Background: The antitumor activity of CLE-10 (4-epi-isoinuviscolide), a sesquiterpene lactone compound, isolated from Carpesium abrotanoides L. has rarely been reported. The aim of this study is to investigate the antitumor activity of CLE-10 and give a greater explanation of its underlying mechanisms.
Methods: The cytotoxicity of CLE-10 was evaluated using MTT assay. Autophagy was detected by the formation of mRFP-GFP-LC3 fluorescence puncta and observed using transmission electron microscopy, while flow cytometry was employed to detect apoptosis. The protein expressions were detected through Western blotting.
Results: CLE-10 induced pro-death autophagy and apoptosis in MDA-MB-231 cells by increasing the protein expression of LC3-II, p-ULK1, Bax, and Bad, as well as downregulating p-PI3K, p-Akt, p-mTOR, p62, LC3-I, Bcl-2, and Bcl-xl. CLE-10 that was pretreated with 3-methyladenine (3-MA) or chloroquine (CQ) weakened the upregulation of the protein expression of p-ULK1, or the downregulation of p62, p-mTOR, and decreased the level of cytotoxicity against MDA-MB-231 cells. Meanwhile, rapamycin enhanced the effect of CLE-10 on the expression of autophagy-related protein and its cytotoxicity, with the IC50 value of CLE-10 decreasing from 4.07 µM to 2.38 µM.
Conclusion: CLE-10 induced pro-death autophagy and apoptosis in MDA-MB-231 cells by upregulating the protein expressions of LC3-II, p-ULK1, Bax, and Bad and downregulating p-PI3K, p-Akt, p-mTOR, p62, Bcl-2, and Bcl-xl.
Keywords: CLE-10; LC3; MDA-MB-231; PI3K/AKT/mTOR; apoptosis; autophagy.