EM-2, a natural sesquiterpene lactone from Elephantopus mollis H.B.K., enhanced the sensitivity of breast cancer cells to epirubicin by blocking protective autophagy

Abstract

Background: EM-2, a natural sesquiterpene lactone isolated from Elephantopus mollis H.B.K., showed a good anti-breast cancer effect when combined with epirubicin (EPI). However, its synergistic sensitization mechanism remains unclear.

Purpose: This study aimed to determine the therapeutic effect and possible synergistic mechanism of EM-2 with EPI in vivo and in vitro and to provide an experimental basis for the treatment of human breast cancer.

Methods: Cell proliferation was measured with MTT and colony formation assays. Apoptosis and reactive oxygen species (ROS) levels were examined through flow cytometry, and the expression levels of proteins related to apoptosis, autophagy, endoplasmic reticulum stress, and DNA damage were detected through Western blot analysis. Moreover, the caspase inhibitor Z-VAD-FMK, autophagy inhibitors bafilomycin A1 and chloroquine, ER stress inhibitor 4-phenylbutyric acid, and ROS scavenger N-acetyl cysteine were applied to verify signaling pathways. Breast cancer cell lines were used to evaluate the antitumor functions of EM-2 and EPI in vitro and in vivo.

Results: We demonstrated that in MDA-MB-231 and SKBR3 cells, the IC₅₀ of EPI combined with EM-2 (IC₂₀) was 37.909 and 33.889 times lower than that of EPI alone, respectively. Further study verified that in EPI-resistant lines (MDA-MB-231/EPI), the IC₅₀ of EPI combined with EM-2 (IC₂₀) was 26.305 times lower than that of EPI alone. Mechanistically, EM-2 could reverse the protective effect of EPI against autophagy in SKBR3 and MDA-MB-231 cells. EM-2 and EPI could trigger ER stress. When EM-2 and EPI were used in combination, ER stress was continuously activated, and ER stress-mediated apoptosis was induced. Meanwhile, EM-2 combined with EPI promoted DNA damage then induced apoptosis. In vivo, the volume of breast cancer xenografts in the combination group was smaller than that in the control, EM-2, and EPI groups. Immunohistochemical experiments demonstrated that the combination of EM-2 and EPI could block autophagy and promote ER stress in vivo.

Conclusion: EM-2 enhances the sensitivity of MDA-MB-231, SKBR3, and EPI-resistant cells to EPI.

Keywords: Autophagy; DNA damage; EM-2; ER stress; Epirubicin.