and has been inveatigated in many recent studies (Kruyt & Schuringa 2010;von Schwarzenberg & Vollmar 2013). Small bright blue dots, which are representative of condensed or fragmented nuclei, were clearly present in CBF-treated groups on Hoechst 33258 staining, while the nuclei of MCF-7 cells in the control group stained an even blue ( Figure S2). In addition, the analysis of Annexin-V APC/7-AAD staining by flow cytometry revealed that the percentage of apoptosis, especially early and late stage apoptosis in CBF-treated MCF-7 cells, significantly increased in a dose-dependent manner ( Figure S3). Furthermore, the proportions of apoptotic MCF-7 cells treated with 0.2, 0.4 and 0.8 μM CBF were 22.94, 50.68 and 68.29%, respectively, while the control group was 5.87% ( Figure S3 (b)). These data, undoubtedly, demonstrate that MCF-7 cells treated with CBF exhibited clear apoptosis, when compared with the control group, providing strong evidence that CBF is capable of inhibiting growth and inducing apoptosis in MCF-7 cells, and exhibiting promising anti-cancer properties.

CBF caused G1 arrest in MCF-7 cells
The cell cycle is an intricate process of growth and proliferation (Schafer 1998), but its disorder is closely related to oncogenesis (Golias et al. 2004). We used PI staining to test whether or not CBF affected MCF-7 cell cycle progression; and analysis by flow cytometry demonstrated that cell cycle was arrested in the G1 phase ( Figure S4), which prevented cells from undergoing mitosis. Also, the percentage of MCF-7 cells in the G1 phase were 49.11, 57.54 and 63.75% following treatment with 0.2, 0.4 and 0.8 μM CBF, respectively, while the control group was 39.45% ( Figure S4 (b)). Therefore, we conclude that, cell cycle arrest and apoptosis induction may be two important causes of cell growth inhibition in human breast cancer MCF-7 cell lines. However, G1 arrest may stop cell cycle progression from entering into the S phase, which will also affect DNA synthesis, so further investigation is needed.

CBF affected gene and protein expression of Bcl-2 and Bax in MCF-7 cells
Although multiple mechanisms are involved in apoptosis, the dysregulation of the B-cell lymphoma-2 (Bcl-2) protein family members in the process of apoptosis seems to be more common (Oltersdorf et al. 2005). The pro-apoptosis protein Bax and anti-apoptosis protein Bcl-2 are two critical regulators of their family, and the ratio of Bax/Bcl-2 is considered to be a decisive factor in determining whether or not cells will go through apoptosis . Consequently, in order to reveal possible molecular mechanisms involved in CBFinduced apoptosis in MCF-7 cells, we measured the gene and protein expression of Bax and Bcl-2 in our subsequent experiment. RT-qPCR analysis showed that Bax gene expression was up-regulated and Bcl-2 gene expression was down-regulated in MCF-7 cells after treatment with 0.2, 0.4 and 0.8 μM CBF for 48 h ( Figure S5), which were in accordance with the increased Bax expression and decreased Bcl-2 expression under identical conditions as demonstrated by western blot analysis ( Figure S6). In addition, the ratio of Bax to Bcl-2 was increased in a dose-dependent manner when compared with the control group ( Figure S6 (b)). Bax and Bcl-2 protein are two central regulators in the mitochondria-mediated apoptosis pathway, and our RT-qPCR and western blot assays indicated the changed expression of both after exposure to CBF. These results provide evidence that CBF induces apoptosis in human breast cancer MCF-7 cells through the mitochondrial apoptotic pathway.

Conclusion
Our results verify, for the first time, that CBF inhibited growth and induced apoptosis in MCF-7 cells by altering the expression of Bax and Bcl-2 proteins. These findings suggest that CBF has potential for further development and use as an anti-cancer agent.

Supplementary material
Experimental details relating to this paper are available online, with Figures S1-S6.