Exp Neurobiol 2011; 20(2): 100-109
Published online June 30, 2011
© The Korean Society for Brain and Neural Sciences
Byung-Chul Kim1, Youn-Sub Kim1, Jin-Woo Lee2, Jin-Hee Seo2, Eun-Sang Ji 2, Hyejung Lee3, Yong-Il Park4 and Chang-Ju Kim2*
1Department of Anatomy-Pointology, College of Oriental Medicine, Kyungwon University, Seongnam 461-701, 2Department of Physiology, College of Medicine, Kyung Hee University, Seoul 130-701, 3Acupuncture and Meridian Science Research Center, Kyung Hee University, Seoul 130-701, 4Division of Biotechnology and Collaborative Institute of Science and Technology, The Catholic University of Korea, Bucheon 420-717, Korea
Correspondence to: *To whom correspondence should be addressed.
TEL: 82-2-961-0407, FAX: 82-2-964-2195
Nitric oxide (NO) is a reactive free radical and a messenger molecule in many physiological functions. However, excessive NO is believed to be a mediator of neurotoxicity. The medicinal plant
Keywords: Coriolus versicolor, nitric oxide, apoptosis, human neuroblastoma
Nitric oxide (NO) is a reactive free radical gas and a messenger molecule with many physiological functions (Schmidt and Walter, 1994; Yun et al., 1996). NO is generated from L-arginine by nitric oxide synthase (NOS), and it is synthesized in neurons, astrocytes, microglial cells, endothelial cells, and many other cell types (Garthwaite and Boulton, 1995). Moreover, in the mammalian central nervous system, NO modulates many physiological functions including neurotransmission, synaptic plasticity, and memory (Hawkins, 1996; Hölscher, 1997). However, excessive NO formation is now believed to be a mediator of neurotoxicity, and NO is known to induce apoptosis in a variety of disorders, such as Alzheimer disease, acquired immune deficiency syndrome (AIDS) dementia, and multiple sclerosis (Gross and Wolin, 1995; Dawson and Dawson, 1996).
Apoptosis, also known as programmed cell death, is a biological process that plays a crucial role in normal development and tissue homeostasis (Woodle and Kulkarni, 1998). However, this type of cell death also contributes to a variety of human disorders (Thompson, 1995). The characteristic morphological changes associated with apoptosis are cell shrinkage, chromatin condensation, internucleosomal DNA fragmentation, and the formation of apoptotic bodies (Wyllie et al., 1980; Chandra et al., 2000; Jang et al., 2002). Several gene expressions have been demonstrated to be involved in the regulation of apoptosis. P53 is a short-lived transcriptional activator that induces apoptosis (Lowe and Ruley, 1993) and the activation of p53 regulates the expression of Bax (Xiang et al., 1998) which is a proapoptotic member of the Bcl-2 family of intracellular proteins. Bcl-2 family proteins also play important roles in regulation of apoptosis. The Bcl-2 family proteins are classified into anti-apoptotic proteins, including Bcl-2 and Bcl-2XL, and pro-apoptotic proteins, such as Bax and Bid. The balance between pro-apoptotic and anti-apoptotic Bcl-2 family members determines the mitochondrial response to apoptotic stimuli (Kim et al., 2010; Upadhyay et al., 2003). Bax alters the permeability of mitochondrial membranes and triggers caspases cascade activation (Budihardjo et al., 1999; Korsmeyer, 1999; Upadhyay et al., 2003). The caspases are a class of cysteine proteases, and are considered to be central players of the apoptotic process and to trigger a cascade of proteolytic cleavages of many proteins in mammals (Aggarwal, 2000). In particular, the most widely studied member of the caspase family, caspase-3, is a key executioner of apoptosis, and is partially or totally responsible for the proteolytic cleavage of many proteins (Cohen, 1997; Ko et al., 2009).
Citrus fruits contain sugar, organic acids, and a number of physiologically active components, such as citric acid, ascorbic acid, minerals, coumarins, and flavonoids (Tanizawa et al., 1992; Kawaii et al., 1999). Moreover, the aqueous extract of
In the present study, we investigated the protective effects of the aqueous extracts of
The human neuroblastoma SK-N-MC cells were cultured in Dulbecco's Modified Eagle Medium (DMEM; Gibco BRL, Grand Island, NY, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco BRL) at 37℃ in a 5% CO2, 95% O2 humidified cell incubator, and the medium was changed every 2 days.
