Morphological Changes Of PC12 Cells Research Articles

The structurally diverse phytocannabinoids cannabichromene, cannabigerol and cannabinol significantly inhibit amyloid β-evoked neurotoxicity and changes in cell morphology in PC12 cells.

Phytocannabinoids (pCBs) have been shown to inhibit the aggregation and neurotoxicity of the neurotoxic Alzheimer's disease protein beta amyloid (Aβ). We characterized the capacity of six pCBs-cannabichromene (CBC), cannabigerol (CBG), cannabinol (CBN), cannabidivarin (CBDV), cannabidiol (CBD) and Δ9 -tetrahydrocannabinol (Δ9 -THC)-to disrupt Aβ aggregation and protect against Aβ-evoked neurotoxicity in PC12 cells. Neuroprotection against lipid peroxidation and Aβ-induced cytotoxicity was assessed using the MTT assay. Transmission electron microscopy was used to visualize pCB effects on Aβ aggregation and fluorescence microscopy, with morphometrics and principal component analysis to assess PC12 cell morphology. CBD inhibited lipid peroxidation with no significant effect on Aβ toxicity, whilst CBN, CBDV and CBG provided neuroprotection. CBC, CBG and CBN inhibited Aβ1-42 -induced neurotoxicity in PC12 cells, as did Δ9 -THC, CBD and CBDV. CBC, CBN and CBDV inhibited Aβ aggregation, whilst Δ9 -THC reduced aggregate density. Aβ1-42 induced morphological changes in PC12 cells, including a reduction in neuritic projections and rounded cell morphology. CBC and CBG inhibited this effect, whilst Δ9 -THC, CBD and CBDV did not alter Aβ1-42 effects on cell morphology. These findings highlight the neuroprotective activity of CBC, CBG and CBN as novel pCBs associated with variable effects on Aβ-evoked neurite damage and inhibition of amyloid β aggregation.

Combination of Lutein and DHA Alleviate H2O2 Induced Cytotoxicity in PC12 Cells by Regulating the MAPK Pathway.

Docosahexaenoic acid (DHA) and lutein are important nutrients for brain health. Whether there were synergistic effects of DHA and lutein on the protection against neuronal cell damage induced by oxidative stress remained unclear. The present study was designed to investigate the synergistic effects of DHA and lutein against hydrogen peroxide (H2O2)-induced oxidative challenge in PC12 cells. PC12 cells were divided into different groups and received H2O2 (80 μM), lutein (20 μM)+H2O2 (80 μM), DHA (25 μM)+H2O2 (80 μM), and lutein (20 μM)+DHA (25 μM)+H2O2 (80 μM), respectively. The results indicated that pre-treatment of cells with lutein, DHA and DHA+lutein could significantly antagonize the H2O2-mediated growth inhibition and morphological changes in PC12 cells (p<0.05). Molecularlevel studies indicated that the DHA+lutein combination can significantly inhibit the mRNA expression of AMAD10 and BAX. Furthermore, Western blot analysis demonstrated that DHA+lutein synergistically inhibits the phosphorylation of JNK1/2. The results of the present study suggest that DHA and lutein in combination may be utilized as potent antioxidative compounds, with potential preventative or palliative effects on age-related neurodegenerative diseases.

Anemarrhenae asphodeloides rhizoma Extract Enriched in Mangiferin Protects PC12 Cells against a Neurotoxic Agent-3-Nitropropionic Acid.

