EM-2 inhibited autophagy and promoted G₂/M phase arrest and apoptosis by activating the JNK pathway in hepatocellular carcinoma cells

This study aimed to investigate the inhibitory effect of EM-2, a natural active monomer purified from Elephantopusmollis H.B.K., on the proliferation of human hepatocellular carcinoma cells and the molecular mechanism involved. The results from the MTT assay revealed that EM-2 significantly inhibited the proliferation of human hepatocellular carcinoma (HCC) cells in a dose-dependent manner but exhibited less cytotoxicity to the normal liver epithelial cell line LO2. EdU staining and colony formation assays further confirmed the inhibitory effect of EM-2 on the proliferation of Huh-7 hepatocellular carcinoma cells. According to the RNA sequencing and KEGG enrichment analysis results, EM-2 markedly activated the MAPK pathway in Huh-7 cells, and the results of Western blotting further indicated that EM-2 could activate the ERK and JNK pathways. Meanwhile, EM-2 induced apoptosis in a dose-dependent manner and G₂/M phase arrest in Huh-7 cells, which could be partially reversed when treated with SP600125, a JNK inhibitor. Further study indicated that EM-2 induced endoplasmic reticulum stress and blocked autophagic flux in Huh-7 cells by inhibiting autophagy-induced lysosome maturation. Inhibition of autophagy by bafilomycin A1 could reduce cell viability and increase the sensitivity of Huh-7 cells to EM-2. In conclusion, our findings revealed that EM-2 not only promoted G₂/M phase arrest and activated ER stress but also induced apoptosis by activating the JNK pathway and blocked autophagic flux by inhibiting autolysosome maturation in Huh-7 hepatocellular carcinoma cells. Therefore, EM-2 is a potential therapeutic drug with promising antitumor effects against hepatocellular carcinoma and fewer side effects.