Rat PC12 Cell Line Research Articles

The effect of steviol on differentiated rat PC-12 cells induced by MPP+

Background: The 1-methyl-4-phenylpyridinium ion (MPP+) is a neurotoxin. It inhibits mitochondrial complex I by interfering with oxidative phosphorylation in mitochondria. That causes ATP depletion and eventual cell apoptosis. Objective: This study investigates steviol's effect, a major metabolite of stevia obtained from Stevia rebaudiana, on MPP+-induced apoptosis of differentiated rat adrenal pheochromocytoma (PC-12) cells. Methods: The PC12 cells were differentiated into neuronal cells by adding nerve growth factor and then treated with MPP+ and steviol in different concentrations; twenty-four hours later, two in vitro toxicity assays. 3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide (MTT) and Lactate Dehydrogenase Leakage assays (LDH) were conducted to detect the cells’ viability. Results: The present study demonstrated that steviol caused statistically significant cell damage and inhibited the proliferation of the differentiated rat PC-12 cell line in a dose-dependent manner. Furthermore, the results from MTT and LDH assays complemented the outcome of our experiments that steviol acted synergistically in the presence of MPP+ to increase cell death in differentiated rat PC-12 cells. Conclusion: The data obtained from MTT and LDH assays indicated that steviol acted synergistically in the presence of MPP+ free radicals, increasing cytotoxicity. Both assays revealed that steviol aggravated the cytotoxicity produced by MPP+ in differentiated rat PC-12 cells. However, as the MTT assay does not correspond to mitochondrial dysfunction, it may give a poor estimate of cytotoxicity of steviol as compared to the LDH assay that quantifies the damage to the cell membrane, especially when there are small-scale variations in cellular reduction capacity.

Surface-Functionalized Conducting Nanofibers for Electrically Stimulated Neural Cell Function.

Strategies involving the inclusion of cell-instructive chemical and topographical cues to smart biomaterials in combination with a suitable physical stimulus may be beneficial to enhance nerve-regeneration rate. In this regard, we investigated the surface functionalization of poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylenevinylene] (MEH-PPV)-based electroconductive electrospun nanofibers coupled with externally applied electrical stimulus for accelerated neuronal growth potential. In addition, the voltage-dependent conductive mechanism of the nanofibers was studied in depth to interlink intrinsic conductive properties with electrically stimulated neuronal expressions. Surface functionalization was accomplished using 3-aminopropyltriethoxysilane (APTES) and 1,6-hexanediamine (HDA) as an alternative to costly biomolecule coating (e.g., collagen) for cell adhesion. The nanofibers were uniform, porous, electrically conductive, mechanically strong, and stable under physiological conditions. Surface amination boosted biocompatibility, 3T3 cell adhesion, and spreading, while the neuronal model rat PC12 cell line showed better differentiation on surface-functionalized mats compared to nonfunctionalized mats. When coupled with electrical stimulation (ES), these mats showed comparable or faster neurite formation and elongation than the collagen-coated mats with no-ES conditions. The findings indicate that surface amination in combination with ES may provide an improved strategy to faster nerve regeneration using MEH-PPV-based neural scaffolds.

Exposure of CuO Nanoparticles Contributes to Cellular Apoptosis, Redox Stress, and Alzheimer's Aβ Amyloidosis.

Fe2O3, CuO and ZnO nanoparticles (NP) have found various industrial and biomedical applications. However, there are growing concerns among the general public and regulators about their potential environmental and health impacts as their physio-chemical interaction with biological systems and toxic responses of the latter are complex and not well understood. Herein we first reported that human SH-SY5Y and H4 cells and rat PC12 cell lines displayed concentration-dependent neurotoxic responses to insults of CuO nanoparticles (CuONP), but not to Fe2O3 nanoparticles (Fe2O3NP) or ZnO nanoparticles (ZnONP). This study provides evidence that CuONP induces neuronal cell apoptosis, discerns a likely p53-dependent apoptosis pathway and builds out the relationship between nanoparticles and Alzheimer’s disease (AD) through the involvement of reactive oxygen species (ROS) and increased Aβ levels in SH-SY5Y and H4 cells. Our results implicate that exposure to CuONP may be an environmental risk factor for AD. For public health concerns, regulation for environmental or occupational exposure of CuONP are thus warranted given AD has already become a pandemic.

