Cytotoxic effects of gastrodin extracted from the rhizome of Gastrodia elata Blume in glioblastoma cells, but not in normal astrocytes, via the induction of oxidative stress-associated apoptosis that involved cell cycle arrest and p53 activation

Food and Chemical Toxicology 107 (2017) 280e292

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Cytotoxic effects of gastrodin extracted from the rhizome of Gastrodia elata Blume in glioblastoma cells, but not in normal astrocytes, via the induction of oxidative stress-associated apoptosis that involved cell cycle arrest and p53 activation Wei-Zhe Liang a, Chung-Ren Jan a, Shu-Shong Hsu b, c, * a

Department of Medical Education and Research, Kaohsiung Veterans General Hospital, Kaohsiung 81362, Taiwan, ROC Department of Surgery, Kaohsiung Veterans General Hospital, Kaohsiung 81362, Taiwan, ROC c Department of Surgery, National Defense Medical Center, Taipei 11490, Taiwan, ROC b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 25 February 2017 Received in revised form 29 June 2017 Accepted 5 July 2017 Available online 6 July 2017

Researches have been conducted to explore the biological effect of gastrodin, a natural compound extracted from the rhizome of Gastrodia elata Blume, in different models. However, the effects of gastrodin on cytotoxicity, cell cycle distribution and oxidative stress in glia cells have not been explored. The aim of this study was to investigate the cytotoxic effect of gastrodin and its mechanisms in DBTRG-05MG human glioblastoma cells and CTX TNA2 rat astrocytes. In DBTRG-05MG cells but not in CTX TNA2 cells, gastrodin (20-30 mM) induced cytotoxicity, G2/M phase cell cycle arrest and apoptosis. Regarding oxidative stress, gastrodin (20-30 mM) elevated intracellular ROS levels but reduced GSH levels. Treatment with the antioxidant NAC (10 mM) partially reversed gastrodin-altered antioxidant enzymes levels. Furthermore, gastrodin induced mitochondria-associated apoptosis. The apoptotic effects evoked by gastrodin were partially inhibited by the antioxidant NAC and the pancaspase inhibitor Z-VAD-FMK. Together, in DBTRG-05MG cells, but not in CTX TNA2 cells, gastrodin activated ROS-associated mitochondrial apoptotic pathways that involved cell cycle arrest. These data provide insight into the molecular mechanisms governing the ability of gastrodin to induce cytotoxicity in human glioblastoma cells and further suggest that gastrodin is a new potential agent for the treatment of human gliblasoma. © 2017 Elsevier Ltd. All rights reserved.

Keywords: Gastrodin Glia cells Cytotoxicity Cell cycle arrest Oxidative stress Mitochondrial apoptotic pathway

1. Introduction Gliomas are the most common primary brain tumors, and they constitute approximately 30% of all brain and central nervous system tumors, and 80% of all malignant brain tumors (Ohgaki and Kleihues, 2005). The World Health Organization (WHO) classification of tumors of the central nervous system divides glioma into grades I-IV, in which grades I and II are classified as low grade, and grades III and IV are defined as high grade (known as malignant glioma) (Louis et al., 2007). Glioblstoma multiforme (GBM) is the most malignant form of brain tumor in adults and is classified as a grade IV astrocytoma (Ohgaki and Kleihues, 2005). Despite recent advances in treatment options such as surgery, chemotherapy and

* Corresponding author. Department of Surgery, Kaohsiung Veterans General Hospital, Kaohsiung 81362, Taiwan, ROC. E-mail address: [email protected] (S.-S. Hsu). http://dx.doi.org/10.1016/j.fct.2017.07.013 0278-6915/© 2017 Elsevier Ltd. All rights reserved.

radiotherapy, patients with GBM demonstrate a low median survival time of <2 years (Ohgaki and Kleihues, 2005). Temozolomide is currently the mainstream chemotherapeutic agent for GBM, but most patients display chemosensitivity to temozolomide at the beginning of therapy; quickly acquired temozolomide resistance and serious side effects have become major hampers for its application in GBM (Oh et al., 2010). Therefore, it is urgent for clinical practice to discover novel natural compounds with higher efficacy and lower toxicity to overcome the chemotherapeutic resistance of GBM (Cragg and Newman, 2005). The use of natural compounds for the treatment of human diseases begin with the history of humanity, and represents the oldest and most widespread form of medication due to their healing properties (Cragg and Newman, 2005). Some types of natural compounds are important as a source of effective anticancer agents (Cragg and Newman, 2005). Gastrodia elata Blume is a traditional Chinese medicine often used for the treatment of headache, convulsions, hypertension and neurodegenerative diseases (Bulpitt

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et al., 2007). Gastrodin, which is extracted from the rhizome of Gastrodia elata Blume (Zhan et al., 2016), has been shown to cause various physiological effects in different models. In non-brain research, gastrodin was found to stimulate anticancer immune response and repress transplanted H22 hepatic ascitic tumor cell growth (Shu et al., 2013), and to inhibit cardiac hypertrophy and fibrosis (Shu et al., 2012). In brain research, gastrodin was shown to suppress amyloid b-induced neurotoxicity in primarily cultured rat hippocampal neurons (Zhao et al., 2012) and neural progenitor cells (Li and Qian, 2016), to inhibit neuroinflammation in rotenoneinduced Parkinson's disease model rats (Li et al., 2012), and to inhibit hypoxia-induced toxicity in primary cultures of rat cortical neurons (Xu et al., 2007). However, the effect of gastrodin on cyotoxicity in human glioblastoma cells is largely unclear. Apoptosis is a vital mode of programmed cell death, which involves the genetically determined elimination of cells (Hengartner, 2000). Apoptosis occurs normally during development and aging and acts as a mechanism to maintain normal cell populations in tissues (Hengartner, 2000). Previous studies have shown that natural compounds exert their anticancer activity by inducing apoptosis (Amin et al., 2009). The cell cycle is controlled by a complex series of signaling pathways by which a cell grows, replicates its DNA and divides (Nigg, 1995). This process includes mechanisms to ensure errors are corrected, and if the correction cannot be performed, cells enter into the apoptotic process (Adams, 2003). However, in cancer cells, this regulatory process malfunctions and results in uncontrolled cell proliferation (Adams, 2003). Reactive oxygen species (ROS) have important multifaceted roles in regulating cell physiology, including cancer cell death (Circu and Aw, 2010). ROS have been shown to induce oxidative stress-associated apoptosis by directly oxidizing or triggering various downstream molecules in cancer models (Zeng et al., 2013). Therefore, the relationship between induction of apoptosis, modulation of cell cycle and production of ROS is very important in cancer treatment. Gastrodin has been reported to have therapeutic benefits against neurodegenerative diseases (Hu et al., 2014), but whether gastrodin causes cytotoxic effects in human glioblastoma cells and normal astrocytes is unknown. Therefore, the aim of this study was to explore the mechanism underlying effects of gastrodin on apoptosis, modulation of cell cycle and ROS signaling, and to establish their relationship in DBTRG-05MG human glioblastoma cells and CTX TNA2 rat astrocytes. DBTRG-05MG cells were established from tissues of a Caucasian female patient with GBM who had been treated with local brain irradiation and multidrug chemotherapy. This cell is well-differentiated, transformed, tumorigenic, and suitable for research on cultured glioblastoma cells (Kruse et al., 1992). Furthermore, DBTRG-05MG cells were used because they produce measurable cytotoxicity upon pharmacological stimulation. It has been shown that several natural compounds could induce cytotoxicity in this cell line such as nbutylidenephthalide (Tsai et al., 2006) and ursolic acid (Lu et al., 2014). CTX TNA2 cells were established from primary cultures of type 1 astrocytes from brain frontal cortex tissue of 1 day old rats. Previous studies have shown that CTX TNA2 cells maintain the in vivo phenotype of a primary porcine in vitro blood brain barrier model (Cantrill et al., 2012) and can be applied for glutamateinduced mammalian traumatic brain injury in vitro model (Lin et al., 2014). Therefore, this cell is often used as a normal control in glia studies (Cantrill et al., 2012; Lin et al., 2014).

