S. choloroleuca, S. mirzayanii and S. santolinifolia protect PC12 cells from H(2)O (2)-induced apoptosis by blocking the intrinsic pathway

Several studies have shown that neuronal cell death due to apoptosis is the major reason for cognitive decline in Alzheimer's disease. In this study, we report the anti-apoptotic effects of three Salvia species from Iran-S. choloroleuca, S. mirzayanii and S. santolinifolia-against H(2)O(2)-induced cytotoxicity in neuron-like PC12 cells. We showed that these antioxidant species could interfere with the intrinsic pathway of apoptosis by attenuating Bax/Bcl-2 ratio, decreasing outer mitochondrial membrane break and decreasing cytochrome c release to cytoplasm. Interestingly, we found that these species were able to replenish reduced glutathione level which affects cellular redox status and cytochrome c activity. Moreover, the decreased level of caspase-3, the executioner caspase, resulted in decrease of PARP-1 cleavage. Anti-apoptotic effects of these species along with their antioxidant effects, may represent a promising approach for treatment of neurodegenerative diseases.     

N-Propionylmannosamine-induced over-expression and secretion of thioredoxin leads to neurite outgrowth of PC12 cells

The function of the central nervous system largely depends on growth and differentiation (neurite outgrowth) of neural cells and it is well established that growth factors, especially nerve growth factor NGF stimulate neurite outgrowth. However, additional factors are implicated in this process notably the redox state of the cells. For the first time we could demonstrate that the application of recombinant thioredoxin stimulates neurite outgrowth of PC12 cells to the same extend as NGF. Thioredoxin, a small redox protein is a major player in the cellular protein reduction system. An increased expression and secretion of thioredoxin is achieved by the application of the novel sialic acid precursor N-propionylmannosamine (ManNProp). From earlier studies it is known that this N-acylmannosamine analog stimulates significantly the neurite outgrowth in cell cultures. This finding would give new insights into the mechanism of the nerve-stimulatory action of ManNProp and demonstrates the novel role of thioredoxin during the regulation of nerve growth, encouraging further studies.     

Panaxynol induces neurite outgrowth in PC12D cells via cAMP- and MAP kinase-dependent mechanisms

Panaxynol, a polyacetylene ((3R)-heptadeca-1,9-diene-4,6-diyn-3-ol; syn. falcarinol), was isolated from the lipophilic fractions of Panax notoginseng, a Chinese traditional medicinal plant. In the present study, we reported the neurotrophic effects of panaxynol on PC12D cells and mechanism involved in neurite outgrowth of the cells. Panaxynol could morphologically promote neurite outgrowth in PC12D cells, concentration-dependently reduce cell division and up-regulate molecular marker (MAP1B) expression in PC12D cells. Panaxynol induces the elevation of intracellular cAMP in PC12D cells. The neurite outgrowth in PC12D cells induced by panaxynol could be inhibited by the protein kinase A inhibitor RpcAMPS and by MAP kinase kinase 1/2 inhibitor U0126. These observations reveal that panaxynol could induce the differentiation of PC12D cells in a process similar to but distinct from that of NGF and the panaxynol's effects were via cAMP- and MAP kinase-dependent mechanisms.     

Metformin protects PC12 cells and hippocampal neurons from H2O 2-induced oxidative damage through activation of AMPK pathway

