Calycopterin Promotes Survival and Outgrowth of Neuron-Like PC12

Article pubs.acs.org/crt

Calycopterin Promotes Survival and Outgrowth of Neuron-Like PC12 Cells by Attenuation of Oxidative- and ER-Stress-Induced Apoptosis along with Inflammatory Response Mahdi Moridi Farimani,†,§ Nazanin Namazi Sarvestani,‡,§ Niloufar Ansari,‡ and Fariba Khodagholi*,‡ †

Department of Phytochemistry, Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, G. C., Evin, Tehran, Iran Neuroscience Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran

‡

ABSTRACT: There is mounting evidence implicating the role of oxidative stress induced by reactive oxygen species (ROS) in neurodegenerative disease, including Alzheimer’s disease. Herein we investigated the neuroprotective potential of a natural flavonoid, calycopterin, against H2O2-induced cell death in differentiated PC12 cells. We pretreated PC12 cells with 25, 50, and 100 μM calycopterin followed by the addition of H2O2 as an oxidative stress agent. We measured cell viability by the MTT test and found that 50 μM is the best protective concentration of calycopterin. Moreover, we measured six different parameters of neurite outgrowth. Interestingly, we found that calycopterin not only protects PC12 cells against H2O2-induced apoptosis but also defends against the destructive effect of oxidative stress on the criteria of neural differentiation. Calycopterin decreased ER stress-associated proteins including calpain and caspase-12, and suppressed ERK, JNK, and p38 MAPK phosphorylation. Moreover, calycopterin inhibited H2O2-induced nuclear translocation of nuclear factor-κB, a known regulator of a host of genes involved in specific stress and inflammatory responses. This observation was perfectly in agreement with the decrease of COX-2 and TNF-α levels. Calycopterin reduced intracellular ROS levels and increased catalase activity. The protective effect of this compound could represent a promising approach for the treatment of neurodegenerative diseases.

1. INTRODUCTION Oxidative stress is the major cause of neuronal death observed in neurodegenerative diseases, such as Alzheimer’s disease (AD).1 This imbalance between prooxidant and antioxidant factors in favor of prooxidants can contribute to the accumulation of intracellular reactive oxygen species (ROS). Owing to their high reactivity, ROS represent a serious hazard for the cell, as they can oxidize macromolecules, thus damaging proteins, lipids, and DNA.2 ROS appear to play a critical role also in apoptosis.3 To cope with these harmful effects, cells evolved powerful systems aimed at preventing accumulation of these species, including antioxidant enzymes. The development of these safeguard systems is key for enabling cells to survive. So, one possible strategy for the development of neuroprotective drugs is to search for low-molecular weight compounds that can regulate the redox state and thereby counter oxidative damage. The use of drug substances derived from plants has a long tradition in medicine. In modern medicine, they are still a major source of new drug development and represent a major part of today’s pharmaceutical market.4 Calycopterin, 5,4′-dihydroxy-3,6,7,8-tetramethoxyflavone (Figure 1), is a member of the flavonoids first isolated from leaves of Digitalis thapsi L. and Calycopteris floribunda Lamk., two traditional Asian medicinal plants.5,6 Different biological activities have been reported for calycopterin including © 2011 American Chemical Society

Figure 1. Chemical structure of calycopterin.

immunoinhibitory and antihelminthic6,7 effects. In the present study, we isolated the calycopterin from Dracocephalum kotschyi Boiss., an endemic Iranian plant, and examined its effect on oxidative stress-induced cell death in neuron-like PC12 cells. To further study the mechanism of its action, we monitored the effect on ER stress-associated factors, as well as NF-κB and MAPK signaling pathways. Moreover, the current study highlights yet another novel function of calycopterin in the protection of neurite outgrowth against the toxicity of H 2O2.

2. MATERIALS AND METHODS 2.1. Materials. Antibodies directed against caspase-3, calpain, NFκB, phospho-p38 MAP kinase (Thr180/Tyr182), phospho- SAPK/ JNK (Thr183/Tyr185), p38 MAP kinase, SAPK/JNK, PARP-1, Bax, Bcl-2, and β-actin were obtained from Cell Signaling Technology. Received: October 1, 2011 Published: November 14, 2011 2280

dx.doi.org/10.1021/tx200420a | Chem. Res. Toxicol. 2011, 24, 2280−2292

Chemical Research in Toxicology

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Figure 2. 1H NMR of calycopterin (acetone-d6, 500 MHz). 2,5-diphenyl tetrazolium bromide) reduction assay. The dark blue formazan crystals formed in intact cells were solubilized in dimethyl sulfoxide, and the absorbance was measured at 550 nm. Results were expressed as the percentages of reduced MTT, assuming the absorbance of control cells as 100%. 2.7. Morphological Analysis of Differentiated PC12 Cells. For morphological analysis, random images were acquired from each well, taking two images per well. A minimum of 50 cells per treatment were quantified. Criteria for selection were that the cell body and processes were completely within the field of view and that the cell body was distinct from neighboring cell bodies. Cells fitting these criteria were analyzed and their cell body area, average neurite length, average neurite width, number of primary neurites, and bipolar morphology were quantified. Data analysis was done by using the Cell A program. Cell body area was defined as the area of the cell exclusive of neurite processes. Neurite length was calculated by summing the lengths of the primary process and all associated branches. To establish the average neurite width, the outlines of individual primary neurites were traced, and the area was calculated and then divided by the length of the neurite. Primary neurites were defined as clear protrusions from the cell body greater than 10 μM length. Cells were considered ‘‘bipolar’’ if they displayed a cell body with one process at either end. To evaluate neurite networks, images were analyzed using the cell counter plugin to score all branching nodes in each image. Nodes were defined as sites at which individual neurites branched or separate neurites contacted each other. All measurements expressed as proportions used the number of cells displaying the characteristic as a subpopulation of the total number of cells that met the selection criteria described above. 2.8. Hoechst Staining. PC12 cells (1 × 106 cells/mL) were treated with different concentrations of calycopterin for 3 h followed by the addition of H2O2 (150 μM) for 24 h. Nuclear morphological changes were assessed using the Hoechst dye 33342 (Invitrogen, H3570). Cells were washed by PBS and incubated with Hoechst 33342 (1:1000) for 5 min at room temperature. Nuclei were visualized using an Olympus microscope. 2.9. Western Blot Analysis. Total proteins were electrophoresed in 12% SDS−PAGE gel, transferred to polyvinylidene fluoride membranes, and probed with specific antibodies. Immunoreactive polypeptides were detected by chemiluminescence using enhanced ElectroChemiLuminescence (ECL) reagents (Amersham Bioscience,

