Hindawi Publishing Corporation Evidence-Based Complementary and Alternative Medicine Volume 2013, Article ID 354840, 9 pages http://dx.doi.org/10.1155/2013/354840 Research Article Evodiamine Induces Transient Receptor Potential Vanilloid-1-Mediated Protective Autophagy in U87-MG Astrocytes Ann-Jeng Liu,1,2 Sheng-Hao Wang,3 Sz-Ying Hou,3,4 Chien-Ju Lin,3 Wen-Ta Chiu,1,5 Sheng-Huang Hsiao,2 Thay-Hsiung Chen,6,7 and Chwen-Ming Shih3,4,8 1 Graduate Institute of Clinical Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan 2 Department of Neurosurgery, Taipei City Hospital Ren-Ai Branch, Taipei, Taiwan 3 Department of Biochemistry, School of Medicine, Taipei Medical University, 250 Wu-Hsing Street, Taipei 110, Taiwan 4 Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei, Taiwan 5 Department of Neurosurgery, Taipei Municipal Wan-Fang Hospital, Taipei, Taiwan 6 Department of Surgery, College of Medicine, Taipei Medical University, Taiwan 7 Division of Cardiac Surgery, Cathy General Hospital, Taipei, Taiwan 8 Traditional Herbal Medicine Research Center, Taipei Medical University Hospital, Taipei, Taiwan Correspondence should be addressed to Thay-Hsiung Chen; [email protected] and Chwen-Ming Shih; [email protected] Received 21 October 2013; Accepted 23 November 2013 Academic Editor: Joen-Rong Sheu Copyright © 2013 Ann-Jeng Liu et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Cerebral ischemia is a leading cause of mortality and morbidity worldwide, which results in cognitive and motor dysfunction, neurodegenerative diseases, and death. Evodiamine (Evo) is extracted from Evodia rutaecarpa Bentham, a plant widely used in Chinese herbal medicine, which possesses variable biological abilities, such as anticancer, anti-inflammation, antiobesity, anti- Alzheimer’s disease, antimetastatic, antianoxic, and antinociceptive functions. But the effect of Evo on ischemic stroke is unclear. Increasing data suggest that activation of autophagy, an adaptive response to environmental stresses, could protect neurons from ischemia-induced cell death. In this study, we found that Evo induced autophagy in U87-MG astrocytes. A scavenger of extracellular calcium and an antagonist of transient receptor potential vanilloid-1 (TRPV-1) decreased the percentage of autophagy accompanied by an increase in apoptosis, suggesting that Evo may induce calcium-mediated protective autophagy resulting from an influx of extracellular calcium. The same phenomena were also confirmed by a small interfering RNA technique to knockdown the expression of TRPV1. Finally, Evo-induced c-Jun N-terminal kinases (JNK) activation was reduced by a TRPV1 antagonist, indicating that Evo-induced autophagy may occur through a calcium/c-Jun N-terminal kinase (JNK) pathway. Collectively, Evo induced an influx of extracellular calcium, which led to JNK-mediated protective autophagy, and this provides a new optionfor ischemic stroke treatment. 1. Introduction that protective autophagy is induced in a stroke mode since blockadeofautophagyresultsinanincreaseincelldeath Brain ischemia, a restriction of the blood supply to tissues, [1] suggests an agent possessing autophagy-inducing ability causing a shortage of oxygen and glucose needed for cellular which may provide a new option for ischemic stroke treat- metabolism, is the leading cause of death and disability ment. worldwide. Tissue plasminogen activator (tPA) therapy is the Evodiamine(Evo)isaquinozolealkaloidisolatedfrom major treatment for ischemic stroke. However, the window Evodia rutaecarpa Bentham which is widely used in Chinese for tPA treatment is within 0∼3hafteronsetofastroke,which herbal medicine with variable effects, such as anticancer, limits its clinical use. Accumulating evidence demonstrating anti-inflammation, anti-obesity, anti-Alzheimer’s disease, 2 Evidence-Based Complementary and Alternative Medicine antimetastatic, antianoxic, and anti-nociceptive functions 2. Materials and Methods [2]. An in vitro study showed that Evo has an endothelium- dependent vasodilatory effect on isolated rat mesenteric 2.1. Cell Culture, Treatment, and Chemicals. Human U87-MG arteries [3]. Furthermore, Evo may serve as an antiatheroscle- astrocytes (obtained from a Caucasian strain) classified as grade IV glioblastoma were purchased from the American rosis agent since it can inhibit oxidative stress-induced ∘ production of chemokine receptor (CCR)-1, CCR2, and intra- TypeCultureCollection(Manassas,VA)andgrownat37C cellular adhesion molecule (ICAM)-1 [4, 5], suggesting that in culture medium consisting of Dulbecco’s modified Eagle’s Evo may possess anticardiovascular disease activity. It was medium (DMEM) supplemented