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purity of gastrodin (purity
98%) was isolated from the rhizome of Gastrodia elata Blume and determined by HPLC densitometry (Fig. 1A). The reagents for cell culture were from Gibco® (Gaithersburg, MD, USA). N-acetylcysteine (NAC) was from Molecular Probes® (Eugene, OR, USA). The pancaspase inhibitor N-Benzyloxycarbonyl-Val-Ala-Asp (O-Me) fluoromethyl ketone (Z-VAD-FMK) was from Calbiochem® (La Jolla, CA, USA). 2.2. Cell culture DBTRG-05MG human glioblastoma cells and CTX TNA2 rat astrocytes purchased from Bioresource Collection and Research Center (Taiwan) were cultured in RPMI-1640 medium (for DBTRG05MG cells) or Dulbecco's modified Eagle's medium (DMEM, for CTX TNA2 cells) supplemented with 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin and 100 mg/ml streptomycin at 37  C in a humidified 5% CO2 atmosphere. 2.3. Experimental solutions Lysis buffer (pH 7.5) contained 20 mM Tris, 150 mM NaCl, 1 mM ethylene diaminetetraaceticacid (EDTA), 1 mM ethylene glycolbis(b-aminoethyl ether)-N,N,N0 ,N'-tetraacetic acid (EGTA), 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM b-glycerophosphate, 1 mM Na3VO4, 1 mg/ml leupeptin and 1 mM phenylmethylsulfonyl fluoride (PMSF). Tris-buffered saline Tween 20 (TBST, pH 7.5) contained 25 mM Tris, 150 mM NaCl and 0.1% (v/v) Tween 20. Phosphate buffer saline (PBS, pH 7.4) contained 137 mM NaCl, 10 mM phosphate, 2.7 mM KCl. A 20 mg/ml (~70 mM) stock solution of gastrodin was prepared in dimethyl sulfoxide (DMSO) and stored at 80  C. The other reagents were dissolved in water, ethanol or DMSO. The concentration of organic solvents in the experimental solution was less than 0.1%, and did not alter cell viability. 2.4. Cell viability assays The assay was based on cleavage of the tetrazolium salt WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3benzene disulfonate) by active mitochondria to produce a colored formazan salt. The intensity of color correlated with the percentage of live cells. Measurements were conducted following manufacturer's instructions (Roche Molecular Biochemical, Indianapolis, IN, USA). Cells were seeded in 96-well plates at a density of 1  104 cells/well in culture medium for 24 h in the presence of gastrodin. The cell viability detecting reagent WST-1 (10 ml pure solution) was added to samples after treatment with gastrodin, and cells were incubated for 2 h at 37  C in a humidified atmosphere with 5% CO2. In experiments using the antioxidant NAC to scavenger intracellular ROS production or the pancaspase inhibitor ZVAD-FMK to block proteolytic caspase activation, cells were treated with 10 mM NAC or 10 mM Z-VAD-FMK for 1 h before addition of gastrodin. The cells were washed once with PBS and incubated with/without gastrodin for 24 h. The absorbance of samples (A450) was determined using a 96-well microplate reader (model MRX II, Dynex Technologies, Chantily, VA, USA). Absolute optical density was normalized to the absorbance of unstimulated cells in each plate and expressed as a percentage of the control value, which was treated with vehicle only (0.1% DMSO), taken as 100% growth. 2.5. Cell cycle distribution by PI staining

2. Materials and methods 2.1. Chemicals Gastrodin was from Sigma-Aldrich® (St. Louis, MO, USA). The

The PI dye (Molecular Probes, Eugene, OR, USA) was used to measure the cell cycle distribution as instructed by the manufacturer. Briefly, PI dye can bind and label DNA and make it possible to obtain a rapid and precise evaluation of cellular DNA content by

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flow cytometric analysis and subsequent identification of hypodiploid cells (Nicoletti et al., 1991). For flow cytometric analysis of subG1 cell counting with fragmented DNA and cell cycle distribution, 1  106 cells per dish were collected after treatments with different concentrations of gastrodin (0, 10 mM, 20 mM or 30 mM) for 24 h. The cells were harvested and incubated with 1 ml of 70% cold ethanol for 2 h at 20  C and then washed with PBS. Cell pellets were incubated with 0.1 mg/ml RNase at 37  C for 30 min before adding 20 mg/ml PI. After incubation with PI for 1 h, the cells were analyzed in a FACScan flow cytometry analyzer (Becton Dickinson, Mountain View, CA, USA). PI was excited at 488 nm, and fluorescence was analyzed at 620 nm wavelength. Data were later analyzed using the flow cytometry analysis software WinMDI 2.8 (by Joe Trotter, freely distributed software), which is also used to determine the percentage of subG1 phase, G0/G1 phase, S phase and G2/M phase. 2.6. Detection of intracellular ROS and glutathione (GSH) levels Cells cultured on 6-well plates were treated with 0e30 mM gastrodin for 24 h. Subsequently, cells were trypsinized and made into suspensions (1  106 cells/ml). For measuring intracellular ROS levels, cells were treated with the oxidation-sensitive fluorescent probe dye dichlorofluorescein diacetate (DCFH-DA, Molecular Probes, Eugene, OR, USA) that responds to changes in intracellular redox status. Cells were incubated with 10 mM membranepermeable DCFH-DA for 30 min at 37  C. Inside cells, the acetate moieties of DCFH-DA were cleaved and oxidized, primarily by H2O2, to green fluorescent 20 -7-’-dichlorofluorescein (DCF) (excitation/ emission, 488/530 nm) (Rothe and Valet, 1990). Intracellular GSH levels were analyzed using 5-chloromethylfluorescein diacetate (CMFDA, Molecular Probes, Eugene, OR, USA). CMFDA is a useful membrane-permeable dye (excitation/emission, 492/517 nm) for determining levels of intracellular GSH as previously described
et al., 2003). Cells were incubated with 5 mM CMFDA at (Sebastia 37  C for 30 min according to the manufacturer's instructions. Cytoplasmic esterases convert nonfluorescent CMFDA to fluorescent 5-chloromethylfluorescein (CMF), which can then react with GSH (excitation/emission, 522/595 nm). Flow cytometry was performed by using a FACScan flow cytometry analyzer. A 15-mm aircooled argon-ion laser was used to excite fluorescent DCF or CMF, and the emitted fluorescence was measured using a band-pass optical filter. Samples were run using 1  104 cells per test sample. Data were later analyzed using the flow cytometry analysis software WinMDI 2.8. For intracellular ROS levels, M1 represents the percentage of intracellular ROS production. A baseline level of ROS counts was obtained when cells were incubated with PBS alone (without 10 mM DCFH-DA) for 30 min at 37  C. The baseline level of ROS counts was 0.05%. For intracellular GSH levels, M1 represents the percentage of reduced GSH content. The baseline level of reduced GSH counts was obtained when cells were incubated with 5 mM CMFDA only for 30 min at 37  C. The baseline level of reduced GSH counts was 100%. M2 represents the percentage of depleted GSH content. The baseline level of depleted GSH counts was obtained when cells were