Metformin, a first line anti type 2 diabetes drug, has recently been shown to extend lifespan in various species, and therefore, became the first antiaging drug in clinical trial. Oxidative stress due to excess reactive oxygen species (ROS) is considered to be an important factor in aging and related disease, such as Alzheimer's disease (AD). However, the antioxidative effects of metformin and its underlying mechanisms in neuronal cells is not known. In the present study, we showed that metformin, in clinically relevant concentrations, protected neuronal PC12 cells from H₂O₂-induced cell death. Metformin significantly ameliorated cell death due to H₂O₂ insult by restoring abnormal changes in nuclear morphology, intracellular ROS, lactate dehydrogenase, and mitochondrial membrane potential induced by H₂O₂. Hoechst staining assay and flow cytometry analysis revealed that metformin significantly reduced the apoptosis in PC12 cells exposed to H₂O₂. Western blot analysis further demonstrated that metformin stimulated the phosphorylation and activation of AMP-activated protein kinase (AMPK) in PC12 cells, while application of AMPK inhibitor compound C, or knockdown of the expression of AMPK by specific small interfering RNA or short hairpin RNA blocked the protective effect of metformin. Similar results were obtained in primary cultured hippocampal neurons. Taken together, these results indicated that metformin is able to protect neuronal cells from oxidative injury, at least in part, via the activation of AMPK. As metformin is comparatively cheaper with much less side effects in clinic, our findings support its potential to be a drug for prevention and treatment of aging and aging-related diseases.     

Okadaic Acid, a Protein Phosphatase Inhibitor, Inhibits Nerve Growth Factor-Directed Neurite Outgrowth in PC 12 Cells

The biochemical mechanisms involved in neurite outgrowth in response to nerve growth factor (NGF) have yet to be completely resolved. Several recent studies have demonstrated that protein kinase activity plays a critical role in neurite outgrowth. However, little information exists about the role of protein phosphatases in the process. In the present study, okadaic acid, a phosphatase inhibitor (specific for types 2A and 1) and tumor promoter, was used to investigate the role of protein phosphatases in neurite outgrowth in PC12 cells. PC12 cells cultured in the presence of 50 ng/ml of NGF started to extend neurites after 1 day. After 3 days, 20-25% of the cells had neurites. Okadaic acid inhibited the rate of neurite outgrowth elicited by NGF with an IC50 of approximately 7 nM. This inhibition was rapidly reversed after washout of okadaic acid. Okadaic acid also enhanced the neurite degeneration of NGF-primed PC12 cells, indicating that continual phosphatase activity is required to maintain neurites. Taken together, these results reveal the presence of an okadaic acid-sensitive pathway in neurite outgrowth and imply that protein phosphatase plays a positive role in regulating the neuritogenic effects of NGE.     

Protection afforded by quercetin against H2O2-induced apoptosis on PC12 cells via activating PI3K/Akt signal pathway

Cell damage and apoptosis induced by oxidative stress have been involved in various neurodegenerative diseases. This study aims to explore the neuro-protective effects of quercetin on PC12 cells apoptosis induced by hydrogen peroxide (H₂O₂) and the underlying mechanisms. The cell viability was detected, as well as the production of reactive oxygen species (ROS), lactate dehydrogenase (LDH) leakage, and the activities of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and malondialdehyde (MDA) of the cells in control, H₂O₂ and quercetin groups. It finally turned out that quercetin might protect PC12 cells against the negative effect of H₂O₂ by decreasing of LDH release, ROS concentration and MDA level and regaining the GSH-Px and SOD activities. To investigate the mechanism, LY294002 was introduced, the phosphatidylinositol-3-kinase (PI3K) inhibitor. Bax/Bcl-2 ratio and Akt phosphorylation (p-Akt) were examined by Western blot analysis. The data showed that LY294002 almost had the same effects with H₂O₂, which was also significantly reversed by quercetin could enhance Bax/Bcl-2 ratio and adjust the p-Akt expression, which indicated quercetin might protect PC12 cells against the negative effect of H₂O₂ via activating the PI3K/Akt signal pathway.     