Advanced glycation endproducts and polysialylation affect the turnover of the neural cell adhesion molecule (NCAM) and the receptor for advanced glycation endproducts (RAGE).

The balance between protein synthesis and degradation regulates the amount of expressed proteins. This protein turnover is usually quantified as the protein half-life time. Several studies suggest that protein degradation decreases with age and leads to increased deposits of damaged and non-functional proteins. Glycation is an age-dependent, non-enzymatic process leading to posttranslational modifications, so-called advanced glycation endproducts (AGE), which usually damage proteins and lead to protein aggregation. AGE are formed by the Maillard reaction, where carbonyls of carbohydrates or metabolites react with amino groups of proteins. In this study, we quantified the half-life time of two important receptors of the immunoglobulin superfamily, the neural cell adhesion molecule (NCAM) and the receptor for advanced glycation end products (RAGE) before and after glycation. We found, that in two rat PC12 cell lines glycation leads to increased turnover, meaning that glycated, AGE-modified proteins are degraded faster than non-glycated proteins. NCAM is the most prominent carrier of a unique enzymatic posttranslational modification, the polysialylation. Using two PC12 cell lines (a non-polysialylated and a polysialylated one), we could additionally demonstrate, that polysialylation of NCAM has an impact on its turnover and that it significantly increases its half-life time.

Curcumin Treatment is Associated with Increased Expression of the N-Methyl-D-Aspartate Receptor (NMDAR) Subunit, NR2A, in a Rat PC12 Cell Line Model of Alzheimer's Disease Treated with the Acetyl Amyloid-β Peptide, Aβ(25-35).

BackgroundThe aim of this study was to investigate the effect of curcumin treatment on the expression of the N-methyl-D-aspartate receptor (NMDAR) subunit, NR2A, in a rat PC12 cell line treated with the acetyl amyloid-β peptide, Aβ(25–35), in an in vitro model of Alzheimer’s disease.Material/MethodsPC12 cells, derived from rat phaeochromocytoma, were treated for 24 hours with increasing concentrations of curcumin (5, 10, 20, 30 μM/L) in the presence of the acetyl amyloid-β peptide, Aβ(25–35). A Cell Counting Kit-8 (CCK-8) assay was used to determine cell viability, and flow cytometry was used to measure cell apoptosis. In the supernatant of the treated PC12 cells, Western blotting was used to measure the cell injury biomarker, lactate dehydrogenase (LDH), and the biomarker for oxidative stress, malondialdehyde (MDA). Expression of the N-methyl-D-aspartate receptor (NMDAR) subunit, NR2A, was analyzed by Western blotting and quantitative reverse transcription-polymerase chain reaction (qRT-PCR).ResultsCurcumin treatment protected the rat PC12 cells from Aβ(25–35)-induced reduction in cell viability, apoptosis, release of LDH, and MDA production. Curcumin treatment of PC12 cells was associated with increased expression of the NMDAR subunit, NR2A.ConclusionsThe findings of this study showed a neuroprotective effect of curcumin treatment in an in vitro model of Alzheimer’s disease that was associated with the increased expression of the NMDAR subunit, NR2A.

Algal Toxin Azaspiracid-1 Induces Early Neuronal Differentiation and Alters Peripherin Isoform Stoichiometry.

Azaspiracid-1 is an algal toxin that accumulates in edible mussels, and ingestion may result in human illness as manifested by vomiting and diarrhoea. When injected into mice, it causes neurotoxicological symptoms and death. Although it is well known that azaspiracid-1 is toxic to most cells and cell lines, little is known about its biological target(s). A rat PC12 cell line, commonly used as a model for the peripheral nervous system, was used to study the neurotoxicological effects of azaspiracid-1. Azaspiracid-1 induced differentiation-related morphological changes followed by a latter cell death. The differentiated phenotype showed peripherin-labelled neurite-like processes simultaneously as a specific isoform of peripherin was down-regulated. The precise mechanism behind this down-regulation remains uncertain. However, this study provides new insights into the neurological effects of azaspiracid-1 and into the biological significance of specific isoforms of peripherin.