Phospho-ERK1/2, ERK1/2, and tumor necrosis factor-α (TNF-α) antibodies were obtained from ABCAM. Caspase-12 was obtained from Assay Designs. Lamin B2 was purchased from Santa Cruz Biotechnology. Silica gel 70−230 and 230−400 mesh were from Merck. All the other reagents, unless otherwise stated, were from Sigma Aldrich (St. Louis, MO). 2.2. Plant Material. The aerial parts of D. kotschyi were collected from Dizin region in the north of Tehran, Iran, in June 2010 and identified by Dr. A. Sonboli. A voucher specimen number (MPH1631) was deposited at the herbarium of Medicinal Plants and Drug Research Institute, Shahid Beheshti University, Tehran, Iran. 2.3. Isolation of Calycopterin. The air-dried, powdered aerial parts of D. kotschyi (750 g) were extracted successively with n-hexane (3 × 3 L), ethyl acetate (3 × 3 L), and methanol (3 × 3 L) by maceration at room temperature. Extracts were concentrated in vacuo, to afford dark gummy residues. The ethyl acetate extract (35 g) was separated on a silica gel column (230−400 mesh, 400 g) eluted with a gradient of n-hexane−EtOAc (100:0 to 0:100), followed by increasing concentrations of MeOH (up to 5%) in EtOAc. After screening by TLC, fractions with similar compositions were pooled, to yield 12 combined fractions (A−L). Fraction G was subjected to further column chromatography over silica gel (70−230 mesh) to afford four fractions (G1−G4). Fraction G2 was triturated with Me2CO to give a crude solid, which was recrystallized from Me 2CO to afford calycopterin (400 mg). 1H- and 13C NMR spectra were recorded on a Bruker DRX 500 spectrometer, using the residual acetone-d6 (δH 2.08/δC 29.8) signals as references. 2.4. Cell Culture and Differentiation. Rat pheochromocytoma (PC12) cells obtained from Pasteur Institute (Tehran, Iran) were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma, Aldrich), supplemented with 10% horse serum, 5% fetal bovine serum, and 1% antibiotic mixture comprising penicillin−streptomycin, in a humidified atmosphere at 37 °C with 5% CO2. Growth medium was changed three times a week. PC12 cells were differentiated by treating with nerve growth factor (NGF) (50 ng/mL) every other day for 6 days. 2.5. Treatment Conditions. Cells were treated with 25, 50, and 100 μM calycopterin for 3 h, followed by the addition of H2O2 (150 μM) for 24 h. 2.6. Measurement of Cell Viability. Cell viability was determined by the conventional MTT (3-[4,5-dimethylthiazol-2-yl]2281



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Figure 3. 13C NMR of calycopterin (acetone-d6, 125 MHz). USA) and subsequent autoradiography. Quantification of results was performed by densitometric scan of films. Data analysis was done by Image J, measuring integrated density of bands after background subtraction. Protein concentrations were determined according to Bradford’s method.8 A standard plot was generated by using bovine serum albumin. Nuclear and cytoplasmic proteins were isolated as described by Kutuk and Basaga.9 2.10. Measurement of Intracellular Reactive Oxygen Species (ROS). The fluorescent probe 2′,7′-dichlorofluorescein diacetate (DCF-DA) was used to monitor intracellular accumulation of ROS. For this purpose, DCFH-DA solution (10 μM) was added to the suspension of the cells (1 × 106/mL), and the mixture was incubated at 37 °C for 1 h. Cells were then washed twice with PBS, and the fluorescence intensity was measured by the Varian Cary Eclipse spectrofluorometer with excitation and emission wavelengths of 485 and 530 nm, respectively. 2.11. Measurement of Lipid Peroxidation. Malondialdehyde (MDA) levels were measured by the double heating method.10 The method is based on spectrophotometric measurement of the purple color generated by the reaction of thiobarbituric acid (TBA) with MDA. Briefly, 0.5 mL of cell lysate was mixed with 2.5 mL of tricholoroacetic acid (TCA, 10% w/v) solution followed by boiling in a water bath for 15 min. After cooling to room temperature, the samples were centrifuged at 3000 rpm for 10 min, and 2 mL of each sample supernatant was transferred to a test tube containing 1 mL of TBA solution (0.67% w/v). Each tube was then placed in boiling water for 15 min. After cooling to room temperature, the absorbance was measured at 532 nm with respect to the blank solution. 2.12. Catalase Activity Assay. Catalase (CAT) activity was measured by the method of Aebi.11 Briefly, 200 μL of cell lysate was added to a cuvette containing 1.995 mL of 50 mM phosphate buffer

(pH 7.0). Reaction was started by the addition of 1.0 mL of freshly prepared 30 mM H2O2. The rate of decomposition of H2O2 was measured spectrophotometrically at 240 nm. 2.13. Data Analysis. All data are represented as the mean ± SEM. Comparison between groups was made by one-way analysis of variance (ANOVA) followed by a specific posthoc test to analyze the difference. The statistical significances were achieved when P < 0.05 (*P < 0.05, **P < 0.01, and *** P < 0.001).

3. RESULTS 3.1. Identification of Calycopterin. Compound 1 was obtained as yellow crystals, and its 1H NMR (Figure 2) and 13C NMR (Figure 3) data were used to identify it as 5,4′-dihydroxy3,6,7,8-tetramethoxyflavone.12,13 Calycopterin (1): 1H NMR (acetone-d6) δ: 12.58 (1H, s, OH-5), 9.37 (1H, s, OH-4′), 8.11 (2H, dd, J = 8.9, 1.8 Hz, H-2′, H-6′) 7.06 (2H, dd, J = 8.9, 1.8 Hz, H-3′, H-5′), 4.09, 3.97, 3.91, 3.90 (each 3H, s, 4OMe). 13C NMR (acetone-d6) δ: 156.8 (C2), 138.6 (C-3), 179.8 (C-4), 149.4 (C-5), 136.5 (C-6), 153.3 (C-7), 133.4 (C-8), 145.2 (C-9), 107.7 (C-10), 122.1 (C-1′), 130.8 (C-2′, C-6′), 116.1 (C-3′, C-5′), 160.8 (C-4′), 59.7 (3OMe), 60.6 (6-OMe), 61.9 (7-OMe), 61.5 (8-OMe). 3.2. Calycopterin protected PC12 cells against H2O2induced cell death. To assess the effect of calycopterin on PC12 cells, differentiated PC12 cells were seeded in 96-well plates and treated with different concentrations (25, 50, 100, and 150 μM) of calycopterin for 24 h. Cell viability was determined by the MTT assay. As shown in Figure 4A, lower doses of calycopterin (25 and 50 μM) had no significant effect 2282