过氧化氢预处理对抗氧化应激诱导的PC12细胞凋亡

氧化应激可明显地诱导细胞凋亡。本研究旨在探讨H2O2预处理能否对H2O2诱导的PC12细胞凋亡生产保护作用及ATP敏感性K^ （ATP-sensitive potassinm,KATP）通道在其中的作用。采用PI染色流式细胞仪（flow cytometry, FCM）检测PC12细胞凋亡。结果表明,经10μmol/L H2O2预处理90min的PC12细胞,分别在20、30、50和100μmol/L H2O2作用24h后,其细胞凋亡率明显下降,与未经H2O2的预处理的PC12细胞相比,差异极显著（P＜0.01）,表明H2O2预处理对H2O2诱导PC12细胞凋亡具有保护作用。用10μmol/L的KATP通道激动齐pinacidil(Pin)可显著减少30和50μmol/L H2O2诱导的PC12细胞凋亡,10μmol/L的KATP通道拮抗齐glybenclamide（Gly）则可显著地抑制甚至取消KATP通道激动剂Pin对H2O3诱导PC12细胞凋亡的保护作用,但并不影响H2O2预处理对H2O2诱导PC12细胞凋亡的保护作用;然而,当联合应用H2O2预处理与Pin时,对PC12细胞凋亡的保护作用显大于各自的细胞凋亡作用。提示KATP通道开放不仅对H2O2诱导PC12细胞凋亡具有保护作用,而且与H2O2预处理一起产生抗PC12细胞凋亡的协同作用。但KATP通道开放可能不参与H2O2预处理的适应性保护作用。     

Bcl-2 protects against Abeta(25-35)-induced oxidative PC12 cell death by potentiation of antioxidant capacity

A substantial body of data indicates that reactive oxygen intermediates (ROIs) are implicated in pathogenesis of diverse human diseases. Oxidative stress induced by ROIs often causes cell death via apoptosis that is regulated by a plenty of functional genes and their protein products. Bcl-2 is one such protein that blocks apoptosis induced by various death stimuli. In spite of extensive research, the molecular mechanisms underlying antiapoptotic function of Bcl-2 are not fully clarified. In the present work, we have investigated the role of bcl-2 in protecting against beta-amyloid (Abeta)-induced oxidative death in rat pheochromocytoma (PC12) cells. Transfection with the antiapoptotic bcl-2 gene rescued PC12 cells from apoptotic death induced by Abeta. Addition of an NF-kappaB inhibitor, such as pyrrolidine dithiocarbamate or N-tosyl-l-phenylalanine chloromethyl ketone, to the media aggravated Abeta-induced PC12 cell death. PC12 cells overexpressing bcl-2 exhibited higher levels of constitutively activated NF-kappaB compared with vector-transfected controls, which appear to be mediated by the elevated activation of Akt/protein kinase B. The ectopic expression of bcl-2 enhanced both the expression and the activity of catalase, which were attenuated by NF-kappaB blockers. These results suggest that NF-kappaB plays a role in bcl-2-mediated protection against Abeta-induced apoptosis in PC12 cells through augmentation of cellular antioxidant capacity.     

Chromatographic Resolution and Characterization of a Nerve Growth Factor-Dependent Kinase That Phosphorylates Microtubule-Associated Proteins 1 and 2 in PC12 Cells

When the supernatant fractions from extracts of control and nerve growth factor (NGF)- or dibutyryl cyclic AMP-treated PC12D cells were applied to DEAE-Sepharose columns and proteins were eluted with a gradient of NaCl, three separate peaks of kinase activity that phosphorylated microtubule-associated proteins (MAPs) were recovered. Enhancement of the kinase activity in peak 1 was noted in the case of dibutyryl cyclic AMP-treated cells. In contrast, the kinase activity in the third peak was markedly elevated, in terms of the ability to phosphorylate MAP1 and MAP2, in the case of the extract from NGF-treated cells. This activity was designated previously as NGF-dependent MAP kinase. The apparent molecular mass of the active kinase was 45-50 kDa. The apparent Km value was 35 microM for ATP with either MAP1 or MAP2 as substrate. When the kinase activity in the fractions from the DEAE-Sepharose column was assayed in the presence of Mn2+ instead of Mg2+, another NGF-stimulated kinase activity was detected in the fractions eluted by a lower concentration of NaCl than that which eluted the Mg(2+)-activated kinase. Other growth factors, namely, epidermal growth factor and basic fibroblast growth factor, also stimulated the activity of NGF-dependent MAP kinase. Possible involvement of the kinase in the outgrowth of neurites has been suggested. The NGF-induced activation of NGF-dependent MAP kinase was blocked by the presence of K-252a. In contrast, the activation of NGF-dependent MAP kinase by basic fibroblast growth factor and by epidermal growth factor was not blocked, but actually stimulated by K-252a, a result that correlates well with the analogous actions of the drug on the outgrowth of neurites that is induced by these growth factors. The latter observation strengthens the possibility of a close relationship between the outgrowth of neurites and the activation of NGF-dependent MAP kinase.     

