OncoTargets and Therapy (Oct 2019)
Methyloleanolate Induces Apoptotic And Autophagic Cell Death Via Reactive Oxygen Species Generation And c-Jun N-terminal Kinase Phosphorylation
Abstract
Myoung Seok Jeong,* Ji Hoon Jung,* Hyemin Lee, Chang Geun Kim, Sung-Hoon Kim College of Korean Medicine, Kyung Hee University, Dongdaemun-Gu, Seoul 02447, Republic of Korea*These authors contributed equally to this workCorrespondence: Sung-Hoon KimCollege of Korean Medicine, Kyung Hee University, 26, Kyungheedae-ro, Dongdaemun-gu, Seoul 02447, Republic of KoreaTel +82-2-961-9233Fax +82-2-961-9598Email [email protected]: To develop a potent anticancer agent similar to oleanolate, the underlying mechanisms of its derivative, methyloleanolate, in the apoptosis and autophagy of A549 and H1299 cells were elucidated.Purpose: The aim of the present study was to investigate the effect of methyloleanolate in inducing apoptotic and autophagic cell death in cancer cells.Materials and methods: Flow cytometric analysis with Annexin V/PI staining, Western blot analysis, and immunofluorescence analysis were conducted in A549 and H1299 cells.Results: Methyloleanolate increased the fraction of Annexin V/PI apoptotic cells and activated caspase-8, caspase-3, and death receptor 5 (DR5) more than oleanolate in A549 and H1299 cells pretreated with pancaspase inhibitor z-VAD-fmk and DR5 depletion. Also, methyloleanolate induced autophagic features of microtubule-associated protein light chain 3 3BII (LC3BII) conversion and puncta in A549 and H1299 cells, along with autophagosomes and vacuoles. Methyloleanolate blocked autophagy flux for impaired autophagy and chloroquine (CQ)-enhanced microtubule-associated protein LC3BII accumulation and cytotoxicity in A549 and H1299 cells, although 3-methyladenine (3-MA) did not. Interestingly, LC3BII accumulation was detected only in methyloleanolate-treated autophagy-related gene 5 (ATG5)+/+ mouse embryonic fibroblast (MEF) cells but not in ATG5−/- MEF cells. Methyloleanolate reduced p-mTOR but activated p-c-Jun N-terminal kinases and reactive oxygen species production in A549 and H1299 cells. Conversely, n-acetyl-l-cysteine and SP600125 blocked apoptotic and autophagic cascades caused by methyloleanolate in A549 and H1299 cells.Conclusion: Overall, the findings suggest that methyloleanolate induces apoptotic and autophagic cell death in non–small cell lung cancers via reactive oxygen species generation and c-Jun N-terminal kinase phosphorylation.Keywords: methyloleanolate, apoptosis, autophagy, JNK, ROS