Establishment of a cellular model to study TrkC-dependent neuritogenesis.

The rat PC12 cell line has become a widely used research tool for many aspects of neurobiology. Nerve growth factor (NGF)-responsive PC12 cells were engineered to drive expression of doxycycline (Dox)-induced gene of interest in the Tet-On expression system that resulted in obtaining PC12-Tet-On cells. TrkA and TrkC are neurotrophin receptors derived from the tropomyosin-related kinase (Trk) family of receptor tyrosine kinases. TrkA receptor binds and is activated mainly by NGF, while TrkC receptor binds and is activated by neurotrophin 3 (NT3). The purpose of this research was to design and describe PC12-based neuronal cell model to study TrkC-triggered versus TrkA-triggered neurite outgrowth. The second-generation tetracycline-responsive promoter (P tight) was used in order to provide low basal expression in the absence of Dox and high-level Dox-induced expression of TrkC. The main advantage of presented model system is dependence of TrkC level on Dox concentration. It also allows to compare activation of intracellular signaling proteins and neurite outgrowth following activation of TrkA and TrkC receptors by NGF and NT3, respectively, in the context of the same quality and quantity of intracellular adaptor proteins, Ras proteins, protein kinases and phosphatases, and phospholipase Cγ1, as a difference in the activation of intracellular signaling network by these two distinct although related receptor tyrosine kinases is expected. The results of our studies suggest that despite slightly weaker activation of ERK1/2 mitogen-activated protein kinases, NT3-triggered TrkC seems to provide apparently stronger than NGF-triggered TrkA signal for neurite elongation in differentiating PC12 cells.

Baicalin protects PC-12 cells from oxidative stress induced by hydrogen peroxide via anti-apoptotic effects

Primary objective: To examine the neuroprotection of baicalin, a flavonoid compound derived from the dried root of Scutellaria baicalensis Georgi, on neurons.Research design: A rat PC12 cell line was used to study the neuroprotection and possible mechanisms of baicalin on H2O2-induced neuron damage.Methods: Three anti- and one pro-apoptosis genes in PC12 cells were examined. Cell apoptosis was induced by H2O2 and apoptotic rate was obtained by flow cytometry. MTT for cell viability, immunofluorescence microscopy for promoter activity and western blot for gene expression were also employed.Results: Data of MTT reduction assay and flow cytometry revealed that viability loss and apoptotic rate were reduced by pre-treatment of PC12 cells with baicalin for 24 hours. Baicalin was also found to increase SOD, GSH-Px activities and to decrease MDA level. Results from Western blot and immunofluorescence microscopy showed baicalin increased the expressions of survivin, Bcl-2 and p-STAT3 and decreased caspase-3 expression which were attenuated by AG-490.Conclusions: The results point to the possibility of the neuroprotective effects of baicalin on neuronal apoptosis induced by oxidative stress and indicate that activation of the JAK/STAT signalling pathway might involve the anti-apoptotic effect of baicalin.

Nm23-M2/NDP kinase B induces endogenous c- myc and nm23-M1/NDP kinase A overexpression in BAF3 cells. Both NDP kinases protect the cells from oxidative stress-induced death

The nm23 gene family encodes nucleoside diphosphate kinases (NDPKs) which supply the cell with (d)NTPs. The human NDPKB, also known as the PuF protein, binds the c- myc promoter and transactivates the c- myc protooncogene. We have now studied the effects of mouse NDPKA and NDPKB overexpression on endogenous c- myc transactivation in the mouse BAF3 and the rat PC12 cell lines. c- myc transcripts were found to be up-regulated by NDPKB only in the BAF3 line. This suggests that c- myc transcriptional control via NDPKB depends on the presence of cell-specific co-factors. Unexpectedly, NDPKB also induced NDPKA expression. This new effect was found in both cell lines, suggesting that NDPKB-dependent nm23-M1 gene transactivation requires cis and/or trans elements different from those involved in c- myc transactivation. Moreover, the BAF3 cell proliferation capacities were found to be independent of NDPKA or B cell contents. Interestingly, cell death induced by c- myc overexpression or H 2O 2 exposure was decreased in nm23-transfected compared to control BAF3 cells. These data collectively suggest that NDPKs might improve cell survival by a mechanism coupling DNA repair and transcriptional regulation of genes involved in DNA damage response.