Protein deacetylase SIRT1 in the cytoplasm promotes nerve growth factor-induced neurite outgrowth in PC12 cells

SIRT1, a NAD⁺-dependent protein deacetylase, is known to have neural functions. However, despite its cytoplasmic expression in some neural cells, its cytoplasmic function, if any, is unknown. Here we found that PC12 (pheochromocytoma) cells expressed SIRT1 in the cytoplasm. Nerve growth factor (NGF)-induced neurite outgrowth of these cells was promoted by activators of SIRT1, while inhibitors of SIRT1 or SIRT1-siRNA significantly inhibited it. The overexpression of a mutant SIRT1 that localised to the cytoplasm but not the nucleus enhanced the NGF-dependent neurite outgrowth, and a cytoplasmic dominant-negative SIRT1 suppressed it. Thus, cytoplasmic SIRT1 increases the NGF-induced neurite outgrowth of PC12 cells.     

Inhibition by epigallocatechin gallate of CoCl2-induced apoptosis in rat PC12 cells

Epigallocatechin-3-gallate (EGCG) is a major constituent of green tea polyphenols. This study was aimed to investigate the possible mechanisms of EGCG-mediated inhibition against apoptosis in rat pheochromocytoma PC12 cells by exposure to CoCl(2). Exposure to CoCl(2) caused the generation of ROS and induced cell death with appearance of apoptotic morphology and DNA fragmentation. However, EGCG rescued the loss of viability in the cells exposed to CoCl(2) and led the reduction of DNA fragmentation and sub-G(1) fraction of cell cycle. Also, EGCG attenuated the CoCl(2)-induced disruption of mitochondrial membrane potential (DeltaPsim), release of cytochrome c from the mitochondria to cytosol and abolished the CoCl(2)-stimulated activities of the caspase cascades, caspase-9 and caspase-3. In addition, EGCG ameliorated the increase in the Bax to Bcl-2 ratio, a marker of apoptosis proceeding, induced by CoCl(2) treatment. Taken together, the present results suggest that EGCG inhibit the CoCl(2)-induced apoptosis of PC12 cells through the mitochondria-mediated apoptosis pathway involved in modulating the Bcl-2 family.     

Protective effects of aloe-emodin on scopolamine-induced memory impairment in mice and H2O2-induced cytotoxicity in PC12 cells