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Figure 4. Neuroprotective effects of calycopterin against H2O2-induced cell death in PC12 cells. Differentiated PC12 cells were treated with different concentrations (25, 50, and 100 μM) of calycopterin for 3 h. After 24 h, cell viability was determined by the MTT assay in the absence (A) and/or presence (B) of H2O2 (150 μM). Viability was calculated as the percentage of living cells in treated cultures compared to those in control cultures. Each value represents the mean ± SD (n = 3). ‡Significantly different from untreated cells. *Significantly different from H2O2-treated cells. The statistical significances were achieved when P < 0.05 (**P < 0.01).

on cell viability, while higher concentrations (100 and 150 μM) decreased cell viability, compared to that of the control. To evaluate the neuroprotective effect of calycopterin, differentiated PC12 cells were seeded in 96-well plates, pretreated with 25, 50, and 100 μM calycopterin for 3 h, and then exposed to H2O2 (150 μM) for 24 h. Cell viability was determined by the MTT assay. As shown in Figure 4B, pretreatment of cells with 25 and 50 μM calycopterin increased survival against H2O2-induced cell death by 2.9- and 3.2-fold, respectively, compared to that of H2O2-treated cells. The effect of 100 μM calycopterin was not significantly different from H2O2-treated cells. 3.3. Morphological Evaluation of Apoptosis. Hoechst 33342 is a DNA stain that binds preferentially to A-T basepairs. Hoechst staining showed that DNA fragmentation and condensation of chromatin, which cause bright fluorescence, were decreased in the presence of 25 and 50 μM calycopterin, compared to those in H2O2-treated cells (Figure 5). At the

concentration of 100 μM, calycopterin had no beneficial effect, compared to that of H2O2-treated cells. 3.4. Effect of Calycopterin on Neurite Outgrowth in Differentiated PC12 Cells. Cell body area, average neurite length, and the average neurite width were evaluated to monitor cell growth (Figure 6A). As shown in Figure 6D, in H2O2-exposed cultures cell body area was increased, compared to that in control cells. Pretreatment of cells with 25 and 50 μM calycopterin resulted in a significant decrease in cell body area. Moreover, neurites exposed to 150 μM H2O2 were not only longer but also wider than those in control cultures (Figure 6C and E). We found that calycopterin, at the concentrations of 25 and 50 μM, could significantly increase average neurite length by 3.4- and 3.8-fold, respectively, compared to that in H 2O2treated cells (Figure 6E). Specific parameters of morphological complexity were also measured. The number of primary neurites (>10 μm) emanating from individual cell bodies was measured. We 2283



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Figure 5. AO/EB double staining. The cells were exposed to 25, 50, and 100 μM calycopterin for 3 h followed by exposure to 150 μM H2O2 for 24 h. The cells were harvested, resuspended in PBS, and incubated with AO/EB (A) and/or Hoechst 33342 (B) stain. The morphological patterns of apoptotic cells are described in the text. All experiments were repeated three times.

found that bipolar morphology of a cell body was increased in H2O2-treated cells by 2.0-fold, compared to that in control cultures. Pretreatment of cells with 25 and 50 μM calycopterin increased the number of neurites per cell and thus decreased the proportion of bipolar cells (Figure 6F and G). We also calculated the ratio of total neurite branching nodes to total number of primary neuritis. The ratio of nodes to primary neurites was reduced in H2O2-treated cells to 48.6%, compared to that in the control, whereas 25 and 50 μM calycopterin increased this ratio by 1.8- and 2.0-fold, respectively, compared to that in H2O2-treated cells (Figure 6H). Moreover, we detected no significant effect in the presence of 100 μM calycopterin on criteria of differentiation, compared to that in H2O2-treated cells (Figure 6A−H). 3.5. Decrease of Bax/Bcl-2 Ratio and Caspase-3 Level by Calycopterin in Neuron-Like PC12 Cells. The ratio of proapoptotic Bax to antiapoptotic Bcl-2 has been reported to be correlated to the initiation of a cascade which leads to the activation of caspases, such as caspase-3.14 To investigate the effects of calycopterin on Bax/Bcl-2 ratio, PC12 cells were

treated with different doses of calycopterin for 3 h and then exposed to H2O2 for 24 h. Our results showed that H2O2 significantly increased the Bax/Bcl-2 ratio, whereas 25 and 50 μM calycopterin could significantly decrease this ratio (Figure 7A and B). Moreover, in the cells pretreated with 25 and 50 μM calycopterin, the level of cleaved caspase-3 was decreased to 35.0 and 23.4%, respectively, compared to that in H 2O2treated cells, while at the concentration of 100 μM no protective effect was observed (Figure 7A and C). 3.6. Effect of Calycopterin on PARP-1 Cleavage in PC12 Cells. In order to confirm caspase-3 activation, we examined the caspase-3 substrate PARP-1 and its cleaved fragment using Western blot analysis. To confirm that calycopterin decreases apoptosis, we measured the level of cleaved PARP-1 by Western blot analyses in the presence of different concentrations of calycopterin. As shown in Figure 8, we found that pretreatment of PC12 cells with 25 and 50 μM calycopterin significantly decreased the level of 89 kDa and 24 kDa bands, compared to that in H2O2-treated cells. The effect 2284