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incubated with PBS only (without 5 mM CMFDA) for 30 min at 37  C. The baseline level of depleted GSH counts was 0.05%. 2.7. Detection of mitochondrial ROS levels Mitochondrial ROS levels were analyzed using dihydrorhodamine 123 (DHR123, Molecular Probes, Eugene, OR, USA). The lipophilicity of DHR123 facilitates its diffusion across cell membranes. Upon oxidation of DHR to the fluorescent rhodamine 123, one of the two equivalent amino groups tautomerizes into an imino, effectively trapping rhodamine 123 within cells (Crow, 1997). Briefly, cells cultured on 6-well plates were treated with 0e30 mM gastrodin for 24 h. Subsequently, cells were trypsinized and made into suspensions (1  106 cells/ml). Cells were incubated with 1 mM membrane-permeable DH123 for 25 min at 37  C. Inside mitochondria, nonfluorescent DH123 was converted to fluorescent rhodamine 123, which can then react with H2O2 (excitation/emission, 505/529 nm). After being washed twice, flow cytometry was performed by using a FACS420 flow cytometer analyzer (Becton Dickinson, Mountain View, CA, USA). A 15-mm air-cooled argon-ion laser was used to excite fluorescent rhodamine 123, and the emitted fluorescence was measured using a band-pass optical filter. Samples were run using 1  104 cells per test sample. Data were later analyzed using the flow cytometry analysis software WinMDI 2.8. For mitochondrial ROS levels, M1 represents the percentage of mitochondrial ROS production. A baseline level of ROS counts was obtained when cells were incubated with PBS alone (without 1 mM dihydrorhodamine 123, DHR123) for 25 min at 37  C. The baseline level of ROS counts was 0.05%. 2.8. Analyses of antioxidant enzyme activities Cells cultured on 6-well plates were treated with 0e30 mM gastrodin for 24 h. Following incubation, the cells were washed with PBS and detached using a scraper. Cells were then lysed in 1 ml of cold PBS using a sonicator, centrifuged for 10 min at 1  104 rpm at 4  C and the resulting supernatant was used for the antioxidant enzyme assays. 2.8.1. Superoxide dismutase (SOD) assay This assay was performed using the SOD Assay Kit (Cayman Chemicals, MI, USA). The assay uses tetrazolium salt for detection of SOD generated by xanthine oxidase. The assay was performed according to the manufacturer's instructions. Equal amount of protein fractions were incubated with reaction mixture containing the tetrazolium salt for 20 min. The absorbance of each standard and sample was read at 450 nm using a 96-well microplate reader. SOD activity was calculated using the linear regression fit of the standard curve data. All assays were repeated three times. One unit is defined as the amount of enzyme needed to catalyze 50% dismutation of the superoxide radical. SOD activity was expressed in U/ml per 1  106 cells. 2.8.2. Glutathione peroxidase (GPx) assay This assay was performed using the GPx Assay Kit (Cayman

Fig. 1. Effect of gastrodin on cell viability, cell cycle distribution, and cell cycle-related protein expression levels in DBTRG-05MG cells. (A) The chemical structure of gastrodin. (B) Cells were treated with 0e50 mM gastrodin for 24 h, and cell viability assay was conducted in DBTRG-05MG cells. Data are mean ± SEM of three separate experiments. Each treatment had six replicates (wells). Data are expressed as percentage of control that is the increase in cell numbers in gastrodin-free groups. Control had 11,167 ± 567 cells/well before experiments, and 13,338 ± 776 cells/well after incubation for 24 h *P < 0.05 compared to control. (C) Representative flow histograms depicting cell cycle distribution in DBTRG-05MG cells treated with gastrodin (0e30 mM) for 24 h. Numbers in the panels represent the percentage of cells in different phases of the cell cycle. (D) Cell cycle distribution was presented in DBTRG-05MG cells when treated with gastrodin (0e30 mM) for 24 h. Data are presented as mean ± SEM of three experiments. *P < 0.05 compared with control. (E) Protein extracts were prepared 24 h after exposure to various concentrations of gastrodin. The effect of gastrodin on CDK1/CDC2, cyclin B1, p53, and p21 (Waf1/Cip1) levels as quantified by densitometry. The figure normalized intensities of the bands of CDK1/CDC2, cyclin B1, p53, p27, and p21 (Waf1/Cip1) levels against the bands of b-actin using NIH image 1.61. *P < 0.05 compared to control.

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Chemicals, MI, USA). This experiment measures GPx activity through a coupled reaction with glutathione reductase. The assay was performed according to the manufacturer's instructions. After the addition of assay buffer and co-substrate mixture, the absorbance of each standard and sample was read at 340 nm using a 96well microplate reader. All assays were repeated three times. One unit is defined as the amount of enzyme that caused the oxidation of 1.0 nmol of NADPH to NADPþ per minute at 25  C. GPx activity was expressed in nmol/min/ml per 1  106 cells.