Aloe-emodin (AE) is one of the most important active components of Rheum officinale Baill. The present study aimed to investigate that AE could attenuate scopolamine-induced cognitive deficits via inhibiting acetylcholinesterase (AChE) activity and modulating oxidative stress. Kunming (KM) mice were received intraperitoneal injection of scopolamine (2 mg/kg) to induce cognitive impairment. Learning and memory performance were assessed in the Morris water maze (MWM). After behavioral testing, the mice were sacrificed and their hippocampi were removed for biochemical assays (superoxide dismutase (SOD), glutathione peroxidase (GPx), malondialdehyde (MDA), AChE and acetylcholine (ACh)). In vitro, we also performed the AChE activity assay and H₂O₂-induced PC12 cells toxicity assay. After 2 h exposure to 200 μM H₂O₂ in PC12 cells, the cytotoxicity were evaluated by cell viability (MTT), nitric oxide (NO)/lactate dehydrogenase (LDH) release and intracellular reactive oxygen species (ROS) production. Our results confirmed that AE showed significant improvement in cognitive deficit in scopolamine-induced amnesia animal model. Besides, it increased SOD, GPx activities and ACh content, while decreased the level of MDA and AChE activity in AE treated mice. In addition, AE was found to inhibit AChE activity (IC₅₀ = 18.37 μg/ml) in a dose-dependent manner. Furthermore, preincubation of PC12 cells with AE could prevent cytotoxicity induced by H₂O₂ and reduce significantly extracellular release of NO, LDH and intracellular accumulation of ROS. The study indicated that AE could have neuroprotective effects against Alzheimer’s disease (AD) via inhibiting the activity of AChE and modulating oxidative stress.     

Cyclosporine A- and FK506-induced apoptosis in PC12 cells

The purpose of this study was to examine, using glycogen synthase kinase (GSK) inhibitors, whether GSK-3 is involved in cyclosporine A (CsA)- and FK506-induced apoptosis in PC12 cells. CsA and FK506 increased apoptotic cell death with morphological changes characterized by cell shrinkage and nuclear condensation or fragmentation. Nerve growth factor (NGF) completely blocked cell death. Caspase-3 activation was accompanied by CsA- and FK506-induced cell death and inhibited by NGF. GSK-3 inhibitors such as alsterpaullone and SB216763 prevented CsA- and FK506-induced apoptosis. These results suggest that CsA and FK506 induce caspase-dependent apoptosis and that GSK-3 activation is involved in CsA- and FK506-induced apoptosis in PC12 cells.     

Phospholipase D2 activity suppresses hydrogen peroxide-induced apoptosis in PC12 cells

Phospholipase D (PLD) plays an important role as an effector in the membrane lipid-mediated signal transduction. However, the precise physiological functions of PLD are not yet well understood. In this study, we examined the role of PLD activity in hydrogen peroxide (H(2)O(2))-induced apoptosis in rat pheochromocytoma (PC12) cells. Treatment of PC12 cells with H(2)O(2) resulted in induction of apoptosis in these cells, which is accompanied by the activation of PLD. This H(2)O(2)-induced apoptosis was enhanced remarkably when phosphatidic acid production by PLD was selectively inhibited by pretreating the PC12 cells with 1-butanol. Expression of PLD2, but not of PLD1, correlated with increased H(2)O(2)-induced PLD activity in a concentration- and time-dependent manner. Concomitant with PLD activation, the PLD2 activity suppressed H(2)O(2)-induced apoptosis in PC12 cells. Expression of PLD2 lipase-inactive mutant (K758R) had no effect on either PLD activity or apoptosis. PLD2 activity also suppressed H(2)O(2)-induced cleavage and activation of caspase-3. Taken together, the results suggest that PLD2 activity is specifically up-regulated by H(2)O(2) in PC12 cells and that it plays a suppressive role in H(2)O(2)-induced apoptosis.     

ALK activation induces Shc and FRS2 recruitment: Signaling and phenotypic outcomes in PC12 cells differentiation

Activation of the neuronal receptor tyrosine kinase ALK (anaplastic lymphoma kinase) promoted the neuron-like differentiation of PC12 cells through specific activation of the ERK MAP-kinase pathway. However, the nature of primary signaling events initiated is still poorly documented. Here, we established that Shc and FRS2 adaptors were recruited and phosphorylated following antibody-based ALK activation. We further demonstrated that Shc was recruited to the consensus phosphotyrosine site NPTpY(1507) and FRS2 was likely recruited to a novel non-orthodox phosphotyrosine site within ALK. Finally, we characterized a functional role for Shc and likely FRS2 in ALK-dependant MAP-kinase activation and neuronal differentiation of PC12 cells. These findings hence open attractive perspectives concerning specific characteristics of ALK in the control of the mechanisms driving neuronal differentiation.     

