Abstract Purpose Primary liver disease is one of the major health problems in the world, and the prognosis of liver cancer is very poor. Liver cancer cells develop strong resistance to clinical chemotherapy drugs, leading to repeated liver cancer. Materials and Methods RNA-sequence was applied to related signal pathways that significantly altered. Flow cytometry and Western blot were performed to detect the changes of cell cycle, apoptosis and MAPK pathways. Immunofluorescence and flow cytometry were used to detect changes in cell mitochondrial membrane potential and intracellular ROS levels. Western blot, immunofluorescence, qRT-PCR and mCherry-GFP-LC3 were used to detect the effect of autophagy. Western blot and qRT-PCR were utilized to detect the effect of ER Stress. Results EM-6 is a novel monomer purified from Elephantopus mollis H.B.K. Mechanistically, compared with cisplatin (CDDP), EM-6 significantly inhibited the proliferation of human hepatocellular cancer cell lines and had less toxicity to human normal epithelial cells. EM-6 can induce mitochondrial membrane potential disruption, which leads to the accumulation of ROS, S-phase arrest and activation of the IRE1α-ASK1-JNK/p38 pathway to promote apoptosis in Huh-7 cells. In addition, EM-6 blocked protective autophagy by inhibiting the initiation of autophagy, and inhibiting the formation of autophagolysosomes triggered Huh-7 cell apoptosis. Conclusion Taken together, our findings suggest that EM-6 activates mitochondrial apoptosis through the ROS/MAPK pathway and promotes the activation of ER stress and the inhibition of autophagic flux to exacerbate apoptosis. These studies demonstrated the promising future of EM-6 in the clinical treatment of hepatocellular cancer.

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.