(excitation/emission, 638/658 nm) at 37  C in the dark for 30 min. Labeled cells were washed once with PBS, the cells were collected and

2.8.3. Catalase (CAT) assay This assay was performed using the CAT Assay Kit (Cayman Chemicals, MI, USA). The assay is based on the reaction of CAT with methanol in the presence of hydrogen peroxide, producing formaldehyde which is measured colorimetrically using 4-amino-3hydrazino-5-mercapto-1,2,4-triazole (Purpald) as the chromogen. Purpald forms a bicyclic heterocycle with aldehydes, which upon oxidation changes from colorless to a purple color. The assay was performed according to the manufacturer's instructions. After the addition of reaction mixture, the absorbance of each standard and sample was read at 540 nm using a 96-well microplate reader. Enzyme activity was then standardized to mg protein. All assays were repeated three times. CAT activity in each sample was expressed in nmol/min/ml. One unit is defined as the amount of enzyme that caused the formation of 1.0 nmol of formaldehyde per minute at 25  C. The CAT activity was expressed in nmol/min/ml per 1  106 cells. 2.9. Alexa ®Fluor 488 annexin V/propidium iodide (PI) staining for apoptosis Cells were treated with 10 mM NAC or 10 mM Z-VAD-FMK for 1 h prior to incubation with gastrodin. Cells were subsequently exposed to gastrodin at concentrations of 0 mM, 20 mM or 30 mM for 24 h. Cells were harvested after incubation and washed in 4  C PBS. Cells were resuspended in 400 ml solution with 10 mM of 4-(2hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 140 mM of NaC1, 2.5 mM of CaC12 (pH 7.4). Alexa ®Fluor 488 annexin V/PI staining solution (Probes Invitrogen, Eugene, OR, USA) was added in the dark. After incubation for 15 min, the cells were collected and analyzed in a FACScan flow cytometry analyzer. Excitation wavelength was at 488 nm and the emitted green fluorescence of annexin V (FL1) and red fluorescence of PI (FL2) were collected using 530 nm and 575 nm bands pass filters, respectively. A total of at least 2  104 cells were analyzed per sample. Light scatter was measured on a linear scale of 1024 channels and fluorescence intensity was on a logarithmic scale. The amount of early apoptosis and late apoptosis/necrosis were determined, respectively, as the percentage of annexin Vþ/PI or annexin Vþ/PIþ cells. Data were later analyzed using the flow cytometry analysis software WinMDI 2.8. The x and y coordinates refer to the intensity of fluorescence of annexin V and PI, respectively. 2.10. Measurements of mitochondrial membrane potential To measure the mitochondrial membrane potential, the MitoProbe™ 1,1 0,3,3,30 ,3'-hexamethylindodicarbocyanine iodide (DiIC1) (5) assay kit (catalog number M34151, Molecular Probes, Eugene, OR, USA) was used as instructed by the manufacturer. Briefly, cells were treated with 10 mM NAC for 1 h prior to incubation with gastrodin. Cells were then exposed to gastrodin at concentrations of 0 mM, 20 mM or 30 mM for 24 h. Cells (including floating cells) grown in 6-well plates were collected following mild trypsinization. Trypsinized cells were washed once with PBS, and the cells were resuspended in 500 ml of PBS. Resuspended cells were labeled with 50 nM DiIC1 (5)

Fig. 2. Effect of gastrodin on intracellular/mitochondrial ROS level, and intracellular GSH level in DBTRG-05MG cells. (A) A representative histogram showing differences between DCF fluorescence intensity in untreated cells and cells treated with 0e30 mM gastrodin for 24 h *P < 0.05 compared to control. Data are mean ± SEM of three separate experiments. (B) A representative histogram showing differences between rhodamine 123 fluorescence intensity in untreated cells and cells treated with 0e30 mM gastrodin for 24 h *P < 0.05 compared to control. Data are mean ± SEM of three separate experiments. (C) A representative histogram showing differences between CMF fluorescence intensity in untreated cells and cells treated with 0e30 mM gastrodin for 24 h *P < 0.05 compared to control. Data are mean ± SEM of three separate experiments.

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analyzed in a FACSCalibur flow cytometry analyzer (Becton Dickinson, Mountain View, CA, USA). Data were later analyzed using the flow cytometry analysis software WinMDI 2.8. For mitochondrial membrane potential levels, M1 represents the percentage of total mitochondrial transmembrane potential cells. The baseline level of total mitochondrial transmembrane potential cells was obtained when cells were incubated only with 10 mM DiIC1 (5) for 30 min at 37  C. The baseline level of total mitochondrial transmembrane potential cells was 100%. M2 represented the percentage of low-mitochondrial transmembrane potential cells. The baseline of low-mitochondrial transmembrane potential cells was 0.05%. 2.11. Assessment of cell cycle-related proteins and apoptotic-related proteins by Western blotting analysis Cells were seeded on 6 cm culture dishes (3  106 cells/dish). After cells were grown to confluence, cells were treated with gastrodin for 24 h. The treatments were terminated by aspirating the supernatant and then washing the dishes with PBS. The cells were lysed on ice for 5 min with 70 ml of lysis buffer. The cell lysates were centrifuged to remove insoluble materials, and the protein concentration of each sample was measured. Approximately 50 mg of supernatant protein from each sample was used for gel electrophoresis analysis on a 10% SDS-polyacrylamide gel. The fractionated proteins on gel were transferred to PVDF membranes (NEN™ Life Science Products, Inc., Boston, MA, USA). For Western blotting, the membranes were blocked with 5% nonfat milk in TBST and incubated overnight with the primary antibody [rabbit anti-human Bax (catalog number 5023) (working concentration, 1 mg/ml), Bcl-2 (catalog number 2872) (working concentration, 1 mg/ml), cytosolic cytochrome c (catalog number 11940) (working concentration,1 mg/ml), cleaved caspase-9 (catalog number 9505) (working concentration, 1 mg/ml), cleaved caspase-3 (catalog number 9661) (working concentration, 1 mg/ml), cyclin-dependent kinase 1 (CDK1/CDC2) (catalog number 4539) (working concentration, 1 mg/ml), cyclin B1 (catalog number 12231) (working concentration,1 mg/ml), p53 (catalog number 2527) (working concentration, 1 mg/ml), p21 (Waf1/Cip1) (catalog number 2947) (working concentration, 1 mg/ml), or mouse anti-human b-actin (catalog number 3700) (working concentration, 0.2 mg/ml); Cell Signaling Technology, Beverly, MA, USA]. Then the membranes were extensively washed with TBST and incubated for 1 h with the secondary antibody [goat anti-rabbit antibody (catalog number 386325) (working concentration, 2 mg/ml) or goat anti-mouse antibody (catalog number 384924) (working concentration, 2 mg/ml); Transduction Laboratories, Lexington, KY, USA]. After extensive washing with TBST, the immune complexes were detected by chemiluminescence using the Renaissance™ Western Blot Chemiluminescence Reagent Plus kit (NEN™ Life Science Products, Inc., Boston, MA, USA). 2.12. Statistics

Fig. 3. Effect of combination of NAC and gastrodin on antioxiadtive enzyme activity in DBTRG-05MG cells. The antioxidant NAC (10 mM) was added to cells followed by treatment with gastrodin in the medium. Antioxiadtive enzyme activity (SOD, GPx or CAT) was determined using commercial assay kits. (A) Data showing SOD activity in control and gastrodin-treated cells pretreated with and without NAC. SOD activity is expressed in U/ml per 1  106 cells. (B) Data showing GPx activity in control and gastrodin-treated cells pretreated with and without NAC. GPx activities are expressed in nmol/min/ml per 1  106 cells (C) Data showing CAT activity in control and

Data are reported as mean ± SEM of three separate experiments. Data were analyzed by one-way analysis of variances (ANOVA) using the Statistical Analysis System (SAS®, SAS Institute Inc., Cary, NC, USA). Multiple comparisons between group means were performed by post-hoc analysis using the Tukey's HSD (honestly significantly difference) procedure. A P-value less than 0.05 were considered significant. gastrodin-treated cells pretreated with and without NAC. CAT activities are expressed in nmol/min/ml per 1  106 cells. *P < 0.05 compared to control. #P < 0.05 compared to the pairing group. Data are mean ± SEM of three separate experiments.