The cell viability was determined using the MTT assay kit according to the manufacturer's instructions (Boehringer Mannheim GmbH, Mannheim, Germany)(Lee et al., 2005). The cells were treated with SNP at concentrations of 0.01 mM, 0.05 mM, 0.1 mM, 0.5 mM, and 1.0 mM for 24 h. To investigate the protective effect of
After treatment with SNP, the cells were washed three times in phosphate-buffered saline (PBS) and fixed with 100% methanol at -20℃ for 10 min. The cells were then observed under a phase-contrast microscope (Olympus, Tokyo, Japan) as a previously described method (Jang et al., 2002).
To detect apoptotic cells
To determine whether SNP induces apoptosis, DAPI staining was performed as a previously described method (Lee et al., 2005). Briefly, the cells were cultured on 4-chamber slides, washed twice with PBS, fixed by incubating with 4% PFA for 30 min, washed with PBS, incubated with 1 µg/ml DAPI for 30 min in the dark, and analyzed under a fluorescence microscope (Zeiss, Oberköchen, Germany).
DNA fragmentation assay was performed using ApopLadder EX™ DNA fragmentation assay kit (TaKaRa, Shiga, Japan) (Lee et al., 2005). The cells were pre-treated for 1 h with CVEcitrus and treated with SNP, lysed with 100 µl of lysis buffer, incubated with 10 µl of 10% sodium dodecyl sulfate (SDS) solution containing 10 µl of Enzyme A at 56℃ for 1 h, and then incubated with 10 µl of Enzyme B at 37℃ for another 1 h. This mixture was added with 70 µl of precipitant and 500 µl of ethanol and centrifuged for 15 min. DNA was extracted by washing the pellet in ethanol and resuspending it in Tris-EDTA (TE) buffer. DNA fragmentation was visualized by 2% agarose gel electrophoresis and staining with ethidium bromide.
Flow cytometric analysis was performed as a previously described method (Jang et al., 2002). Briefly, after pre-treatment for 1 h with CVEcitrus and the treating them with SNP, the cells were collected, fixed by incubating with 75% ethanol in PBS at -20℃ for 1 h, then incubated with 100 µg/ml RNase and 20 µg/ml propidium iodide in PBS for 30 min at 37℃, and analyzed using FACScan (Becton Dickinson, San Jose, CA, USA).
Total RNA was isolated from the SK-N-MC cells using easy-BLUE™ total RNA extraction kit according to the manufacturer's instructions (iNtRON, INC., Seoul, Korea) (Lee et al., 2005). Two µg of RNA and 2 µl of random hexamers (Promega, Madison, WI, USA) were added together and the mixture was heated at 65℃ for 10 min. To the mixture, 1 µl of AMV reverse transcriptase (Promega), 5 µl of 10 mM dNTP (Promega), 1 µl of RNasin (Promega), and 5 µl of 10×AMV RT buffer (Promega) were added and the final volume was adjusted to 50 µl with dimethyl pyrocarbonate (DEPC)-treated water. The reaction mixture was then incubated at 42℃ for 1 h.
PCR amplification was performed in a reaction volume of 40 µl containing 1 µl of the appropriate cDNA, 1 µl of each set of primers at a concentration of 10 pM, 4 µl of 10×reaction buffer, 1 µl of 2.5 mM dNTP, and 2 units
Western blot was performed as a previously described method (Kim et al., 2010). The cells were lysed in the lysis buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.5% deoxycholic acid, 1% nonidet-P40 (NP40), 0.1% SDS, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 100 µg/ml leupeptin. Protein concentration was measured using a Bio-Rad colorimetric protein assay kit (Bio-Rad). Protein of 50 µg was separated on SDS-polyacrylamide gels and transferred onto a nitrocellulose membrane (Schleicher & Schuell GmbH, Dassel, Germany). Mouse anti-p53 antibody (1 : 500; Santa Cruz Biotech, Santa Cruz, CA, USA), rabbit anti-phospho p53 (Thr 18) antibody (1 : 200; Santa Cruz Biotech), and mouse anti-Bax antibody (1 : 1,000; Santa Cruz Biotech) were used as primary antibody. Horseradish peroxidase-conjugated anti-mouse antibody for p53 and Bax, and anti-rabbit antibody for phospho p53 (1 : 1,000; Santa Cruz Biotech) were used as secondary antibody. The detection of the band was performed using the enhanced chemiluminescence (ECL) detection system (Amersham Pharmacia Biothech GmbH, Freiburg, Germany).