The rhizome of Anemarrhena asphodeloides Bunge, used in Traditional Chinese Medicine as a brain function-improving herb, is a promising source of neuroprotective substances. The aim of this study was to evaluate the protective action of xanthones from A. asphodeloides rhizomes on the PC12 cell line exposed to the neurotoxic agent—3-nitropropionic acid (3-NP). The xanthone-enriched fraction of the ethanolic extract of A. asphodeloides (abbreviated from now on as XF, for the Xanthone Fraction), rich in polyphenolic xanthone glycosides, in concentrations from 5 to 100 μg/mL, and 3-NP in concentrations from 2.5 to 15 mM, were examined. After 8, 16, 24, 48, and 72 h of exposure of cells to various combinations of 3-NP and XF, the MTT viability assay was performed and morphological changes were estimated by confocal fluorescence microscopy. The obtained results showed a significant increase in the number of cells surviving after treatment with XF with exposure to neurotoxic 3-NP and decreased morphological changes in PC12 cells in a dose and time dependent manner. The most effective protective action was observed when PC12 cells were pre-incubated with the XF. This effect may contribute to the traditional indications of this herb for neurological and cognitive complaints. However, a significant cytotoxicity observed at higher XF concentrations (over 10 µg/mL) and longer incubation time (48 h) requires caution in future research and thorough investigation into potential adverse effects.

The Efficacy of Electrospun PAN/Kefiran Nanofiber and Kefir in Mammalian Cell Culture: Promotion of PC12 Cell Growth, Anti-MCF7 Breast Cancer Cells Activities, and Cytokine Production of PBMC.

BackgroundKefiran is a useful polysaccharide made of branched glucogalactose which is produced by microorganisms. Here the anti-MCF-7 breast cancer cells activity of kefiran and cytokine productions (IL-6) of peripheral blood mononuclear cells (PBMC) treated by kefiran was studied. Also, the effect of using kefiran as a useful and cost-effective scaffold in neural stem cell culture (PC12 cell culture) was investigated.Material and MethodsKefiran was produced from raw milk with 0.5% fat and 10 g of kefir grains. After incubation for 48 hrs at room temperature, the solvent collected (crude kefiran). These samples were kept at 100°C for 1 hr (boiled kefiran) and the supernatant was precipitated by ethanol (pure kefiran). Then, the electrospun nanofibers, pure polyacrylonitrile (PAN), PAN/kefiran 5%, and PAN/kefiran 10% were fabricated and used as scaffolds in the cell culture. The structure of fabricated was studied by SEM and the cytokine production (IL-6) in vitro in the cell culture supernatant of PBMC line after treatment with kefiran (1mg/mL, 5 mg/mL) and kefiran-PAN 5% and 10% were carried out by enzyme-linked immunosorbent assay (ELISA). The attachment of PC12 cells was examined by inverted microscope. Also, cytotoxicity of kefiran for PC12 and MCF7 cells and morphological changes of PC12 cells were evaluated by MTT and Cresyl violet staining (Nissl staining) respectively.ResultsThe mean diameter of fabricated PAN/kefiran 5% and 10% nanofibers were 310.2±43.97 nm. The contact angle measurement results (26.9± 1.9 for the pure PAN scaffold vs 12.3± 1.13 for the PAN/kefiran) revealed enhanced hydrophilicity of scaffolds upon the incorporation of kefiran and PAN. Seeding of PC12 cells on the scaffolds showed that fabrication of kefiran into PAN led to the enhancement of cell attachment, proliferation, and morphological changes. Also, the promotion of PBMC growth and decreasing of MCF7 cell lines viability were shown through MTT assay. No significant changes were measured for the level of IL-6 in PAN/kefiran 5% treated cells compared to the control (p ≥ 0.05).ConclusionThese results suggest superior properties of kefiran/PAN nanofibrous scaffolds for the neural stem cell culture especially for repairing injured spinal cord. Also, the pure kefiran could be used for the enhancement of PBMC growth and reducing the MCF7 cancerous cells growth. So, using biocompatible, anti-bacterial, and anti-tumor kefiran/PAN nanofibers for regenerative medicine seems promising.

Protective effect of nitronyl nitroxide against hypoxia-induced damage in PC12 cells.