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3. Results 3.1. Gastrodin concentration-dependently induced cytotoxicity and cell cycle arrest, and modulated cell cycle arrest-related protein expressions in DBTRG-05MG cells To investigate the effect of gastrodin on cell viability, DBTRG05MG cells were incubated with various concentrations of gastrodin for 24 h. Cell viability was subsequently measured by the WST-1 assay. In Fig. 1B, in comparison with the control group (without gastrodin), gastrodin (10e50 mM) significantly induced cytotoxicity after 24 h treatment in DBTRG-05MG cells (P < 0.05) (n ¼ 3). However, gastrodin (10e50 mM) did not affect cell viability after treatment for 24 h in CTX TNA2 cells (P > 0.05) (n ¼ 3) (Suppl. 1A). The IC50 (half maximal inhibitory concentration) value of gastrodin was approximately 25 mM in DBTRG-05MG cells. Furthermore, at the concentration of 50 mM, gastrodin caused cell death by 99.2% ± 0.6% in DBTRG-05MG cells. Therefore, the concentrations of 20 mM and 30 mM of gastrodin were chosen as optimal concentrations in subsequent analyses. To further investigate the cytotoxic effects of gastrodin on DBTRG-05MG cells, the cell cycle distribution was analyzed by flow cytometry. Representative flow histograms depicting cell cycle distribution following a 24 h exposure to gastrodin (0e30 mM) are shown in Fig. 1C. At the concentrations of 20 mM and 30 mM, gastrodin significantly increased the percentage of subG1 and G2/M phase cells, which was accompanied by the decreased percentage of G0/G1 phase cells in DBTRG-05MG cells (P < 0.05) (n ¼ 3) (Fig. 1D) but did not affect cell cycle distribution in CTX TNA2 cells (P > 0.05) (n ¼ 3) (Suppl. 1B and 1C). Therefore, cytotoxicity induced by gastrodin was related to subG1 cell counting with fragmented DNA and G2/M phase arrest in DBTRG-05MG cells. Because Fig. 1D shows that gastrodin induced G2/M phase arrest in DBTRG-05MG cells, the next issue was to detect the expression levels of G2/M phase cell cycle-related proteins in gastrodintreated cells. The G2/M phase arrest-related protein levels of CDK1/CDC2, cyclin B1, p53 and p21 (Waf1/Cip1) (Taylor and Stark, 2001) were investigated (Fig. 1E). Compared with control, treatment with gastrodin (20 mM or 30 mM) resulted in a concentrationdependent decrease (P < 0.05) (n ¼ 3) in the levels of CDK1 by 2.5 ± 0.2 folds or 4.4 ± 0.3 folds and cyclin B1 by 2.3 ± 0.2 folds or 3.9 ± 0.3 folds, respectively, but in a concentration-dependent increase (P < 0.05) (n ¼ 3) in the levels of p53 by 6.1 ± 0.2 folds or 7.5 ± 0.3 folds, and p21 by 5.1 ± 0.2 folds or 8.2 ± 0.3 folds, respectively. These findings suggest that gastrodin-induced G2/M phase cell arrest was associated with the modulation of CDK1, cyclin B1, p53 and p21 expressions. Because the major difference between CTX TNA2 cells and DBTRG-05MG cells is the status of p53 protein levels, experiments were performed to examine the effect of gastrodin on p53 status in CTX TNA2 cells, and the effect of combination of gastrodin and p53 siRNA on p53 expressions in DBTRG-05MG cells. The data show that CTX TNA2 cells did not express p53 protein levels. Treatment with gastrodin (20 or 30 mM) did not activate p53 expressions in CTX TNA2 cells (Suppl. 2). Furthermore, p53 knockdown reduced gastrodin-activated p53 expressions in DBTRG-05MG cells (Suppl. 3). Therefore, the results suggest that the difference of p53 expression is the probable key factor to cause gastodin-induced different sensitivity between DBTRG-05MG and CTX-TNA2 cells. 3.2. Gastrodin increased intracellular ROS levels without increasing mitochondrial ROS levels, and reduced intracellular GSH levels in DBTRG-05MG cells Since ROS, a natural byproduct of the normal metabolism of oxygen, plays a significant role in many biochemical functions

Fig. 4. Relationship between gastrodin-induced cytotoxicity and ROS production in DBTRG-05MG cells. (A) The antioxidant NAC (10 mM) was added to cells followed by treatment with gastrodin in the medium. Cell viability assay was subsequently performed. *P < 0.05 compared to control. #P < 0.05 compared to the pairing group. Data are mean ± SEM of three separate experiments. (BC) NAC (10 mM) was added to cells followed by treatment with gastrodin in the medium. Alexa ®Fluor 488 annexin V/PI staining for apoptosis was subsequently performed. *P < 0.05 compared to the pairing group. Data are mean ± SEM of three separate experiments.

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Fig. 5. Effect of combination of NAC and gastrodin on mitochondrial membrane potential in DBTRG-05MG cells. (A) Cells were treated with various concentrations of gastrodin for 24 h. In NAC group, NAC (10 mM) was added to cells followed by treatment with gastrodin in medium and staining with (DiIC1) (5) followed by incubation at 37  C for 30 min. The mean (DiIC1) (5) fluorescence intensity was detected with flow cytometry. (B) Data are expressed in percentage of cells displaying mitochondrial depolarization in gastrodin-treated groups compared with controls. *P < 0.05 compared to control. #P < 0.05 compared to the pairing group. Data are mean ± SEM of three separate experiments.

including cytotoxicity in cancer cells (Pelicano et al., 2004), efforts were made to explore the effect of gastrodin on intracellular/ mitochondrial ROS levels, and intracellular GSH levels. Fig. 2A shows that DCF fluorescence intensity (intracellular ROS levels) was increased by treatment with 20 or 30 mM gastrodin for 24 h in a concentration-dependent manner by 24.61 ± 0.65% or 49.67 ± 0.83%, respectively. However, at the concentrations of 20 or 30 mM gastrodin, DCF fluorescence intensity did not increase in CTX TNA2 cells (data not shown). In Fig. 2B, the exposure of cells to 20 or 30 mM gastrodin for 24 h did not increase the rhodamine 123 fluorescence intensity (mitochondrial ROS levels) in DBTRG-05MG cells. It suggests that gastrodin increased ROS production in cytoplasm but not in mitochondria in DBTRG-05MG cells. Fig. 2C shows that the M1 population of DBTRG-05MG cells had higher reduced