Caspase-3 enzyme activity was measured using the ApoAlert® caspase-3 assay kit (Clontech, Palo Alto, CA, USA) according to the manufacturer's protocols (Kim et al., 2003). In brief, the cells were lysed with 50 µl of chilled Cell Lysis Buffer. A 50 µl aliquot of 2×reaction buffer (containing DTT) and 5 µl of the appropriate conjugated substrate at a concentration of 1 mM were added to each lysate. The mixture was incubated in a water bath at 37℃ for 1 h, and the absorbance was measured using a microtiter plate reader at a test wavelength of 405 nm.
The results are expressed as the mean±standard error of the mean (SEM). The data were analyzed by one-way ANOVA followed by Duncan's post-hoc test using SPSS. The difference was considered statistically significant at p<0.05.
The viability of cells incubated with SNP for 24 h at concentrations of 0.01 mM, 0.05 mM, 0.1 mM, 0.5 mM, or 1.0 mM was 95.50±0.64%, 88.09±0.60%, 78.20±0.77%, 52.72±1.13%, or 34.14±0.69%, respectively (Fig. 1A). As the SNP concentration was increased, the cell viability was decreased. The viability of cells exposed to the 0.5 mM SNP for 24 h was 52.72±1.13%.
The viability of the cells pre-treated for 1 h with the CVEsynthetic at concentrations of 0.05 mg/ml, 0.1 mg/ml, 0.5 mg/ml, or 1.0 mg/ml, and then exposed to 0.5 mM SNP for 24 h was 52.69±0.81%, 53.47±0.54%, 55.67±1.02%, or 53.42±1.08%, respectively (Fig. 1B).
The viability of the cells pre-treated for 1 h with the CE at concentrations 0.05 mg/ml, 0.1 mg/ml, 0.5 mg/ml, or 1.0 mg/ml, and then exposed to 0.5 mM SNP for 24 h was 58.39±0.64%, 60.07±1.26%, 66.48±0.77%, or 66.14±2.01, respectively (Fig. 1C).
The viability of the cells pre-treated for 1 h with the CVEcitrus at concentrations of 0.05 mg/ml, 0.1 mg/ml, 0.5 mg/ml, or 1.0 mg/ml, and then exposed to 0.5 mM SNP for 24 h was increased to 56.22±1.39%, 64.03±3.01%, 77.24±0.70%, or 83.37±1.36%, respectively (Fig. 1D).
The above data demonstrated that the cell viability was reduced by SNP and that CVEsynthetic showed no significant protective effect against NO-induced cytotoxicity in SK-N-MC cells and that CE had a little protective effect against NO-induced cytotoxicity. The most potent protective effect against the SNP-induced cytotoxicity was observed for CVEcitrus. Therefore, we selected CVEcitrus for further study (Fig. 1).
To characterize SNP-induced changes in cell morphology, the cells were examined by phase-contrast microscopy. The cells treated with 0.5 mM SNP for 24 h detached from the culture dish, and became rounded and irregular in shape with cytoplasmic blebbings. The cells pre-treated for 1 h with the 1.0 mg/ml CVEcitrus and exposed to 0.5 mM SNP for 24 h were indistinguishable from the normal cells (Fig. 2, Upper).
In the DAPI assay, nuclear condensation, DNA fragmentation, and perinuclear apoptotic bodies were detected in the cells treated with the 0.5 mM SNP for 24 h. The cells pre-treated for 1 h with the 1.0 mg/ml CVEcitrus and exposed to 0.5 mM SNP for 24 h were comparable to the normal cells (Fig. 2, Middle).