with 10% heat-inactivated reported that Evo protected rats from myocardial ischemia- fetal bovine serum (FBS), 200 mM L-glutamine, 100 U/mL reperfusion injury [6].However,theeffectofEvoonischemic penicillin, 100 g/mL streptomycin, 100 mM sodium pyru- stroke in the brain is not well understood. vate, and 1% nonessential amino acids. The mixture was Evo is considered a transient receptor potential vanilloid- kept in a humidified atmosphere containing 5%2 CO .For 1 (TRPV1) agonist [7]. TRPV1, a ligand-gated calcium ion the drug-response experiments, cells were either pretreated channelactivatedbyvanilloids,protons,andvariousenviron- (treatment group) or not (control group) with the indicated mental stresses, was implicated as a pain-sensing transducer inhibitor for 1 h and then incubated with 6 MEvofortherest and plays a key role in regulating cell death. Capsaicin, a of the experimental period [16]. DMEM, FBS, and nonessen- well-known TRPV1 agonist, extracted from hot chili peppers, tial amino acids were purchased from Hyclone (Logan, UT), was shown to lead to human nasopharyngeal carcinoma and phenol red-free RPMI, L-glutamine, penicillin-strepto- cell death through mitochondrial and endoplasmic reticular my-cin, and sodium pyruvate were obtained from Gibco (ER) stress [8]. Furthermore, capsaicin induced apoptosis (Grand Island, NY). Evo, bovine serum albumin (BSA), in glioma cells through a p38-mediated signal pathway [9]. acridine orange (AO), Fluo-3 AM, 5,5,6,6,-tetrachloro- However, capsazepine (CPZ), a TRPV1 antagonist potenti- 1,1,3,3,-tetraethylbenzimidazolylcarbocyanine iodide (JC-1), ated the anticancer effect of tumor necrosis factor-related ethylene glycol tetra-acetic acid (EGTA), CPZ, and 3- apoptosis-inducing ligand (TRAIL) through upregulation (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide of death receptors [10]. Therefore, the role of TRPV1 in (MTT)werepurchasedfromSigma(St.Louis,MO).Pro- regulating apoptosis is controversial, and the effect of TRPV1 pidium iodide (PI) was purchased from Calbiochem (San onautophagyneedstobeinvestigated. Diego, CA). The Annexin-V-FITC reagent was supplied by Autophagy, an evolutionarily conserved mechanism reg- Biovision (Mountain View, CA). Rabbit polyclonal anti-LC3 ulating the turnover of long-lived proteins and damaged was obtained from MBL International (Nagoya, Japan). Rab- organelles, is also considered type II programmed cell death. bit polyclonal anti-GAPDH and anti-TRPV1 were obtained Autophagy is characterized by the formation of double- from Cell Signaling (Beverly, MA). Secondary anti-bodies, membrane vesicles (autophagosomes) and the processing including horseradish peroxidase- (HRP-) conjugated goat of microtubule-associated protein 1 light chain 3 (LC3). anti-rabbit immunoglobulin G (IgG), were purchased from Dysfunction of autophagy may lead to cancer development, Pierce (Rockford, IL). Polyvinylidene difluoride (PVDF) bacterial and viral infections, neurodegenerative disorders, membranes were supplied by Millipore (Bedford, MA), and and cardiovascular diseases [11, 12]. The role of autophagy the Protein Assay Dye Reagent was from Bio-Rad (Hercules, in ischemia has been widely investigated. Rapamycin, an CA). autophagy inducer, protected against hypoxia-induced brain injury through induction of autophagy [13]. Superoxide dismutase (SOD)-2 knockdown led to ischemic brain dam- 2.2. Measurement of Acidic Vesicular Organelles (AVOs). age under a hyperglycemic condition through induction of Autophagy was analyzed by flow cytometry with AO dye oxidative stress, which was associated with a reduction in according to published procedures [17]. After the indicated autophagy [14]. Furthermore, melatonin-induced neuronal treatment, cells were stained with AO (1 g/mL) for a period protection against cell death resulting from glucose-oxygen of 20 min. Trypsinized adherent cells and cells suspended deprivation was abolished by the autophagy inhibitor, 3- in the medium were collected in phenol red-free RPMI medium. Green (510∼530 nm) and red (650 nm) fluorescence methyladenine (3-MA) [15]. All of the above suggest that 4 autophagy may play a protective role in ischemia-induced emissions from 10 cells illuminated with blue (488 nm) neuronal damage. excitation light were measured on a flow cytometer using Human U87-MG astrocytes (obtained from a Caucasian CellQuest software (Becton Dickinson, San Jose, CA). The strain) classified as grade IV glioblastoma were employed percentage of autophagy was summed across the upper-left as a cell model in this study. We found that Evo induced and upper-right quadrants. protective autophagy of astrocytes.