GSH content, as revealed by high CMF fluorescence, whereas M2 population exerted lower reduced form of GSH. Exposure of DBTRG-05MG cells to gastrodin for 24 h markedly increased the percentage of cells residing in the M2 population of 20.91% ± 0.51% (20 mM gastrodin) and 47.16% ± 0.71% (30 mM gastrodin), respectively. 3.3. Gastrodin regulated antioxidant enzyme activities in DBTRG05MG cells Since gastrodin increased ROS levels but reduced GSH levels in DBTRG-05MG cells (Fig. 2), efforts were made to examine whether gastrodin affected antioxidative enzyme activity (SOD, GPx or CAT levels) (Punnonen et al., 1994) in DBTRG-05MG cells. The data show

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that SOD levels (Fig. 3A) were increased by treatment with 20 mM or 30 mM gastrodin for 24 h but GPx and CAT levels were decreased (Fig. 3B and C). The following experiments were aimed to explore the effect of combination of NAC and gastrodin on SOD, GPx or CAT levels in DBTRG-05MG cells. NAC, served as an antioxidant to study the cytotoxicity in cancer cell research, was used to inhibit ROS production during gastrodin treatment (Zafarullah et al., 2003). NAC (10 mM) loading inhibited gastrodin (20e30 mM)-induced increases in ROS levels in medium (data not shown). This suggests that NAC effectively inhibited gastrodin-induced ROS production. In NAC-treated groups, NAC (10 mM) partially reversed 20 mM or 30 mM gastrodin-evoked increases in SOD levels by 1.9 ± 0.3 folds or 2.3 ± 0.3 folds (Fig. 3A) but partially reversed gastrodin-evoked decreases in GPx levels by 1.5 ± 0.3 folds or 2.2 ± 0.3 folds (Fig. 3B), in CAT levels by 1.6 ± 0.3 folds or 2.1 ± 0.3 folds (Fig. 3C), respectively. 3.4. Gastrodin induced ROS-associated cytotoxicity in DBTRG05MG cells Because acute incubation with gastrodin significantly increased intracellular ROS levels, experiments were performed to examine whether gastrodin-induced cytotoxicity involved ROS production. Fig. 4A shows that 10 mM NAC treatment did not change the control value of cell viability. In the presence of 10e50 mM gastrodin, NAC treatment partially prevented gastrodin-induced cell death by 4.2 ± 0.8%, 23.4 ± 0.8%, 23.8 ± 0.8%, 16.8 ± 0.7%, or 1.1 ± 0.7%, respectively (P < 0.05). Because in Fig. 4A, in the presence of 20 mM and 30 mM gastrodin, NAC significantly inhibited gastrodin-induced cytotoxic responses, this concentration range was chosen for apoptotic experiments. Annexin V/PI staining was applied to detect apoptotic cells after gastrodin treatment. Fig. 4B shows that the total percentage of apoptotic cells (early and late apoptotic cells) was increased by treatment with 20 or 30 mM gastrodin for 24 h in a concentration-dependent manner by 41.9 ± 0.8% or 70.5 ± 0.6%, respectively. However, at the concentration of 20 or 30 mM gastrodin, the total percentage of apoptotic cells did not increase in CTX TNA2 cells (data not shown). In NAC-treated groups, NAC (10 mM) loading did not alter the control value of apoptosis but partially inhibited 20 mM or 30 mM gastrodin-induced increases in the total percentage of apoptotic cells by 26.3 ± 1.5% or 43.7 ± 1.5%, respectively (P < 0.05) (n ¼ 3) (Fig. 4B and C). The data suggest that gastrodin induced cytotoxicity that involved ROS production in DBTRG-05MG cells. 3.5. Gastrodin decreased ROS-associated mitochondrial membrane potential levels in DBTRG-05MG cells Mitochondrial dysfunction has been shown to participate in the induction of apoptosis. The decrease of mitochondrial membrane potential could trigger the process of apoptotic pathway (Kroemer and Reed, 2000). In Fig. 4, because gastrodin induced ROSassociated apoptosis in DBTRG-05MG cells, efforts were made to examine whether gastrodin decreased mitochondrial membrane potential levels and, if so, whether this response was associated with intracellular ROS levels. In Fig. 5A, (DiIC1) (5) fluorescence intensity was decreased by treatment with 20 mM or 30 mM gastrodin for 24 h but the percentage of depolarized cells was increased in gastrodin-treated cells. In NAC-treated groups, NAC

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(10 mM) loading did not alter the control value of mitochondrial membrane potential, but partially inhibited 20 mM or 30 mM gastrodin-induced decreases in mitochondrial membrane potential levels by 10.3 ± 1.5% or 33.8 ± 1.7%, respectively (P < 0.05) (n ¼ 3) (Fig. 5B). The data suggest that gastrodin induced ROS-associated apoptosis that involved mitochondrial membrane potential reduction in DBTRG-05MG cells. 3.6. Gastrodin activated ROS-associated mitochondrial apoptotic pathways in DBTRG-05MG cells Since Fig. 5 shows that gastrodin decreased mitochondrial membrane potential levels, it suggests that gastrodin induced mitochondrial-associated apoptotic pathway. It has been shown that the levels of Bcl-2 protein family such as Bax and Bcl-2, cytosolic cytochrome c, cleaved caspase-9/caspase-3 regulated the process of mitochondrial apoptotic pathway (Hengartner, 2000). Therefore, the expression levels of these protein in gastrodininduced apoptosis were examined in DBTRG-05MG cells. Treatment with 20 mM or 30 mM gastrodin, Bax, cytosolic cytochrome c, and cleaved caspase-9/caspase-3 levels were increased but Bcl-2 levels were decreased (n ¼ 3) (Fig. 6A). In NAC-treated groups, NAC (10 mM) partially inhibited 20 mM or 30 mM gastrodin-evoked increases in Bax levels by 2.6 ± 0.3 folds or 2.7 ± 0.3 folds (Fig. 6B), in cytosolic cytochrome c levels by 2.6 ± 0.3 folds or 2.5 ± 0.3 folds (Fig. 6D), in cleaved caspase-9 levels by 2.4 ± 0.3 folds or 2.9 ± 0.3 folds (Fig. 6E) and in cleaved caspase-3 levels by 2.3 ± 0.3 folds or 2.9 ± 0.3 folds (Fig. 6F), respectively. Furthermore, NAC treatment partially prevented 20 mM or 30 mM gastrodin-evoked decreases in Bcl-2 levels by 1.7 ± 0.3 folds or 3.0 ± 0.3 folds (Fig. 6C), respectively. The findings imply that gastrodin-induced apoptosis was associated with the modulation of Bax, Bcl-2, cytosolic cytochrome c, and cleaved caspase-9/caspase-3 expressions. 3.7. Gastrodin induced caspase-associated cytotoxicity in DBTRG05MG cells Because acute incubation with gastrodin significantly increased cleaved caspase-9/caspase-3 levels, experiments were performed to examine whether gastrodin induced caspases-associated cytotoxicity. Z-VAD-FMK, served as a pancaspase inhibitor, was used to prevent protelytic caspases activation during gastrodin treatment (Ekert et al., 1999). Fig. 7A shows that 10 mM Z-VAD-FMK treatment did not change the control value of cell viability. In the presence of 10e50 mM Z-VAD-FMK, Z-VAD-FMK treatment partially prevented gastrodin-induced cell death by 4.1 ± 0.8%, 23.5 ± 0.8%, 24.0 ± 0.8%, 16.7 ± 0.7%, or 1.2 ± 0.7%, respectively (P < 0.05). In apoptotic experiments, Z-VAD-FMK (10 mM) loading did not alter the control value of apoptosis but partially inhibited 20 mM or 30 mM gastrodininduced total percentage of apoptotic cells (early and late apoptotic cells) by 20.7 ± 1.5% or 28.1 ± 1.5%, respectively (P < 0.05) (n ¼ 3) (Fig. 7B and C). The data suggest that gastrodin induced apoptosis that involved caspase activation in DBTRG-05MG cells. 4. Discussion Studies have shown that some chemotherapy drugs such as temozolomide are being introduced in therapy of glioblastomas, but the curative efficacy is unsatisfactory (Oh et al., 2010).