To further confirm the induction of apoptosis by SNP in the SK-N-MC cells, the 0.5 mM SNP-treated cells were analyzed
In order to ascertain the protective effect of CVEcitrus against SNP-induced apoptosis, DNA fragmentation, reflecting the endonuclease activity characteristic of apoptosis, was analyzed. SNP treatment at 0.5 mM for 24 h resulted in the formation of definite fragments which could be seen
Through flow cytometric analysis of DNA content using the DNA-specific dye PI, we assessed the protective effect of CVEcitrus against SNP-induced cell death. The population of cells in the sub-G1 phase in the 0.5 mM SNP-treated group increased from 12.90% (control level) to 24.77%, whereas this figure was reduced by CVEcitrus pre-treatment at 0.5 mg/ml or 1 mg/ml for 1 h and exposed to 0.5 mM SNP for 24 h to 18.50% or 16.38%, respectively (Fig. 4).
The RT-PCR was performed to estimate the relative expressions of the p53 and Bax mRNA. In the present study, the mRNA level of p53 in the control was set at 1.00. The level of p53 mRNA following treatment with 0.5 mM SNP for 24 h increased to 13.79±4.21, but it was only 3.30±0.57 or 2.28±0.27 in the cells pre-treated with CVEcitrus at 0.5 mg/ml or 1.0 mg/ml for 1 h and exposed to 0.5 mM SNP for 24 h, respectively.
The mRNA level of Bax in the control was set at 1.00. The level of Bax mRNA following treatment with 0.5 mM SNP increased to 6.19±0.54, but it was only 4.49±0.43 or 1.30±0.06 in the cells pre-treated with CVEcitrus at 0.5 mg/ml or 1.0 mg/ml for 1 h and exposed to 0.5 mM SNP for 24 h, respectively (Fig. 5).
When the cells were treated with 0.5 mM SNP for 24 h, Bax (21 kDa) and p53 protein (53 kDa) expressions were up-regulated. Compared to these cells, those of cells pre-treated with CVEcitrus at 0.5 mg/ml or 1.0 mg/ml for 1 h and exposed to 0.5 mM SNP for 24 h showed lower Bax and p53 protein expressions.
When the expressions of p53, phosphor-p53, and Bax in the control cells were set at 1.00, p53 expression after treatment with 0.5 mM SNP for 24 h increased to 13.20±1.29, but it was only 5.43±1.28 or 1.72±0.25 in the cells pre-treated with CVEcitrus at 0.5 mg/ml or 1.0 mg/ml for 1 h and exposed to 0.5 mM SNP for 24 h, respectively. The phpspho-p53 (Thr 18) expression after treatment with 0.5 mM SNP for 24 h increased to 11.60±1.28, but it was only 5.99±0.86 or 3.92±0.83 in the cells pre-treated with CVEcitrus at 0.5 mg/ml or 1.0 mg/ml for 1 h and exposed to 0.5 mM SNP for 24 h, respectively. The Bax expression after treatment with 0.5 mM SNP for 24 h increased to 4.23±0.40, but it was only 2.08±0.17 or 1.43±0.25 in the cells pre-treated CVEcitrus at 0.5 mg/ml or 1.0 mg/ml for 1 h and exposed to 0.5 mM SNP for 24 h, respectively (Fig. 6).
Caspase-3 enzyme activity was measured using DEVD-peptide-nitroanilide (
In the brain, NO is synthesized by neuronal nitric oxide synthase (NOS) and acts as an intercellular messenger at the physiological level. However, the high concentrations of NO induced by certain pathological conditions, such as brain ischemia, inflammation, neurodegenerative diseases, and may result in neuronal dysfunction (Heales et al., 1999; Murphy, 1999; Lee et al., 2010). NO also causes apoptotic neuronal cell death (Lee et al., 2005). In the present study, we investigated whether
Our MTT assay results showed that SK-N-MC cell viability was significantly reduced by SNP treatment, and that CVEcitrus exerted a significant protective effect against NO-induced cytotoxicity. However, CVEsynthetic showed no protective effect and CE has a little protective effect. Flow cytometric analysis of DNA contents showed an increase in the population of cells in the sub-G1 phase after SNP treatment, whereas the cells pre-treated with CVEcitrus prior to SNP showed a decrease in the sub-G1 phase. Under the phase-contrast microscope, the cells treated with SNP only showed apoptotic morphologies, i.e., cell shrinkage, cytoplasmic condensation, and irregularity in shape. Moreover, apoptotic bodies were observed in the SNP-treated cells stained with DAPI. However, the cells pre-treated with CVEcitrus prior to SNP showed lower levels of apoptotic morphologic changes.