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Figure 6. Effect of calycopterin on H2O2-induced disruption of neurite outgrowth in differentiated PC12 cells. (A) The criteria of PC12 differentiation is shown on two neurons (left image) of a sample image. The “P” on right image indicates the primary neuritis of a neuron 1. The yellow arrow shows the length of a neurite, extent of elongation, and membrane-enclosed protrusions of cytoplasm. The green circle in the right image shows the cell body. Neurite width is not equal in all parts of the neurons; thus, the average neurite width must be calculated by dividing cell body area to average neurite length. The blue arrows shows two bipolar cells. The letter “N” indicates the nodes, the sites at which individual neurites branched or separate neurites contacted each other. (B) NGF-differentiated PC12 cells were pretreated with 25, 50, and 100 μM calycopterin for 3 h and then exposed to H2O2 (150 μM). Criteria were quantified at 24 h; (C) average neurite length; (D) cell body area; (E) average neurite width; (F) number of primary neurites per cell; (G) percent of bipolar cells; and (H) the ratio of nodes to primary neurites. ‡Significantly different from untreated cells. *Significantly different from H2O2-treated cells. The statistical significances were achieved when P < 0.05 (*P < 0.05, **P < 0.01).

respectively, compared to those in H2O2-treated cells. We could not detect any difference from H2O2-treated cells in those cells pretreated with 100 μM calycopterin (Figure 9). 3.8. Calycopterin Significantly Reduced ROS Generation in Differentiated PC12 Cells. Intracellular free radicals, such as ROS, are key messengers for the interaction among ER stress, oxidative stress, and inflammation.17 To examine whether calycopterin affects H2O2-induced apoptosis through reducing intracellular ROS production, we measured the ROS level in the presence and/or absence of different concentrations of calycopterin. As shown in Table 1, while H2O2 increased H2DCFDA fluorescence, treatment of PC12 cells with 25 and 50 μM calycopterin significantly decreased the intracellular oxidation of the fluorescent probe.

of 100 μM calycopterin was not significantly different from H2O2-treated cells (Figure 8). 3.7. Calycopterin Reduced Caspase-12 and Calpain Levels in PC12 Cells. Caspase-12 is an ER-specific caspase that participates in apoptosis under ER stress.15 Calpain, a cytoplasmic cysteine protease that requires calcium ions for activity, is implicated in a variety of cellular processes such as signal transduction, cell proliferation, differentiation, and apoptosis.16 To determine whether calycopterin affects ER stress factors, we measured caspase-12 and calpain levels by Western blot analysis. As shown in Figure 9, pretreatment of cells with 25 and 50 μM calycopterin reduced the calpain level to 42.9 and 33.1% (Figure 9A and B) and decreased the caspase-12 level to 38.6 and 29.7% (Figure 9A and C), 2285



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In addition, 150 μM H2O2 decreased CAT activity to 37.6%, whereas pretreatment with 25 and 50 μM calycopterin significantly increased CAT activity by about 2.77- and 2.93fold, respectively (Table 2). 3.10. Calycopterin Decreased the NF-κB Nuclear Level in Differentiated PC12 Cells. NF-κB is a transcription factor widely expressed in neurons, which has roles in regulating inflammation, control of cell division, and apoptosis.18 Several factors including ROS generation and ER stress, lead to the phosphorylation of NF-κB cytoplasmic inhibitor, IκB-α, and subsequent proteosomal degradation of IκB-α.19 NF-κB is thus liberated and transported to the nucleus to initiate the transcription of downstream inflammatory mediators, such as COX-2. As shown in Figure 10, 25 and 50 μM calycopterin significantly decreased the NF-κB nuclear level to 37.7 and 31.0%, respectively, compared to that in H2O2-treated cells. We found that 100 μM calycopterin did not significantly affect NFκB nuclear translocation, compared to that in H 2O2-treated cells. 3.11. Calycopterin Decreased COX-2 and TNF-α Expression in H2O2-Treated PC12 Cells. TNF-α is a pleiotropic cytokine which is involved in the blood−brain barrier, inflammatory, thrombogenic, and vascular changes associated with brain injury.20 We investigated whether calycopterin could prevent the activation of TNF-α. We found that H2O2 exposure increased the TNF-α level by 2.03-fold, as determined by Western blot analysis (Figure 11A and B). This level was decreased to 62.3 and 56.7%, when cells were pretreated with 25 and 50 μM calycopterin, respectively, compared to the H2O2-treated cells. Cyclooxygenase or prostaglandin−endoperoxide synthase catalyzes the conversion of arachidonic acid and oxygen into prostaglandin H2, suggesting that COX-2 is involved in the mechanism of neuronal death/survival.21 As shown in Figure 11A and C, pretreatment of cells with 25 and 50 μM calycopterin decreased the COX-2 level to 30.0 and 23.4%, respectively, compared to H2O2-treated cells. Not surprisingly, no significant change was detected in the presence of 100 μM calycopterin, compared to that in H2O2-exposed cells (Figure 11). 3.12. Calycopterin Decreased MAPK Phosphorylation in PC12 Cells. In order to further investigate the molecular mechanisms of neuroprotection exerted by calycopterin, we examined its effect on the H2O2-induced phosphorylation of MAPKs. MAPK cascades are key signaling pathways involved in the regulation of normal cell proliferation, survival, and differentiation.22 As shown in Figure 12, phosphorylation of p38, ERK1/2, and JNK were decreased in the presence of 25 and 50 μM calycopterin in H2O2-stimulated PC12 cells. No significant changes were detected in the level of nonphosphorylated ERK, JNK, and p38 MAPK in the cells treated with H2O2, in the presence and/or absence of calycopterin. Moreover, no significant change was detected in the presence of 100 μM calycopterin (Figure 12).

Figure 7. Bax/Bcl-2 ratio and caspase-3 levels in PC12 cells pretreated with calycopterin. (A) PC12 cells were pretreated with 25, 50, and 100 μM calycopterin for 3 h and then exposed to H2O2 (150 μM) for 24 h. Twenty micrograms of proteins were separated on SDS−PAGE, Western blotted, probed with anti-Bax, anti-Bcl-2, and/or anticaspase3 antibodies and reprobed with anti-β-actin antibody (one representative Western blot is shown; n = 3). (B) The ratio of densities of Bax and Bcl-2 bands to β-actin and the ratio of normalized Bax/Bcl-2 were measured. (C) The densities of Procaspase-3 and Caspase-3 bands were measured, and the ratio to β-actin were calculated. The mean of three independent experiments is shown. ‡ Significantly different from untreated cells. *Significantly different from H2O2-treated cells. The statistical significances were achieved when P < 0.05 (*P < 0.05, **P < 0.01).