Fig. 6. Effect of combination of NAC and gastrodin on mitochondrial apoptotic protein levels in DBTRG-05MG cells. (A) Protein extracts were prepared 24 h after exposure to various concentrations of gastrodin. In NAC group, NAC (10 mM) was added to cells followed by treatment with gastrodin in the medium. (BCDEF) The effect of gastrodin on Bax (B), Bcl-2 (C), cytosolic cytochrome c (D), cleaved caspase-9 (E) or cleaved caspase-3 (F) levels as quantified by densitometry. The figure normalized intensities of the bands of Bax, Bcl-2, cytosolic cytochrome c, cleaved caspase-9 or cleaved caspase-3 levels against the bands of b-actin using NIH image 1.61. *P < 0.05 compared to control. #P < 0.05 compared to the pairing group. Data are representative of three separate experiments.

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Fig. 7. Effect of combination of Z-VAD-FMK and gastrodin on cytotoxicity in DBTRG05MG cells. (A) The pancaspase inhibitor Z-VAD-FMK (10 mM) was added to cells followed by treatment with gastrodin in the medium. Cell viability assay was subsequently performed. *P < 0.05 compared to control. #P < 0.05 compared to the pairing group. Data are mean ± SEM of three separate experiments. (BC) Z-VAD-FMK (10 mM) was added to cells followed by treatment with gastrodin in the medium. Alexa ®Fluor 488 annexin V/PI staining for apoptosis was subsequently performed. *P < 0.05 compared to the pairing group. Data are mean ± SEM of three separate experiments.

Therefore, there is a pressing need to search for novel strategies to develop improved therapeutics against glioblastomas. In this context, one promising direction for research concerns the use of naturally occurring compounds as preventive agents. Our study was aimed to investigate the cytotoxic effects and possible apoptotic mechanisms induced by the rhizome of Gastrodia elata Blume extract gastrodin on human glioblastoma cells as well as to determine whether this compound caused cytotoxicity in normal rat astrocytes. Our findings show that gastrodin concentrationdependently inhibited cell viability and evoked cell cycle arrest in DBTRG-05MG cells but not in CTX TNA2 cells (Suppl. 1). In DBTRG05MG cells, gastrodin inhibited cell viability with an IC50 of approximately 25 mM after 24 h treatment. The results suggest that at the concentration range of 20e30 mM, gastrodin selectively caused cytotoxicity in DBTRG-05MG cells but not in CTX TNA2 cells. Cell cycle plays an important role in regulating cell physiology. Blockade of the cell cycle is regarded as an effective strategy in the development of novel cancer therapies (Sandal, 2002). This study shows that in DBTRG-05MG cells, gastrodin caused an increase in the percentage of subG1 and G2/M phase cells. Therefore, the data suggest that gastrodin induced cytotoxicity partly via its ability to cause G2/M phase cell arrest. The molecular mechanism of G2/M phase cell arrest evoked by gastrodin treatment in DBTRG-05MG cells was also explored. In eukaryotes, cyclin-dependent kinases 1 (CDK1) and cyclin B1 are the two specific regulators in G2/M phase (Nigg, 1995). Because gastrodin evoked G2/M phase cell cycle arrest, the protein expression levels of cyclin B1 and CDK1 were investigated. Our data show that gastrodin decreased cyclin B1 and CDK1 levels, implying that those proteins may be involved in the G2/M phase arrest. In addition to cyclins and CDKs, the cyclin-dependent kinase inhibitors (CDKIs) also play important roles in the cell cycle progression through inhibiting the activity of CDK-cyclin complex. As one of the most important CDKIs, p21 protein accounts for sustaining G2/M phase cell arrest after DNA damage (Bunz et al., 1998) and interacting with CDK1-cyclin B1 complex. Furthermore, p53 protein is in the upstream of p21 protein, which acts as an inhibitor of G2/M phase-associated protein (Bunz et al., 1998). The data show that gastrodin significantly increased p53 and p21 levels during the G2/M phase arrest in DBTRG-05MG cells. Therefore it suggests that gastrodin could lead to cell cycle arrest at the G2/M phase, which was accompanied by an inhibition of cell cycle-regulatory proteins (p53 and p21) controlling the G2/M phase mitotic check point. The major difference between DBTRG-05MG cells and CTX TNA2 cells is the status of p53 protein levels. p53 protein was shown to be expressed normally in DBTRG-05MG cells (Kruse et al., 1992) but not in CTX TNA2 cells (Cantrill et al., 2012). Previous studies have shown that p53 activation was involved in the sensitivity of cytotoxicity in human glioblastoma cells to the naturally-occurring compound such as n-butylidenephthalide (Tsai et al., 2006) or dioscin (Lv et al., 2013). Similarly, our data show that gastrodin significantly increased p53 levels during cell cycle arrest in DBTRG05MG cells but not in CTX TNA2 cells (Suppl. 2). Therefore, the data suggest that at least one of the possible targets of gastodin, which is p53 expression, plays a role in gastrodin-induced cytotoxicity in human glioblastoma cells but not in normal rat astrocytes. However, gastrodin was shown to block the expression of p53 phosphorylation, caspase-3 and cytochrome c, reduce bax/bcl-2 ratio induced by glutamate in PC12 rat pheochromocytoma cells (Jiang et al., 2014). These findings indicate that gastrodin protects PC12 cells from the apoptosis induced by glutamate through a mechanism of inhibiting p53 pathway (Jiang et al., 2014). Therefore, the mechanisms underlying gastrodin-induced cytotoxicity appear to vary among different cancer cell types. ROS are highly reactive ions or free radicals containing oxygen

