Hydroxyl Radical (·OH) Played a Pivotal Role in Oridonin-Induced Apoptosis and Autophagy in Human Epidermoid Carcinoma A431 Cells

• Published in: Biological & pharmaceutical bulletin 2012/12/01, Vol.35(12), pp.2148-2159
• Main Authors: Yu, Yang, Fan, Si Miao, Song, Jun Ke, Tashiro, Shin-ichi, Onodera, Satoshi, Ikejima, Takashi
• Format: Article
• Language: English
• Subjects: Acetylcysteine - pharmacology; Antineoplastic Agents, Phytogenic - pharmacology; Antineoplastic Agents, Phytogenic - therapeutic use; apoptosis; Apoptosis - drug effects; autophagy; Autophagy - drug effects; Carcinoma, Squamous Cell - drug therapy; Carcinoma, Squamous Cell - metabolism; Carcinoma, Squamous Cell - physiopathology; Cell Line, Tumor; Diterpenes, Kaurane - pharmacology; Diterpenes, Kaurane - therapeutic use; Dose-Response Relationship, Drug; Down-Regulation; Free Radical Scavengers - pharmacology; Glutathione - pharmacology; Humans; Hydrogen Peroxide - pharmacology; hydroxyl radical; Hydroxyl Radical - metabolism; Iron - pharmacology; Isodon - chemistry; Membrane Potential, Mitochondrial - drug effects; Membrane Potential, Mitochondrial - physiology; oridonin; Oxidative Stress - drug effects; Phytotherapy; Plant Extracts - pharmacology; Plant Extracts - therapeutic use; Protein-Tyrosine Kinases - metabolism; reactive oxygen species; Signal Transduction
• ISSN: 0918-6158; 1347-5215
• DOI: 10.1248/bpb.b12-00405

Oridonin, a diterpenoid compound extracted and purified from Rabdosia rubescen, has been reported to induce tumor cell apoptosis through tyrosine kinase pathway. To further examine the mechanism of oridonin, we selected human epidermoid carcinoma A431 cell as a test object. Besides apoptosis, oridonin also induced A431 cell autophagy, and this autophagy antagonized apoptosis and played a protective role for A431 cells. Reactive oxygen species (ROS) played a pivotal role in induction of cytotoxicity. Therefore, a ROS scavenger, N-acetylcysteine (NAC) combined with oridonin was appiled. Results of morphologic observation, flow cytometric analysis and Western blot analysis showed that NAC could significantly reverse both ROS generation and down-regulation of mitochondrial membrane potential in oridonin treated cells. NAC inhibited oridonin induced apoptosis through both the intrinsic and extrinsic apoptotic pathways. NAC effectively inhibited both oridonin-induced apoptosis and autophagy by reducing intracellular oxidative stress. To further examine the mechanism of ROS, exogenous enzyme antioxidants (superoxide dismutase (SOD), catalase (CAT)) and non-enzyme antioxidants (glutathione (GSH)) were applied to detect the effect of oridonin on ROS generation. Only GSH exerted a similar role with NAC, suggesting that hydroxyl radical (·OH) played the major role in oridonin-induced cell death. Oridonin could decrease the GSH level in A431 cells in a dose-dependent manner. In addition, after treatment with ·OH donor, Fenton reagent, the changes in A431cells were similar to the results of oridonin treatment. All the results proved that ·OH played the pivotal role in oridonin induced apoptosis and autophagy in A431 cells.