Honokiol Activates Reactive Oxygen Species-Mediated Cytoprotective Autophagy in Human Prostate Cancer Cells

BACKGROUND. Honokiol (HNK), derived from the bark of an oriental medicinal plant (Magnolia officinalis), is a promising anticancer agent with preclinical in vitro (PC-3 and LNCaP cells) and in vivo (PC-3 xenografts) efficacy against prostate cancer. However, the mechanisms affecting anticancer response to HNK are not fully understood. METHODS. Human (androgen-independent PC-3 and androgen-responsive LNCaP) and murine (Myc-CaP) prostate cancer cells, and PC-3 tumor xenografts were used for various assays. Autophagy was assessed by transmission electron microscopy, immunofluorescence (LC3 puncta), and immunoblotting (LC3BII detection). Cell viability was determined by trypan blue assay. Apoptosis was quantitated by DNA fragmentation detection and Annexin V/propidium iodide assay. Reactive oxygen species (ROS) were detected by electron paramagnetic resonance spectrometry and flow cytometric/microscopic analysis of MitoSOX red fluorescence. RESULTS. Exposure of PC-3, LNCaP, and Myc-CaP cells to pharmacologic doses of HNK resulted in autophagy induction. The PC-3 tumor xenografts from HNK-treated mice contained higher levels of LC3BII protein compared with control tumors. Cell viability inhibition and apoptosis induction resulting from HNK exposure were significantly augmented by pharmacological inhibition of autophagy using 3-methyladenine as well as RNA interference of autophagy regulator ATG5. HNK-mediated increase in levels of LC3BII protein was partially but markedly diminished in the presence of antioxidants, including N-acetylcysteine, polyethylene glycol-conjugated (PEG)-superoxide dismutase, and PEG-catalase. On the other hand, antioxidants had no impact on HNK-induced apoptosis. CONCLUSIONS. In conclusion, the present study demonstrates, for the first time, that HNK induces ROS-mediated cytoprotective autophagy in prostate cancer cells. (C) 2014 Wiley Periodicals, Inc.