Hypoxia induces cellular oxidative stress that is associated with neurodegenerative diseases. HPN (4'-hydroxyl-2-substituted phenyl nitronyl nitroxide), a stable nitronyl nitroxide, has excellent free radical scavenging properties. The purpose of this study was to investigate the protective effects of HPN on hypoxia-induced damage in PC12 cells. It was shown that HPN significantly attenuated hypoxia-induced loss of cell viability, release of lactate dehydrogenase (LDH), and morphological changes in PC12 cells. Moreover, hypoxic PC12 cells had increased levels of reactive oxygen species (ROS), malondialdehyde (MDA), and expression of HIF-1α and VEGF, but had reduced levels of superoxide dismutase (SOD) and catalase (CAT), and HPN reversed these changes. HPN also inhibited hypoxia-induced cell apoptosis via suppressing the expression of Bax, cytochrome c, and caspase-3, and inducing the expression of Bcl-2. These results indicate that the protective effects of HPN on hypoxia-induced damage in PC12 cells is associated with the suppression of hypoxia-induced oxidative stress and cell apoptosis. HPN could be a promising candidate for the development of a novel neuroprotective agent.

Cyanidin suppresses amyloid beta-induced neurotoxicity by inhibiting reactive oxygen species-mediated DNA damage and apoptosis in PC12 cells.

Amyloid beta (Aβ)-induced oxidative stress is a major pathologic hallmark of Alzheimer's disease. Cyanidin, a natural flavonoid compound, is neuroprotective against oxidative damage-mediated degeneration. However, its molecular mechanism remains unclear. Here, we investigated the effects of cyanidin pretreatment against Aβ-induced neurotoxicity in PC12 cells, and explored the underlying mechanisms. Cyanidin pretreatment significantly attenuated Aβ-induced cell mortality and morphological changes in PC12 cells. Mechanistically, cyanidin effectively blocked apoptosis induced by Aβ, by restoring the mitochondrial membrane potential via upregulation of Bcl-2 protein expression. Moreover, cyanidin markedly protected PC12 cells from Aβ-induced DNA damage by blocking reactive oxide species and superoxide accumulation. These results provide evidence that cyanidin suppresses Aβ-induced cytotoxicity, by preventing oxidative damage mediated by reactive oxide species, which in turn inhibits mitochondrial apoptosis. Our study demonstrates the therapeutic potential of cyanidin in the prevention of oxidative stress-mediated Aβ neurotoxicity.

Neuroprotective effects of 20(S)-protopanaxadiol against glutamate-induced mitochondrial dysfunction in PC12 cells.

Ginseng (Panax ginseng C.A. Mey.) is commonly used in traditional oriental medicine for its wide spectrum of medicinal properties, including anti-inflammatory, antitumorigenic, adaptogenic and anti-aging properties. 20(S)-Protopanaxadiol (PPD), the main intestinal metabolite of ginsenosides, is one of the active ingredients in ginseng. In this study, we aimed to investigate the neuroprotective effects of PPD on PC12 cells; however, the underlying mechanisms remain elusive. We examined cell viability by MTT assay and the morphological changes of PC12 cells following glutamate-induced cell damage and evaluated the anti-apoptotic effects of PPD using Hoechst 33258 staining, western blot analysis and Muse™ Cell Analyzer and the antioxidant effects of PPD using FACS analysis and immunofluorescence. Furthermore, PPD exerted protective effects on PC12 cells via the inhibition of mitochondrial damage against glutamate-induced excitotoxicity using immunofluorescence, electron microscopy and FACS analysis. We demonstrate that treatment with PPD suppresses apoptosis, which contributes to the neuroprotective effects of PPD against glutamate-induced excitotoxicity in PC12 cells. Treatment with PPD inhibited nuclear condensation and decreased the number of Annexin V-positive cells. In addition, PPD increased antioxidant activity and mitochondrial homeostasis in the glutamate-exposed cells. These antioxidant effects were responsible for the neuroprotection and enhanced mitochondrial function following treatment with PPD. Furthermore, PD inhibited the glutamate-induced morphological changes in the mitochondria and scavenged the mitochondrial and cytosolic reactive oxygen species (ROS) induced by glutamate. In addition, mitochondrial function was significantly improved in terms of mitochondrial membrane potential (MMP) and enhanced mitochondrial mass compared with the cells exposed to glutamate and not treated with PPD. Taken together, the findings of our study indicate that the antioxidant effects and the enhanced mitochondrial function triggered by PPD contribute to the inhibition of apoptosis, thus leading to a neuroprotective response, as a novel survival mechanism.