In addition, TUNEL-positive cells, indicative of apoptotic DNA strand breaks and nicks in DNA molecules, were detected in the SNP-treated cells, but the cells showed lower levels of TUNEL-positive cells. To provide evidence supporting the involvement of apoptosis in the SNP-induced cytotoxicity, the DNA fragmentation assay was performed. Distinctive ladder pattern characteristic of apoptotic cell death was detected in the cells treated with SNP, on the other hand pre-treatment with CVEcitrus prior to SNP showed lower SNP-induced DNA laddering intensity.
The present results showed that apoptosis is closely implicated to NO-induced cytotoxicity in human neuroblastoma SK-N-MC cells and that CVEcitrus has a protective effect against this cytotoxicity. Molecular mechanisms underlying the NO-mediated apoptosis involve different pathways which depend on cell type and the cellular environment (Bosca and Hortelano, 1999). Many studies have demonstrated that NO-induced apoptosis occurs through a p53-dependent pathway in various cells including neuronal cells (Yung et al., 2004; Lee et al., 2005). It was demonstrated that NO enhances p53 protein expression and its phosphorylation in myoblast cells (Lee et al., 2005). The present study also showed that NO increased p53 protein expression and its phosphorylation at Thr 18 in SK-N-MC cells.
Apoptosis-regulatory proteins have been repeatedly implicated in the susceptibility of neurons to cell death (Xu et al., 2007; Kim et al., 2010; Baek et al., 2011). Caspase-3 is one of the most widely studied members of the caspase family and it is involved in apoptosis as the principal executor (Cohen, 1997; Kim et al., 2010). Several pathways have been shown to mediate p53-induced apoptosis, and Bax is a well known p53 target gene and a proapoptotic member of the Bcl-2 family (Miyashita and Reed, 1995; McCurrach et al., 1997). Bax promotes the release of cytochrome c into the cytosol from mitochondria, which in turn activates caspase-3 (Reed, 1995; Upadhyay et al., 2003). In the present study, we observed that SNP increased Bax expressions at the mRNA and protein levels in SK-N-MC cells, and that it finally increased caspase-3 enzyme activity. We further investigated whether CVEcitrus inhibits NO-related cell death pathways involving p53, Bax, and caspase. Our results showed that CVEcitrus attenuated NO-induced apoptotic cell death by blocking a p53- and Bax-dependent caspase-3 pathway. Suppression of DNA fragmentation and caspase-3 expression is known to be closely related with inhibition of apoptosis of neurons, resulting in facilitation of memory recovery (Quindry et al., 2007; Ko et al., 2009; Baek et al., 2011).
Under normal conditions, NO modulates many physiological functions including neurotransmission, synaptic plasticity, and memory in the mammalian central nervous system (Hawkins, 1996; Hölscher, 1997). However, under excessive NO formation conditions, NO exerts neurotoxicity inducing apoptosis, and NO-induced apoptosis causes many brain disorders (Gross and Wolin, 1995; Dawson and Dawson, 1996). The present study showed that CVEcitrus reduced NO-induced apoptotic cell death in a neuroblastoma cell line. The pro-apoptotic proteins, p53- and Bax, are known to activate caspase-3, resulting in apoptosis. The possible mechanisms of neuroprotective effect of CVEcitrus can be ascribed to the inhibition of p53- and Bax-dependent caspase-3 activation. Our results suggest that CVEcitrus potentially has therapeutic value in the treatment of a variety of NO-induced brain diseases such as stroke.