4. DISCUSSION AD is a devastating neurodegenerative disease characterized by the progressive decline of cognitive functions. Although the exact cause of AD remains elusive, apoptosis, an evolutionarily conserved form of cell death, has been known as one of the most important signaling pathways, which have been implicated in the pathophysiology of AD.1 Apoptosis is a gene-regulated phenomenon with the characteristic change of cellular structure

3.9. Calycopterin Reduced Lipid Peroxidation and Increased CAT Activity in PC12 Cells. Treatment of PC12 cells with 150 μM H2O2 caused the increase in the intracellular MDA level by 1.67-fold, while preincubation of cells with 25 and 50 μM calycopterin markedly attenuated the change of the MDA level to 67.0 and 58.7%, respectively, compared to that in H2O2-treated cells (Table 2). 2286



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Figure 8. Expression of PARP-1−1 in PC12 cells pretreated with calycopterin. (A) PC12 cells were pretreated with 25, 50, and 100 μM calycopterin for 3 h and then exposed to H2O2 (150 μM) for 24 h. Twenty micrograms of proteins were separated on SDS−PAGE, Western blotted, probed with anti-PARP-1 antibody, and reprobed with anti-Lamin B2 antibody. (One representative Western blot is shown; n = 3.) (B) The densities of the corresponding bands were measured, and the ratio to Lamin B2 were calculated. The mean of three independent experiments is shown. ‡Significantly different from untreated cells. *Significantly different from H2O2-treated cells. The statistical significances were achieved when P < 0.05 (*P < 0.05, **P < 0.01).

myelination, and programmed cell death.27 PC12 cells are derived from a rat adrenal medullarly tumor and exit the cell cycle and differentiate into sympathetic neuron-like cells when treated with NGF, providing a useful model for studying neuronal signaling pathways and other neurobiochemical events.28 In this study, we found that calycopterin protects PC12 cells against H2O2-induced apoptosis, as was evident by the decrease of chromatin condensation in Hoechst staining. In support of this morphological observation, we also detected a decreased level of Bax/Bcl2, caspase-3, and cleaved PARP-1 in the presence of 25 and 50 μM calycopterin. In addition, on the basis of our results calycopterin improves neurite outgrowth that has been impaired by H2O2. Once neurons begin to aggregate into recognizable structures, and sometimes even before, they begin to extend elongated, membrane-enclosed protrusions of cytoplasm that are called processes or neurites. 29 The lack of normal neurite outgrowth might reflect the dysfunction of molecules or proteins that are important for maintaining a normal neuronal process, and rescue of damaged neurons is an attractive strategy for the treatment of neurodegenerative diseases.30 We found that calycopterin at the concentrations of 25 and 50 μM could increase neural

including chromatin condensation, cell and nuclear shrinkage, membrane blebbing, and oligonucleosomal DNA fragmentation.23 Many agents that induce apoptosis are either oxidants or stimulators of cellular oxidative metabolism. Conversely, many inhibitors of apoptosis have antioxidant activities or enhance cellular antioxidant defenses.24,25 Medicinal plants have been widely investigated for their various effects. D. kotschyi Boiss. (Labiatae) is used in Iranian traditional medicine for the treatment of rheumatoid diseases. The inhibitory effect of D. kotschyi on the lectin-induced cellular immune response has been demonstrated previously.7 In the present study, we focused on the effect of calycopterin, which is isolated from D. kotschyi, on oxidative stress-induced apoptosis in neuron-like PC12 cells and determined the signaling pathways involved in its neuroprotective effect. Calycopterin was first isolated both by Karner and Ratnagiriswaran et al. in 1934,5,6 and its chemical structure was discovered by Rodriguez et al. in 1972, 26 but still, little is known about the biological activity and the mechanism of its action. Key cellular processes of a normal brain include proliferation, differentiation of precursor cells into neurons or glia, migration, elaboration of axons and dendrites, synapse formation, 2287



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Table 2. Effect of Calycopterin on Lipid Peroxidation and Antioxidant Enzyme Activities in H2O2-Treated PC12 Cells treatment control H2O2 calycopterin calycopterin H2O2 calycopterin calycopterin H2O2 calycopterin calycopterin H2O2

MDA (nmol/mg protein) ± ± ± ±

0.03 0.06† 0.04 0.03*

CAT (μmol/mg protein)

(25 μM) (25 μM) +

0.58 0.97 0.62 0.65

2.10 0.79 1.91 2.19

± ± ± ±

0.10 0.08† 0.09 0.07*

(50 μM) (50 μM) +

0.60 ± 0.03 0.57 ± 0.04*

1.98 ± 0.10 2.32 ± 0.11*

(100 μM) (100 μM) +

0.72 ± 0.04† 0.78 ± 0.04

1.01 ± 0.070† 0.89 ± 0.08

a†

Significantly different from untreated cells. *Significantly different from H2O2-treated cells.

Figure 9. Calpain and caspase-12 levels in PC12 cells pretreated with calycopterin. (A) PC12 cells were pretreated with 25, 50, and 100 μM calycopterin for 3 h and then exposed to H2O2 (150 μM) for 24 h. Twenty micrograms of proteins were separated on SDS−PAGE, Western blotted, probed with anticalpain, and/or anticaspase-12 antibodies and reprobed with anti-β-actin antibody. (One representative Western blot is shown; n = 3.) The densities of calpain (B) and caspase-12 (C) bands were measured, and the ratio to β-actin was calculated. The mean of three independent experiments is shown. ‡ Significantly different from untreated cells. *Significantly different from H2O2-treated cells. The statistical significances were achieved when P < 0.05 (*P < 0.05, **P < 0.01).

Figure 10. Effects of calycopterin on the nuclear level of NF-κB in PC12 cells. (A) PC12 cells were pretreated with 25, 50, and 100 μM calycopterin for 3 h and then exposed to H2O2 (150 μM) for 24 h. Twenty micrograms of proteins were separated on SDS−PAGE, Western blotted, probed with anti-NF-κB antibody, and reprobed with anti-Lamin B2 antibody (one representative Western blot is shown; n = 3). (B) The densities of corresponding bands were measured, and the ratio to Lamin B2 were calculated. The mean of three independent experiments is shown. ‡Significantly different from untreated cells. *Significantly different from H2O2-treated cells. The statistical significances were achieved when P < 0.05 (*P < 0.05, ** P < 0.01).