In vitro and in vivo angiogenesis in PC12 pheochromocytoma cells is mediated by vascular endothelial growth factor.

Angiogenesis, the formation of new blood vessels from existing vascular endothelium, is essential for tumor growth. Vascular endothelial growth factor (VEGF) is an endotheliumspecific mitogen and regulator of angiogenesis. Angiogenesis has been associated to the malignant phenotype of pheochromocytomas and is readily observed in experimental pheochromocytomas. Although VEGF gene expression has already been demonstrated in the rat PC12 cell line, the detailed mechanisms of action are not known. We have, therefore, studied angiogenesis in the rat PC12 pheochromocytoma cell line in vitro and in vivo. VEGF gene expression and accumulation of VEGF protein in cytoplasm and conditioned medium of PC12 cells was found. Conditioned medium from PC12 cells significantly increased proliferation of VEGF-dependent endothelial cells from human umbilical veins, and this effect reversed upon addition of a neutralizing anti-VEGF antibody. Dexamethasone and nerve growth factor (NGF) increased VEGF mRNA expression and accumulation of VEGF protein of PC12 subclones with established metastatic activity in vivo. PC12 cells xenotransplanted to nude mice had marked VEGF expression and induced host angiogenesis, confirmed by the presence of CD34-positive endothelial cells in the experimental PC12 tumors. When NGF-primed PC12 cells were immobilized in Matrigel supplemented with rising concentrations of the growth factor and xenotransplanted, increasing NGF resulted in tumors with smaller areas of necrosis and increased vital tumor volume. These results suggest that VEGF is a mediator of angiogenesis in the PC12 pheochromocytoma cell line, and that dexamethasone and NGF affect VEGF expression. Our data further suggest that NGF may contribute to angiogenesis in experimental pheochromocytoma.

Hamartin expression and interaction with tuberin in tumor cell lines and primary cultures.

Tuberous sclerosis (TSC) is a neurocutaneous disorder characterized by multi-system hamartomatous lesions, and results from a mutation in TSC1, that encodes hamartin, or TSC2, that encodes tuberin. We have examined hamartin expression in a diverse range of human and rat cell lines and primary cultured cells derived from tissues that express hamartin in vivo. Strong hamartin signal was detected in every cell line of human origin examined, representing neuronal, epithelial, lymphoid, renal, vascular smooth muscle, liver, and prostatic cells. Primary cell cultures of oligodendroglioma, meningioma, and glioblastoma multiforme origin were also found to express hamartin. Hamartin was also detected in the rat PC12 cell line, as well as purified primary cultures of rat cortical neurons, astrocytes, and oligodendroglia, with a stronger signal found in astrocytes. Using co-immunoprecipitation, we have also confirmed the physical interaction of tuberin and hamartin in a diverse range of human and rat cell types. These findings demonstrate that hamartin is widely expressed in human and rat cell lines and cultures, and demonstrate that hamartin expression is not lost during the establishment of tumor cell lines or primary cultures. This suggests that the cell lines and cultures studied may serve as useful in vitro models for biochemical investigations involving hamartin and tuberin both individually and as a complex, as well as studies to elucidate the mechanisms underlying the organ-specific pathology of TSC.

Regulation of neural cell adhesion molecule polysialylation state by cell-cell contact and protein kinase C delta.