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that impair redox homeostasis and cellular functions, leading to cell death. Under physiological conditions, a variety of antioxidant systems scavenge ROS to maintain the intracellular redox homeostasis and normal cellular functions (Circu and Aw, 2010; Kamata and Hirata, 1999). GSH is the most important thiol-containing molecule, as it functions as a redox buffer, antioxidant, and enzyme cofactor against oxidative stress (Circu and Aw, 2010; Kamata and Hirata, 1999). The balance of intracellular ROS and GSH levels could regulate cytotoxicity in cancer research (Estrela et al., 2006). Our study shows that gastrodin concentrationdependently increased intracellular ROS levels while depleted GSH levels in DBTRG-05MG cells, suggesting that gastrodin induced cytotoxicity that involved regulation of ROS/GSH levels. The chemical nature of the substrates fueling the respiratory chain complex of mitochondria, the amplitude of the membrane potential in mitochondria, the pH of the matrix, and the oxygen tension in the surroundings are important factors controlling ROS production in mitochondria (Wallace, 1999). Although the respiratory chain complexes of mitochondria are responsible for augmenting ROS production (Wallace, 1999), in addition to this system, there are other ROS-generating systems to be identified such as peroxisomes and endoplasmic reticulum (Circu and Aw, 2010). Our findings show that gastrodin increased intracellular ROS levels without increasing mitochondrial ROS levels. This suggests that gastrodin induced oxidative stress changes in cytosol but not in mitochondria in human glioblastoma cells. It has been shown that the peroxisomes are considered as the major sites of oxygen consumption in the cell and produce ROS production under physiological conditions (Circu and Aw, 2010). Alternatively, endoplasmic reticular monooxygenases cause augmented level of cellular ROS production, endorse lipid peroxidation and induce cytotoxicity (Circu and Aw, 2010). Therefore, the source of ROS production by gastrodin needs further investigation in future studies. In cancer research, examining the balance between ROS and antioxidant enzyme levels in cancer cells is important for knowing whether cancer progression can be inhibited (Punnonen et al., 1994). Therefore, this study investigated the effect of gastrodin on antioxidant enzyme levels in DBTRG-05MG cells. The levels of antioxidant enzymes, such as SOD, GPx and CAT, are closely linked with cellular responses to various oxidative stresses (Punnonen et al., 1994). SOD catalyses the dismutation of superoxide anion into water and H2O2 whereas CAT and GPx protect against oxidative damage by converting H2O2 into water. The data show that gastrodin concentration-dependently increased SOD levels while decreased GPx and CAT levels in DBTRG-05MG cells. A possible explanation for the decrease in GPx and CAT activity in gastrodintreated cells is via increased H2O2. Most researches in gastrodin focus on the antiapoptotic effect in different models such as glutamate-treated PC12 cells (Jiang et al., 2014), ischemic stroke mice model (Peng et al., 2015) and lipopolysaccharides-treated astrocytes (Wang et al., 2016). Our data concentrated on the mechanism underlying the effect of gastrodin on apoptosis in human glioblastoma cells. The findings show that gastrodin (20e30 mM) caused apoptosis after 24 h treatment in DBTRG-05MG cells but not in CTX TNA2 cells. Therefore, gastrodin at this concentration range can be applied for exploring apoptotic effects in human glioblastoma cells. Mitochondria play a critical role in the regulation of intrinsic apoptotic pathway (Jürgensmeier et al., 1998). In intrinsic pathway, the increased ratio of proapoptotic protein Bax/antiapoptotic protein Bcl-2 can lead to the collapse of mitochondrial membrane potential, which results in release of cytochorme c and subsequent activation of caspase-9 and caspase-3 (Jürgensmeier et al., 1998). In our study, gastrodin enhanced mitochondrial membrane potential reduction and Bax, cytosolic cytochrome c, cleaved caspase-9/

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caspase-3 levels, but decreased Bcl-2 levels. Furthermore, treatment with the antioxidant NAC or the pancaspase inhibitor Z-VADFMK significantly decreased gastrodin-induced apoptosis. Therefore, the data suggest that gastrodin activated ROS-associated mitochondrial pathways of apoptosis that involved caspase activation in DBTRG-05MG cells. NAC is an aminothiol and synthetic precursor of intracellular cysteine and glutathione (Zafarullah et al., 2003). The action of NAC results from its antioxidative or free radical scavenging property as an antioxidant (Zafarullah et al., 2003). Furthermore, NAC has been widely used as a research tool in the field of apoptosis research for investigating the role of ROS in induction of apoptosis (Zafarullah et al., 2003). The role of ROS in cancer is dichotomous. Low levels of ROS have been shown to promote cancer through stimulation of cell proliferation, increase of cell survival and amplification of angiogenesis, but high levels of ROS have been shown to have anticancer effects by inducing cell cycle arrest and apoptosis (Circu and Aw, 2010). Although NAC has been recognized as a “beneficial” drug in the market, the mechanisms underlying NAC-induced protective effects depends on different models. This study shows that NAC has reversible effects against gastrodin-induced cytotoxicity in DBTRG-05MG cells. Gastrodin induced ROS-associated cytotoxicity which targets DBTRG-05MG cells. Therefore, it suggests that NAC has protective effects on gastrodin-induced cytotoxicity in glioblastoma cells. In future clinical trials, it should be cautioned that NAC and gastrodin may be avoided to use simultaneously when glioblastoma patients received treatment. In in vivo studies, gastrodin was shown to have a neuroprotective role in ischemic/reperfused cerebral hippocampus in rats (Zeng et al., 2007) or spinal cord ischemia reperfusion injury rabbits (Fang et al., 2016). The pharmacokinetic behavior of gastrodin in rat plasma and cerebrospinal fluid (CSF) after intranasal and intravenous administration was also investigated (Wang et al., 2007; Tang et al., 2015). No BioResponse (BR) gastroin-related adverse effects were reported at doses up to 100 mg/kg. A single 15 mg/ml dose of BR-gastrodin resulted in a mean Cmax of ~30 mM after 24 h exposure (Wang et al., 2007; Tang et al., 2015). Gastrodin at concentrations between 15 mM and 30 mM passed into blood brain barrier and reached the brain tissue of middle cerebral artery occlusion rats (Wang et al., 2008). Our data show that gastrodin (20e30 mM) caused cytotoxictiy in human glioblastoma cells but not in normal rat astrocytes. Therefore, gastrodin may have a potential for the treatment and prevention of glioblastoma and its complications. 5. Conclusion Together, in DBTRG-05MG human glioblastoma cells, but not in CTX TNA2 rat astrocytes, the natural compound gastrodin activated mitochondrial pathways of apoptosis in a ROS-associated manner that involved cell cycle arrest and caspase activation. The use of natural anticancer agents, in comparison to their synthetic counterparts, has been regarded to be relatively safe pharmaceutically as they are non-toxic or exert minimal cytotoxicity. Because our findings show that gastrodin exhibits selective toxicity toward cancer cells compared to normal cells, gastrodin possesses an anticancer potential to treat human glioblastoma. Since the ROSassociated apopotic pathway and its biological significance have not been examined in gastrodin-treated human glioblastoma cells, our data may be useful for understanding the mechanism of action of gastrodin in human glioblastoma. Because gastrodin has preventive and therapeutic potentials against human brain diseases, in the future, advanced drug delivery systems for enhanced bioavailability and mechanisms to maintain effective therapeutic concentrations in the brain should also be explored.

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