Rapid and sensitive neurotoxicity test based on the morphological changes of PC12 cells with simple computer-assisted image analysis

In order to develop a rapid and sensitive bioassay for the screening of chemicals with possible neurotoxicity, a computer-assisted simple image-analysis system was developed to quantify small changes in the specific morphology of the cultured pheochromocytoma cell line, PC12. This cell forms a neuronlike microfibril network (neurites) in response to a nerve growth factor (NGF) stimulation in vitro. Dichrolvos (DDVP) and methylmercury chloride (MMC) were employed as model neurotoxicants. In DDVP treatment, there was no large difference in the ED 50s (effective dose that reduces the morphological index by 50%) among the toxicities determined from various morphological indices, but they were significantly lower than those observed by whole-cell-area-based toxicity assay using the hepatoblastoma cell line, Hep G2. In contrast, in MMC treatment, neurite-length-based toxicity was observed as early as 2 h, and at 48 h this was lower by over three orders of magnitude compared with whole-cell-area-based one (2.06 × 10 −7 mM vs. 6.42 × 10 −4 mM). These results demonstrate that the developed bioassay using image analysis of nerve-tissue-derived cell morphology allows us to screen possible neurotoxic chemicals very rapidly with highly enhanced sensitivity, particularly for some chemicals that preferentially act on nerve fibers.

Effects of Docosahexaenoic and Arachidonic Acids on the Synthesis and Distribution of Aminophospholipids during Neuronal Differentiation of PC12 Cells

We have shown previously that docosahexaenoic acid (DHA) promotes and arachidonic acid (AA) suppresses neurite outgrowth of PC12 cells induced by nerve growth factor (NGF) and that incorporation of [3H]ethanolamine into phosphatidylethanolamine (PE) is suppressed in PC12 cells by AA while DHA has no effect. In the present study, the effects of these fatty acids on PE synthesis via decarboxylation of phosphatidylserine (PS), another pathway of PE synthesis, and distribution of aminophospholipids were examined. Incorporation of [3H]serine into PS and PE was elevated in the course of NGF-induced differentiation and was further stimulated significantly by DHA, but not by AA. [3H]Ethanolamine uptake by PC12 cells was significantly suppressed by AA but not by DHA while these fatty acids did not affect [3H]serine uptake, indicating that the suppression by AA of [3H]ethanolamine incorporation into phosphatidylethanolamine is attributable, at least in part, to a reduction in [3H]ethanolamine uptake. The distribution of PE in the outer leaflet of plasma membrane decreased during differentiation, which is known to be accompanied by an increase in the surface area of plasma membrane. Supplementation of PC12 cells with DHA or AA did not affect the distribution of aminophospholipids. Thus, DHA and AA affected aminophospholipid synthesis and neurite outgrowth differently, but not the transport and distribution of aminophospholipids, while the PE concentration in the outer leaflet of the plasma membrane decreased in association with morphological changes in PC12 cells induced by NGF.

