The cardiac glycosides oleandrin and odoroside A, polyphenolic monomer compounds extracted from Nerium oleander, have been found to have antitumor effects on various tumors at low doses. However, the mechanisms of anticancer effects of oleandrin and odoroside A are not well known. Therefore, in this study, we aimed to investigate the anticancer effects of oleandrin and odoroside A and their associated mechanisms in highly metastatic MDA-MB-231 breast cancer cells and radiotherapy-resistant (RT-R) MDA-MB-231 cells. Our results showed that oleandrin and odoroside A dose-dependently decreased the colony formation and the invasion of both cell lines at nanomolar ranges. Furthermore, oleandrin (50 nM) and odoroside A (100 nM) reduced octamer-binding transcription factor 3/4 (OCT3/4) and β-catenin levels and matrix metalloproteinase-9 (MMP-9) activity. Finally, we found that phospho-STAT-3 levels were increased in MDA-MB-231 and RT-R-MDA-MB-231, but not in endothelial cells (ECs), and that the levels were significantly decreased by oleandrin (50 nM) and odoroside A (100 nM). Inhibition of phospho-signal transducer and activator of transcription (STAT)-3 significantly reduced OCT3/4 and β-catenin levels and MMP-9 activity, ultimately resulting in reduced invasion. These results suggest that the anticancer effects of oleandrin and odoroside A might be due to the inhibition of invasion through of phospho-STAT-3-mediated pathways that are involved in the regulation of invasion-related molecules.

The key barrier to the effectiveness of radiotherapy remains the radioresistance of breast cancer cells, resulting in increased tumor recurrence and metastasis. Thus, in this study, we aimed to clarify the difference between radiotherapy-resistant (RT-R) breast cancer (BC) and BC, and accordingly, analyzed gene expression levels between radiotherapy-resistant (RT-R) MDA-MB-231 cells and MDA-MB-231 cells. Gene expression array showed that ESM-1 was the most upregulated in RT-R-MDA-MB-231 cells compared to MDA-MB-231 cells. Then, we aimed to investigate the role of ESM-1 in the increased tumorigenesis of RT-R-BC cells. RT-R-MDA-MB-231, which showed an increased expression level of ESM1, exhibited significantly enhanced proliferation, colony forming ability, migration, and invasion compared to MDA-MB-231 cells, and ESM-1 knockdown effectively reversed these effects. In addition, compared to MDA-MB-231 cells, RT-R-MDA-MB-231 cells displayed improved adhesion to endothelial cells (ECs) due to the induction of adhesion molecules and increased MMP-9 activity and VEGF-A production, which were decreased by ESM-1 knockdown. Moreover, the expression of HIF-1α and activation of NF-κB and STAT-3 were increased in RT-R-MDA-MB-231 cells compared to MDA-MB-231 cells, and these effects were abolished by the knockdown of ESM-1. Finally, we confirmed the role of ESM-1 in tumorigenesis in an in vivo mouse model. Tumor volume, lung metastasis, and tumorigenic molecules (VEGF-A, HIF-1α, MMP-9, ICAM-1, VCAM-1, and phospho-NF-κB and phospho-STAT-3) were significantly induced in mice injected with ESM-1-overexpressing 4T1 cells and greatly enhanced in those injected with ESM-1-overexpressing RT-R-4T1 cells. Taken together, these results suggest for the first time that ESM-1 plays a critical role in tumorigenesis of breast cancer cells, especially RT-R-breast cancer cells, through the induction of cell proliferation and invasion.

Cancer stem cells (CSCs) exist in solid tumors and contribute to therapeutic resistance and disease recurrence. Previously, we reported that radiotherapy-resistant (RT-R)-MDA-MB-231 cells from highly metastatic MDA-MB-231 cells produced more CSCs than any other RT-R-breast cancer cells and showed therapeutic resistance and enhanced invasiveness. Hypoxia inducible factor-1α (HIF-1α) induced in the tumor microenvironment leads to the release of lysyl oxidase (LOX), which mediates collagen crosslinking at distant sites to facilitate environmental changes that allow cancer cells to easily metastasize. Therefore, in this study, we investigated whether RT-R-MDA-MB-231 cells induce greater HIF-1α expression, LOX secretion, and premetastatic niche formation than MDA-MB-231 cells do. RT-R-MDA-MB-231 cells increased HIF-1α expression and LOX secretion compared with MDA-MB-231 cells. Mice harboring RT-R-MDA-MB-231 cell xenografts showed enhanced tumor growth and higher expression of the CSC markers, CD44, Notch-4, and Oct3/4. In addition, mice injected with RT-R-MDA-MB-231 cells exhibited a higher level of HIF-1α in tumor tissue, increased secretion of LOX in plasma, higher induced levels of crosslinked collagen, and a higher population of CD11b⁺ BMDC recruitment around lung tissue, compared with those injected with MDA-MB-231 cells. These results suggest that RT-R-MDA-MB-231 cells contribute to tumor progression by enhancing premetastatic niche formation through the HIF-1α-LOX axis.