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Background. Triple-negative breast cancer (TNBC) is a subtype of breast cancer with limited therapeutic options. Eupalinolide O (EO) was reported to inhibit tumor growth. This study is aimed at exploring the role of EO on TNBC both in vivo and in vitro. Methods. In in vitro experiments, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and clonogenic assay were conducted to measure the impact of EO on TNBC cell growth at different concentrations and time points. Flow cytometry was conducted to evaluate cell apoptosis. Mitochondrial membrane potential (MMP) loss, caspase-3 activity, and reactive oxygen species (ROS) generation were assessed. The expressions of apoptosis-related mRNAs and Akt/p38 MAPK signaling pathway-related proteins were measured. In in vivo experiments, by injecting TNBC cells into the nude mice to induce xenograft tumor, mice were treated with EO for 20 days. Then, in vivo bioluminescence imaging system was utilized to monitor the growth and distribution of TNBC cells. Tumor volume and weight were also recorded. Hematoxylin-eosin (HE) staining and ELISA assay were applied to observe tumor tissue morphology and ROS levels. Furthermore, western blotting was conducted to observe the expression of apoptosis-related proteins and Akt/p38 MAPK signaling pathway-associated proteins. Results. EO inhibited the cell viability and proliferation of TNBC cells but not normal epithelial cells. Furthermore, EO induced apoptosis, decreased MMP, and elevated caspase-3 activity and ROS content in TNBC cells. Meanwhile, the expression of apoptosis-related mRNAs and Akt/p38 MAPK pathway-related proteins was regulated by EO treatment. Besides, in vivo experiments demonstrated EO not only suppressed tumor growth, Ki67 expression, ROS generation, and Akt phosphorylation but also upregulated caspase-3 expression and p-38 phosphorylation. Conclusion. EO may induce cell apoptosis in TNBC via regulating ROS generation and Akt/p38 MAPK pathway, indicating EO may be a candidate drug for TNBC. [ABSTRACT FROM AUTHOR]

This study aimed to isolate, prepare and identify the main flavonoids from a standardized Smilax glabra flavonoids extract (SGF) using preparative HPLC, MS, 1H NMR and 13C NMR, determine the contents of these flavonoids using UPLC, then compare their pharmacological activities in vitro. We obtained six flavonoids from SGF: astilbin (18.10%), neoastilbin (11.04%), isoastilbin (5.03%), neoisoastilbin (4.09%), engeletin (2.58%) and (−)-epicatechin (1.77%). The antioxidant activity of six flavonoids were evaluated by determining the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical and 2,2′-Azinobis (3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS+) radical scavenging activity and ferric reducing antioxidant power (FRAP). In addition, the anti-inflammatory activity of six flavonoids were evaluated by determining the production of cytokines (IL-1β, IL-6), nitric oxide (NO) using enzyme linked immunosorbent assay and the NF-κB p65 expression using Western blotting in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. The results showed that (−)-epicatechin, astilbin, neoastilbin, isoastilbin and neoisoastilbin had strong antioxidant activities, not only in DPPH and ABTS+ radicals scavenging capacities, but in FRAP system. Furthermore, all the six flavonoids could significantly inhibit the secretion of IL-1β, IL-6, NO (p < 0.01) and the protein expression of NF-κB p-p65 (p < 0.01) in LPS-stimulated RAW264.7 cells. This study preliminarily verified the antioxidant and anti-inflammatory activities of six flavonoids in S. glabra. [ABSTRACT FROM AUTHOR]