Constitutively Active Gα12, Gα13, and Gαq Induce Rho-dependent Neurite Retraction through Different Signaling Pathways

In neuronal cells, activation of a certain heterotrimeric G protein-coupled receptor causes neurite retraction and cell rounding via the small GTPase Rho. However, the specific heterotrimeric G proteins that mediate Rho-dependent neurite retraction and cell rounding have not yet been identified. Here we investigated the effects of expression of constitutively active Galpha subunits on the morphology of differentiated PC12 cells. Expression of GTPase-deficient Galpha12, Galpha13, and Galphaq, but not Galphai2, caused neurite retraction and cell rounding in differentiated PC12 cells. These morphological changes induced by Galpha12, Galpha13, and Galphaq were completely inhibited by C3 exoenzyme, which specifically ADP-ribosylates and inactivates Rho. The tyrosine kinase inhibitor tyrphostin A25 blocked the neurite retraction and cell rounding induced by Galpha13 and Galphaq. However, tyrphostin A25 failed to inhibit the Galpha12-induced neuronal morphological changes. On the other hand, inhibition of protein kinase C or elimination of extracellular Ca2+ blocked the neurite retraction and cell rounding induced by Galphaq, whereas the morphological effects of Galpha12 and Galpha13 did not require activation of protein kinase C and extracellular Ca2+. These results demonstrate that activation of Galpha12, Galpha13, and Galphaq induces Rho-dependent morphological changes in PC12 cells through different signaling pathways.

Distinct biological properties of two RET isoforms activated by MEN 2A and MEN 2B mutations.

Germline mutations of the RET proto-oncogene, which codes for a receptor tyrosine kinase, cause multiple endocrine neoplasia type 2A (MEN 2A) and 2B (MEN 2B) and familial medullary thyroid carcinoma (FMTC). MEN 2 mutations have been shown to result in RET oncogenic activation. The RET gene encodes several isoforms whose biological properties, when altered by MEN 2 mutations, have not been thoroughly addressed yet. In this study, we have introduced a MEN 2A mutation (Cys634-->Arg) and the unique MEN 2B mutation (Met918-->Thr) in two RET isoforms of 1114 and 1072 amino acids which differ in the carboxy-terminus part. Herein, we report that each RET isoform activated by MEN 2A or MEN 2B mutation was transforming in fibroblasts and induced neuronal differentiation of pheochromocytoma PC12 cells. However, among the different RET-MEN 2 mutants, the long RET isoform activated by the MEN 2B mutation stimulated the most prominent neurite outgrowth in PC12 cells, while the short RET isoform counterpart elicited a very weak differentiation effect in PC12 cells. We further demonstrate that the morphological changes of PC12 cells caused by constitutively activated RET oncoproteins involved the engagement of a Ras-dependent pathway. These findings provide evidence that the biological properties of RET-MEN 2 mutants depend on the interplay between the RET isoforms and the nature of the activating MEN 2 mutation.

Characterization of the Bone Morphogenetic Protein-2 as a Neurotrophic Factor: INDUCTION OF NEURONAL DIFFERENTIATION OF PC12 CELLS IN THE ABSENCE OF MITOGEN-ACTIVATED PROTEIN KINASE ACTIVATION

Rat pheochromocytoma PC12 cells are shown to express a single class of high affinity binding sites for bone morphogenetic protein (BMP)-2 (1,300 receptors/cell, Kd = 31.3 pM). Affinity cross-linking using radiolabeled BMP-2 demonstrated the presence of six components with apparent molecular masses of 170, 155, 105, 90, 80, and 70 kDa. BMP-2 induced morphological changes in PC12 cells with the concomitant expression of three neurofilament proteins. Thus, BMP-2 would appear to be another neurotrophic factor that, like nerve growth factor or basic fibroblast growth factor, stimulates the neuronal differentiation of PC12 cells. Unlike nerve growth factor and basic fibroblast growth factor, however, BMP-2 failed to induce the activation of either 41- and 43-kDa mitogen-activated protein (MAP) kinases or the MAP kinase/extracellular signal-regulated kinase kinase (MEK). Also, BMP-2 did not induce the expression of the c-fos gene in PC12 cells. Activin A was also capable of inducing the neuronal differentiation of PC12 cells without activating MAP kinases and MEK. These findings show a clear dissociation between the requirement for the activation of the MAP kinase cascade and the ability of BMP-2 and activin A to induce PC12 cell neuronal differentiation. In addition, these results suggest that the activation of MAP kinases and MEK is not an absolute requirement for PC12 cell differentiation.