Table 1. Intracellular ROS Levels in PC12 Cells Pretreated with 25, 50, and 100 μM Calycopterin Measured by DCF-DA (520 nm)a treatment

fluorescence intensity (absorbance at 520 nm)

control H2O2 calycopterin (25 μM) + H2O2 calycopterin (50 μM) + H2O2 calycopterin (100 μM) + H2O2

48.8 ± 4.1 335.6 ± 9.8† 160.9 ± 8.6** 106.7 ± 9.3** 310.2 ± 8.1

length and neuronal complexity, the morphological characteristics of neural differentiation which reflect the existence of normal neuronal processes. Apoptotic cell death occurs through the precisely controlled transcription of certain genes.14,23 Many current studies have focused on the analysis of apoptotic factors centered in the mitochondria and the nucleus. However, many others have reported that neuronal death in AD has its origin in ER.31,32 There is a growing body of evidence that the ER can play pivotal roles in regulating cell survival and apoptosis in a variety of cell types including neurons.33 Two important factors involved in ER stress are caspase-12 and calpain. Activation of

a

The mean of three independent experiments is shown. †Significantly different from untreated cells. *Significantly different from H2O2treated cells. The statistical significances were achieved when P < 0.05 (**P < 0.01, ***P < 0.001). 2288



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PARP-1 activity. It is tempting to speculate that calpaindependent PARP-1 cleavage turns the enzyme into a constitutively active form that no longer requires binding to damaged DNA for activity.37 Moreover, calpain is able to be activated via caspase-mediated cleavage of its inhibitor, calpastatin, during the initiation of apoptosis.38 Here, we did not address the signaling pathway associated with ER stress; however, our findings provide evidence that calycopterin protects PC12 cells against death under conditions associated with ER stress. Reduction of intracellular ROS level by calycopterin in H2O2treated cells added another piece to the puzzle of neuroprotection exerted by this compound. Mitochondria are the major generator and also regulator of ROS. Normally, ROS act as key second messengers in numerous biological signaling pathways including differentiation, proliferation, transcriptional regulation, and cell death.17 Overproduction of ROS and insufficiency of scavenging enzymes like CAT, which were observed in this study, lead to ROS accumulation, which causes apoptosis, inflammatory responses, and finally cell death. It is widely accepted that neuroinflammation is a key player in AD.39 The NF-κB transcription factor system is the major signaling pathway evoking the inflammatory response.18 NF-κB is sequestered in the cytoplasm, where it is bound to I-κB. Exposure to diverse stimuli induces the release of NF-κB from I-κB and causes its translocation to the nucleus, allowing activation of transcription.19 Several studies have indicated that ROS could directly, i.e., by phosphorylating I-κB, and indirectly, i.e., by degrading I-κB, activate NF-κB.19,40 Indeed, it is widely accepted that both ROS-scavenging enzymes and antioxidant agents could block NF-κB activation.41,42 Our findings, which showed decreased levels of both nuclear NF-κB level and its downstream molecules, including TNF-α and COX-2, along with the increase of antioxidant CAT enzyme in the presence of calycopterin, indicated that this flavone could interfere with the NF-κB pathway and inflammatory response induced by H2O2 in PC12 cells. Moreover, we found that the phosphorylation of MAPKs was suppressed in the presence of calycopterin. MAPK cascades are other important signaling pathways involved in proliferation, differentiation, and cell death. MAPKs transduce signals via sequential phosphorylation of distinct kinase modules, which ultimately leads to an appropriate biological response to the initial stimuli.22 Evidence shows that the proapoptotic JNK cascade induces apoptosis via the phosphorylation of Bcl-2, which causes interruption of its anti-apoptotic function.43 Once again, ROS could directly, by activating upstream kinases, and indirectly, by inactivating inhibitors, promote prolonged MAPK activation.44,45 Interestingly, Ventura et al. have reported that TNF-α-induced ROS accumulation was blocked in the cells lacking JNK, indicating a central role for JNK in ROS accumulation.46 Moreover, many reports suggest that JNK, ERK1/2, and p-38 MAPK inhibit NF-κB activation,47,48 while other reports indicate a stimulatory role for MAPK toward NFκB activation.49,50 Despite the effort of well-defined interactions between MAPKs, ROS, and NF- κB, review of the existing evidence shows that their interactions are highly dependent on cell type and context. Our results showed that treatment of PC12 cells with 25 and 50 μM calycopterin decreased MAPK phosphorylation, as well as NF-κB activation, resulting in increase of cell survival, compared to that of H2O2-treated cells. In summary, we provided the documentation of the protective effects of calycopterin, a natural flavonoid in PC12

Figure 11. Effects of calycopterin on the level of COX-2 and TNF-α in PC12 cells. (A) PC12 cells were pretreated with 25, 50, and 100 μM calycopterin for 3 h and then exposed to H2O2 (150 μM) for 24 h. Twenty micrograms of proteins were separated on SDS−PAGE, Western blotted, probed with anti-COX-2 and/or anti-TNF-α antibodies, and reprobed with anti-β-actin antibody (one representative Western blot was shown; n = 3). The densities of TNF-α (B) and COX-2 (C) bands were measured, and the ratios to β-actin were calculated. The mean of three independent experiments is shown. ‡ Significantly different from untreated cells. *Significantly different from H2O2-treated cells. The statistical significances were achieved when P < 0.05 (*P < 0.05, **P < 0.01).

caspase-12, which may occur very early prior to any detectable changes in mitochondrial function, appears to be required for ER stress-induced apoptosis because neurons from caspase-12 deficient mice are resistant to apoptosis induced by Aβ.34 Overall, the data in the literature suggest that caspase-12 is initially processed by calpain, but it can also be activated by caspase-3/7. On the contrary, active caspase-12 could cleave procaspase-3 to its active form.35 Thus, active caspase-12 can initiate a positive feedback loop that activates caspase-3 and potentiates apoptosis induction. Besides, in classical apoptosis, PARP-1 activity is completely abolished by caspase-dependent cleavage within PARP-1’s DNA binding domain.36 Interestingly, PARP-1 is also a calpain substrate, and it has been suggested that calpain-dependent cleavage of PARP-1 ablates 2289



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Figure 12. Effects of calycopterin on H2O2-induced phosphorylation of MAPKs in PC12 cells. (A) PC12 cells were pretreated with 25, 50, and 100 μM calycopterin for 3 h and then exposed to H2O2 (150 μM) for 24 h. Twenty micrograms of proteins were separated on SDS−PAGE, Western blotted, probed with phosphorylated MAPK antibodies, and reprobed with anti-β-actin antibody. (One representative Western blot was shown; n = 3.) The densities of phospho-ERK, ERK (B), phospho-JNK, JNK (C), phospho-p38, and p38 (D) bands were measured, and the ratio to total levels was calculated. The mean of three independent experiments is shown. ‡Significantly different from untreated cells. *Significantly different from H2O2-treated cells. The statistical significances were achieved when P < 0.05 (*P < 0.05, **P < 0.01).

