Abstract
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.
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Acknowledgements
This research was supported by the Science and Technology Project of Guangdong Province (No. 2016ZC0044), the Science and Technology Planning Project of Guangdong Province (No. 2018B030320007), the Clinical research projects of the first affiliated hospital of Jinan university (No. 2018008, 2019315). Meanwhile, this research was also supported by the Guangzhou Science and Technology Program (No. 202002030087) and the National Natural Science Foundation of China (No. 82074064).
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JWJ and XFZ contributed to the conception of the study. JY and ZDL contributed to the design, performance, and data analysis of the experiments. CYH and ZYL contributed to the writing and submitting of the paper. CYH contributed to the revise of this paper. XYZ and QL contributed to the collection and assembly of data. SYM and QZ contributed to the typesetting and correction of the paper. XFZ and JWJ contributed to the critical revision and final approval of the article.
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Yang, J., Li, Zd., Hou, Cy. et al. EM-2 inhibited autophagy and promoted G2/M phase arrest and apoptosis by activating the JNK pathway in hepatocellular carcinoma cells. Acta Pharmacol Sin 42, 1139–1149 (2021). https://doi.org/10.1038/s41401-020-00564-6
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DOI: https://doi.org/10.1038/s41401-020-00564-6
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