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.

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.

Sq-2, a biotinylated annonaceous acetogenin, induces apoptosis, autophagy and S-phase arrest by activating the MAPK pathway in breast cancer cells.

Squamocin, an annonaceous acetogenin isolated from plants in the Annonaceae family, has antitumour activity. In this study, we report that Sq-2, a biotinylated squamocin monomer, has a favorable antitumour effect on MDA-MB-231 and SKBR3 breast cancer cells in vitro. MTT assays show that Sq-2 has a better antitumour effect on MDA-MB-231 cells than Sq-5 and Sq-6. Furthermore, RNA-Seq and KEGG enrichment analyses reveal that Sq-2 activates the MAPK signaling pathway, and results of western blot analysis demonstrate that Sq-2 activates the JNK and p38 pathways in MDA-MB-231 and SKBR3 cells. Flow cytometry and western blot analysis reveal that Sq-2 induces cell apoptosis by increasing the expressions of cleaved Caspase-3 and cleaved PARP as well as the ratio of Bax/Bcl-2. Inhibition of the Caspase family by Z-VAD-FMK attenuates the viability of MDA-MB-231 cells, indicating that Sq-2 induces apoptosis in a Caspase-dependent manner. Additionally, pretreatment with the p38 inhibitor SB203580 or JNK inhibitor SP600125 partially reverses the increase in the apoptosis rate and decrease in cell viability prompted by Sq-2. Furthermore, Sq-2 treatment decreases the expression level of CyclinD1 and increases the expression levels of p21, p27, CyclinA1, and CDK2, causing S-phase arrest in MDA-MB-231 and SKBR3 cells. Further study indicates that Sq-2 stimulates autophagy in MDA-MB-231 and SKBR3 cells, and inhibition of autophagy by bafilomycin A1 increases cell viability and promotes cell survival. Sq-2, a novel biotin-squamocin compound, shows a significant inhibitory effect on the propagation of SKBR3 and MDA-MB-231 breast cancer cells. Furthermore, Sq-2 treatment not only induces S-phase arrest and activates the JNK and p38 pathways to trigger apoptosis but also causes autophagy to promote apoptosis in MDA-MB-231 and SKBR3 cells.