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cells. Interestingly, we found that 25 and 50 μM calycopterin could protect neurite outgrowth and neural complexity against H2O2 that could present a highly promising avenue for pharmacological intervention to minimize neuronal cell injury in different pathological conditions. Clearly, further insight into calycopterin function will emerge from a better understanding of its interaction with other ER resistant proteins and their crosstalk with upstream and downstream signaling molecules. A further study of its detailed mechanisms is now in progress in our laboratory.

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REFERENCES

(1) Sims, N. R. (1996) Energy metabolism, oxidative stress and neuronal degeneration in Alzheimer’s disease. Neurodegeneration 5, 435−440. (2) Halliwell, B. (1991) Reactive oxygen species in living systems: source, biochemistry, and role in human disease. Am. J. Med. 91, 14S− 22S. (3) Fleury, C., Mignotte, B., and Vayssière, J. L. (2002) Mitochondrial reactive oxygen species in cell death signaling. Biochimie 84, 131−141. (4) Balunas, M. J., and Kinghorn, A. D. (2005) Drug discovery from medicinal plants. Life Sci. 78, 431−441. (5) Karrer, W., and Venkataraman, K. (1935) Identity of calycopterin and thapsin. Nature 135, 878−878. (6) Ratnagiriswaran, A. N., Sehra, K. B., and Venkataraman, K. (1934) The anthelminthic constituent of the leaves of Calycopteris floribunda. Biochem. J. 28, 1964−1967. (7) Faham, N., Javidnia, K., Bahmani, M., and Amirghofran, Z. (2008) Calycopterin, an immunoinhibitory compound from the extract of Dracocephalum kotschyi. Phytother. Res. 22, 1154−1158. (8) Bradford, M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248−254.

AUTHOR INFORMATION

Corresponding Author *Tel: 0098-21-22429768. Fax: 0098-21-22432047. E-mail: [email protected]. Author Contributions § These authors contributed equally to this work. Funding This work was supported by Research Council of the Shahid Beheshti University and in part by Shahid Beheshti University of Medical Sciences Research Funds. 2290



Article

(1−42) is generated in the endoplasmic reticulum/intermediate compartment of NT2N cells. Nat. Med. 3, 1021−1023. (32) Mattson, M. P., and Guo, Q. (1997) Cell and molecular neurobiology of presenilins: a role for the endoplasmic reticulum in the pathogenesis of Alzheimer’s disease? J. Neurosci. Res. 50, 505−513. (33) Takuma, K., Yan, S. S., Stern, D. M., and Yamada, K. (2005) Mitochondrial dysfunction, endoplasmic reticulum stress, and apoptosis in Alzheimer’s disease. J. Pharmacol. Sci. 97, 312−316. (34) Nakagawa, T., Zhu, H., Morishima, N., Li, E., Xu, J., Yankner, B. A., and Yuan, J. (2000) Caspase-12 mediates endoplasmic-reticulumspecific apoptosis and cytotoxicity by amyloid-beta. Nature 403, 98− 103. (35) Hitomi, J., Katayama, T., Taniguchi, M., Honda, A., Imaizumi, K., and Tohyama, M. (2004) Apoptosis induced by endoplasmic reticulum stress depends on activation of caspase-3 via caspase-12. Neurosci. Lett. 357, 127−130. (36) Wesierska-Gadek, J., Gueorguieva, M., Wojciechowski, J., and Tudzarova-Trajkovska, S. (2004) In vivo activated caspase-3 cleaves PARP-1 in rat liver after administration of the hepatocarcinogen Nnitrosomorpholine (NNM) generating the 85 kDa fragment. J. Cell Biochem. 93, 774−787. (37) McGinnis, K. M., Gnegy, M. E., Park, Y, H, Mukerjee, N., and Wang, K. K. (1999) Procaspase-3 and poly(ADP)ribose polymerase (PARP) are calpain substrates. Biochem. Biophys. Res. Commun. 263, 94−99. (38) Wang, K. K., Posmantur, R., Nadimpalli, R., Nath, R., Mohan, P., Nixon, R. A., Talanian, R. V., Keegan, M., Herzog, L., and Allen, H. (1998) Caspase-mediated fragmentation of calpain inhibitor protein calpastatin during apoptosis. Arch. Biochem. Biophys. 356, 187−196. (39) Agostinho, P., Cunha, R. A., and Oliveira, C. (2010) Neuroinflammation, oxidative stress and the pathogenesis of Alzheimer’s disease. Curr. Pharm. Des. 16, 2766−2778. (40) Hayden, M. S., and Ghosh, S. (2004) Signaling to NF-kappaB. Genes Dev. 18, 2195−2224. (41) Ansari, N., Khodagholi, F., Amini, M., and Shaerzadeh, F. (2011) Attenuation of LPS-induced apoptosis in NGF-differentiated PC12 cells via NF-κB pathway and regulation of cellular redox status by an oxazine derivative. Biochimie 93, 899−908. (42) Packer, L., and Suzuki, Y. J. (1993) Vitamin E and alpha-lipoate: role in antioxidant recycling and activation of the NF-kappa B transcription factor. Mol. Aspects Med. 14, 229−239. (43) Deng, X., Xiao, L., Lang, W., Gao, F., Ruvolo, P., and May, W. S. Jr. (1993) Novel role for JNK as a stress-activated Bcl2 kinase. J. Biol. Chem. 276, 23681−23688. (44) Kamata, H., Honda, S., Maeda, S., Chang, L., Hirata, H., and Karin, M. (2005) Reactive oxygen species promote TNFalpha-induced death and sustained JNK activation by inhibiting MAP kinase phosphatases. Cell 120, 649−661. (45) Bubici, C., Papa, S., Dean, K., and Franzoso, G. (2006) Mutual cross-talk between reactive oxygen species and nuclear factor-kappa B: molecular basis and biological significance. Oncogene 25, 6731−6748. (46) Ventura, J. J., Cogswell, P., Flavell, R. A., Baldwin, A. S. Jr., and Davis, R. J. (2004) JNK potentiates TNF-stimulated necrosis by increasing the production of cytotoxic reactive oxygen species. Genes Dev. 18, 2905−2915. (47) Je, J. H., Lee, J. Y., Jung, K. J., Sung, B., Go, E. K, Yu, B. P., and Chung, H. Y. (2004) NF-kappaB activation mechanism of 4hydroxyhexenal via NIK/IKK and p38 MAPK pathway. FEBS Lett. 566, 183−189. (48) Kim, H. J, Lee, H. S., Chong, Y. H., and Kang, J. L. (2006) p38 Mitogen-activated protein kinase up-regulates LPS-induced NFkappaB activation in the development of lung injury and RAW 264.7 macrophages. Toxicology 225, 36−47. (49) Li, J. D., Feng, W., Gallup, M., Kim, J. H., Gum, J., Kim, Y., and Basbaum, C. (1998) Activation of NF-kappaB via a Src-dependent Ras-MAPK-pp90rsk pathway is required for Pseudomonas aeruginosainduced mucin overproduction in epithelial cells. Proc. Natl. Acad. Sci. U.S.A. 95, 5718−5723.