Iptakalim protects PC12 cell against H2O2-induced oxidative injury via opening mitochondrial ATP-sensitive potassium channel

The final common pathway in the demise of dopaminergic neurons in Parkinson's disease may involve oxidative stress and excitotoxicity. In this study, we examined the neuroprotective effects of a novel ATP-sensitive potassium channel (K(ATP)) opener, iptakalim (IPT), against H(2)O(2)-induced cytotoxicity in rat dopaminergic PC12 cells. Pretreatment with IPT could attenuate increased extracellular glutamate levels and inhibit calcium influxing induced by H(2)O(2). Moreover, IPT regulated the expressions of bcl-2 and bax which were responsible for inhibiting apoptosis in PC12 cells. These protective effects of IPT were abolished by selective mitoK(ATP) channel blocker 5-hydroxydecanoate. Therefore, IPT can protect PC12 cells against H(2)O(2)-induced oxidative injury via activating mitoK(ATP) channel.     

Increased Neurite Outgrowth Induced by Inhibition of Protein Tyrosine Kinase Activity in PC12 Pheochromocytoma Cells

Abstract: Genistein and other inhibitors of protein tyrosine kinases were examined for effects on neurite elongation and growth cone morphology in the rat PC12 pheochromocytoma cell line. Genistein increased the rate of neurite elongation in PC12 cells grown on a collagen/polylysine substratum after priming with nerve growth factor (NGF), but had no effect on undifferentiated cells. Steady-state levels of phosphotyrosine-modified proteins (105, 59, 52, and 46 kDa) were reduced in NGF-primed cells by genistein treatment. The target of genistein action did not appear to be the NGF receptor/ trk tyrosine kinase because the presence of NGF in cultures of NGF-primed cells was not necessary for genistein-stimulated neurite outgrowth. The tyrosine kinase inhibitors tyrphostin RG508964 and herbimycin A also increased the rate of neurite elongation in NGF-primed PC12 cells. Video-enhanced differential interference contrast microscopy revealed that growth cones of genistein-treated cells had less complex morphologies and were less dynamic than untreated cells, with short filopodia restricted to the leading edge, unlike untreated cells whose growth cones exhibited longer, more numerous filopodia and lamellipodia, which remodeled continuously. These results suggest that protein tyrosine kinase activity in PC12 cells negatively regulates neurite outgrowth and directly or indirectly affects growth cone morphology.     

Puerarin protects PC12 cells against beta-amyloid-induced cell injury

beta-Amyloid protein (Abeta), a major protein component of brain senile plaques in Alzheimer's disease, is known to be directly responsible for the production of reactive oxygen species (ROS) and induction of apoptosis. In this study, the protective effect of puerarin, an isoflavone purified from the radix of the Chinese herb Pueraria lobata, on Abeta-induced rat pheochromocytoma (PC12) cultures was investigated. Although exposure of PC12 cells to 50 microM Abeta25-35 caused significant viability loss and apoptotic rate increase, pretreatment of the cells with puerarin for 24h reduced the viability loss and apoptotic rate. Puerarin (1 microM) significantly inhibited Abeta25-35-induced apoptosis of PC12 cells. Preincubation of the cell with puerarin also restored the ROS and mitochondrial membrane potential levels that had been altered as a result of Abeta25-35 treatment. Puerarin was also found to increase the Bcl-2/Bax ratio and reduce caspase-3 activation. These results suggest that puerarin could attenuate Abeta25-35-induced PC12 cell injure and apoptosis and could also promote the survival of PC12 cells. Therefore, puerarin may act as an intracellular ROS scavenger, and its antioxidant properties may protect against Abeta25-35-induced cell injury.