Post-translational modification of neural cell adhesion molecule (NCAM) with alpha2,8-linked polysialic acid, which regulates homophilic adhesion and/or signal transduction events, is crucial to synaptic plasticity in the developing and adult brain. Evidence from in vitro models has implicated polysialylation in the regulation of cell growth, migration, and differentiation. Here, using two in vitro models, we demonstrate that polysialylation is downregulated by cell-cell contact and correlated with a state of neuronal differentiation. Furthermore, we report a role for protein kinase C delta (PKCdelta) in the regulation of NCAM polysialylation. Pharmacological studies using the PKC activator, phorbol myristate acetate, and inhibitors, calphostin-C, and staurosporine, demonstrated PKC activity to be inversely related to NCAM polysialylation in the mouse neuro-2A cell line. Isoform-specific immunoblot studies indicated this effect to be mediated by the calcium-independent PKCdelta isozyme, as its expression was inversely related to NCAM polysialylation state in both neuro-2A and rat PC-12 cell lines. Isoform specificity was further confirmed using the PKCdelta-selective inhibitor rottlerin, which produced a marked increase in PSA expression (36.9+/-5.25 a.u. vs. 24.7+/-0.80 arbitrary units control) coupled with a neuritogenic response. Likewise, decreased expression of PKCdelta was seen in nerve growth factor (NGF)-differentiated PC-12 cells. These findings suggest that the neuronal differentiation process may involve inhibition of PKCdelta, resulting in enhanced morphological plasticity, as evidenced by activation of NCAM polysialylation.

Differentiation in a Rat PC12 Cell Line Induced by Ostruthin and (-)-Bornyl Ferulate, Constituents of a Chinese Herbal Medicine

A search for neuritogenic compounds in Chinese herbs resulted in the isolation of two known substances, ostruthin and (-)-bornyl ferulate, from Notopterygium incisum (and/or N. forbesii). Both compounds induced comparable neurite-like structures in 20% of rat PC12 cells at 2 micrograms/ml, and showed cytotoxicity at concentrations higher than 3 micrograms/ml.

PC12‐E2 cells: A stable variant with altered responses to growth factor stimulation

A variant cell line, designated E2, characterized by more rapid responses to nerve growth factor (NGF) and basic fibroblast growth factor (bFGF) and markedly more robust responses to interleukin-6 and 8-Br-cAMP, has been subcloned from the rat PC12 cell line. The enhanced responsiveness to NGF in E2 cells is not due to receptor overexpression as judged by TrkA protein levels and tyrosine kinase activity, but may be associated with the increased and prolonged tyrosine phosphorylation of ERK1 (extracellular signal regulated kinase 1) and ERK2. The rapid morphological differentiation induced by different growth factors in E2 cells is mediated in a transcription-independent manner suggesting that E2 cells may constitutively express some differentiation-associated molecules that allow direct entry into the neuronal program.

Alpha 3, alpha 5, and beta 4: three members of the rat neuronal nicotinic acetylcholine receptor-related gene family form a gene cluster.

We have identified two additional members of the neuronal nicotinic acetylcholine receptor (nAChR)-related gene family. cDNA clones for one new gene, designated alpha 5, were isolated from rat hippocampus and rat PC12 cell line cDNA libraries. The alpha 5 gene encodes a protein of 48,800 daltons (424 amino acids) which exhibits significant overall amino acid sequence identity with the previously cloned rat nAChR subunits alpha 1 (49%), alpha 2 (55%), alpha 3 (52%), and alpha 4 (49%). Features characteristic of other nAChR alpha-subunits are present such as conserved cysteine residues at positions 127, 141, 191, and 192, and four strongly hydrophobic domains. A second addition to the nAChR-related gene family, designated beta 4, is encoded in overlapping rat genomic clones lambda DD15 and lambda RG518A. The beta 4 gene, encoding a mature protein of 53,300 daltons (475 amino acids), consists of 6 exons and has a transcription unit length of approximately 18 kilobase pairs. The beta 4 gene encoded protein shows considerable amino acid sequence identity with nAChR beta 1 (43%), beta 2 (64%), and beta 3 (44%) subunits. Northern blots showed that, along with alpha 3 and beta 2, transcripts for both the alpha 5 and beta 4 genes are present in the PC12 cell line, while in situ hybridization experiments demonstrated expression of the alpha 5 and beta 4 genes in a small number of nuclei in the central nervous system. Finally, the genes that encode the beta 4, alpha 3, and alpha 5 proteins are transcribed with convergent polarities and form a tightly linked gene cluster spanning approximately 60 kilobase pairs.