(9) Kutuk, O., and Basaga, H. (2003) Aspirin prevents apoptosis and NFkappaB activation induced by H2O2 in HeLa cells. Free Radical Res. 37, 1267−1276. (10) Draper, H. H., and Hadley, M. (1990) Malondialdehyde determination as index of lipid peroxidation. Methods Enzymol. 186, 421−431. (11) Aebi, H. (1984) Catalase in vitro. Methods Enzymol. 105, 121− 126. (12) Gohari, A., Saeidnia, S., Matsuo, K., Uchiyama, N., Yagura, T., and Michiho, K. (2003) Flavonoid constituents of Dracocephalum kotschyi growing in Iran and their trypanocidal activity. Nat. Med. 57, 250−252. (13) El-Ansari, M. A., Barron, D., Abdalla, M. F., Saleh, N. A. M., and Le Quere, J. L. (1991) Flavonoid constituents of Stachys aegyptiaca. Phytochemistry 30, 1169−1173. (14) Ola, M. S., Nawaz, M., and Ahsan, H. (2011) Role of Bcl-2 family proteins and caspases in the regulation of apoptosis. Mol. Cell. Biochem. 351, 41−58. (15) Nakagawa, T., and Yuan, J. (2000) Cross-talk between two cysteine protease families. Activation of caspase-12 by calpain in apoptosis. J. Cell Biol. 150, 887−894. (16) Perrin, B. J., and Huttenlocher, A. (2002) Calpain. Int. J. Biochem. Cell Biol. 34, 722−5. (17) Zhang, K. (2010) Integration of ER stress, oxidative stress and the inflammatory response in health and disease. Int. J. Clin. Exp. Med. 3, 33−40. (18) Karin, M. (1998) The NF-kappa B activation pathway: its regulation and role in inflammation and cell survival. Cancer J. Sci. Am. 4 (Suppl 1), S92−S99. (19) Kaltschmidt, B., Sparna, T., and Kaltschmidt, C. (1999) Activation of NF-kappa B by reactive oxygen intermediates in the nervous system. Antioxid. Redox Signaling 1, 129−144. (20) Clark., I. A., Alleva, L. M., and Vissel, B. (2010) The roles of TNF in brain dysfunction and disease. Pharmacol. Ther. 128, 519−548. (21) O’Banion, M. K., Chang, J. W., Kaplan, M. D., Yermakova, A., and Coleman, P. D. (1997) Glial and neuronal expression of cyclooxygenase-2: relevance to Alzheimer’s disease. Adv. Exp. Med. Biol. 433, 177−180. (22) Choi, W. S., Eom, D. S., Han, B. S, Kim, W. K., Han, B. H., Choi, E. J., Oh, T. H., Markelonis, G. J., Cho, J. W., and Oh, Y. J. (2004) Phosphorylation of p38 MAPK induced by oxidative stress is linked to activation of both caspase-8- and -9-mediated apoptotic pathways in dopaminergic neurons. J. Biol. Chem. 279, 20451−20460. (23) Gerschenson, L. E., and Rotello, R. J. (1992) Apoptosis: a different type of cell death. FASEB J. 6, 2450−2455. (24) Asadi, S., Ahmadiani, A., Esmaeili, M. A., Sonboli, A., Ansari, N., and Khodagholi, F. (2010) In vitro antioxidant activities and an investigation of neuroprotection by six Salvia species from Iran: a comparative study. Food Chem. Toxicol. 48, 1341−1349. (25) Lee, C., Park, G. H., Kim, C. Y., and Jang, J. H. (2011) [6]Gingerol attenuates β-amyloid-induced oxidative cell death via fortifying cellular antioxidant defense system. Food Chem. Toxicol. 49, 1261−1269. (26) Rodriguez, E., Vander Velde, G., Mabry, T. J., Sankara Subramanian, S., and Nair, A. G. R. (1972) The structure of calycopterin. Phytochemistry 11, 2311−2312. (27) Schwartzkroin, P. A. (2001) Mechanisms of brain plasticity: from normal brain function to pathology. Int. Rev. Neurobiol. 45, 1−15. (28) Greene, L. A., and Tischler, A. S. (1982) PC12 pheochromocytoma cells in neurobiological research. Adv. Cell Neurobiol. 3, 373−414. (29) Dotti, C. G., Sullivan, C. A, and Banker, G. A. (1988) The establishment of polarity by hippocampal neurons in culture. J. Neurosci. 8, 1454−68. (30) Radio, N. M., and Mundy, W. R. (2008) Developmental neurotoxicity testing in vitro: models for assessing chemical effects on neurite outgrowth. Neurotoxicology 29, 361−376. (31) Cook, D. G, Forman, M. S, Sung, J. C, Leight, S., Kolson, D. L, Iwatsubo, T., Lee, V. M., and Doms, R. W. (1997) Alzheimer’s A beta 2291



Article

(50) Vanden Berghe, W., Plaisance, S., Boone, E., De Bosscher, K., Schmitz, M. L., Fiers, W., and Haegeman, G. (1998) p38 and extracellular signal-regulated kinase mitogen-activated protein kinase pathways are required for nuclear factor-kappaB p65 transactivation mediated by tumor necrosis factor. J. Biol. Chem. 273, 3285−3290.

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Calycopterin Promotes Survival and Outgrowth of Neuron-Like PC12

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