Importance of ADP-ribosylation in the morphological changes of PC12 cells induced by cholera toxin.

Cholera toxin (CTX) is composed of two subunits, subunit A, which possesses ADP-ribosyltransferase activity, and subunit B, which is responsible for receptor binding. It has previously been shown that agents that increase cyclic AMP (cAMP) levels in cells induce differentiation of PC12 cells into neurite-like cells. In this report, we show that as little as 100 pg of CTX per ml induces such changes. CTX was found to ADP-ribosylate at least four membrane proteins of PC12 cells in vitro and in vivo and to increase intracellular cAMP levels. We have developed an inducible ctx gene expression system in Vibrio cholerae by using the tac promoter. The culture medium of the CTX-producing bacteria was able to induce the morphological changes and the ADP-ribosylation of the PC12 cell membrane proteins. We have constructed two CTX-cross-reactive mutant proteins (CTX-CRM) by site-directed mutagenesis. The choice of glutamic acid 29 as the target amino acid was based on sequence similarities with other bacterial toxins. CTX-CRM-E29 delta, in which the Glu-29 of the A subunit was deleted, showed strongly reduced ADP-ribosyltransferase activity and did not induce significant morphological changes of PC12 cells. In contrast, CTX-CRM-E29D, in which the Glu-29 was replaced by an aspartic acid, was as active as the wild-type protein. We conclude that the ADP-ribosylation activity of CTX is important for the toxin-induced differentiation of PC12 cells. Pertussis toxin, which had no visible effect on PC12 cell morphology, was also able to ADP-ribosylate a membrane-bound protein(s) in vitro and in vivo. Pertussis toxin alone did not significantly increase cAMP levels in PC12 cells, but it acted synergistically with CTX.

Effect of manganese on tyrosylprotein sulfotransferase activity in PC12 cells

Recent studies in our laboratory have revealed that Mn 2+ is capable of promoting cell spreading and neurite outgrowth in PC12 cells, a process which is dependent on Mn 2+ stimulation of the interaction between extracellular matrix (ECM) components and their corresponding integrin receptors. Since the major ECM proteins implicated in the Mn 2+-induced morphogenesis, fibronectin and vitronectin, are both tyrosine sulfated, it was of interest to determine whether Mn 2+ can regulate the activity of the enzyme responsible for tyrosine sulfation, tyrosylprotein sulfotransferase (TPST). Results of the present studies demonstrated that Mn 2+ can suppress TPST activity in PC12 cells in both a time and concentration-dependent manner at concentrations of Mn 2+ (0.1 to 1 mM) that promote morphological changes in PC12 cells. Since uptake of Mn 2+ may occur via the Ca 2+ channel, LaCl 3, an inhibitor of Ca 2+ transport, was examined and found not to prevent the suppression of TPST activity induced by Mn 2+, suggesting that Mn 2+ may function at an extracellular site. Suppression of TPST activity occurred with cells plated in serum-free medium on a substrata consisting of either serum or polylysine, suggesting that attachment to extracellular matrix was not an absolute requirement for regulation of activity. Consistent with this is the fact that an RGD-containing pentapeptide did not prevent suppression of TPST activity. Results from the present study demonstrated that Mn 2+ is capable of promoting the suppression of TPST activity in PC12 cells at concentrations that have been shown to induce cell spreading and neurite outgrowth. However, unlike the Mn 2+-induced morphological changes, the presence of ECM proteins is not an absolute requirement for suppression of TPST activity.

Growth of tumour cell lines in polymer capsules: ultrastructure of encapsulated PC12 cells.

Recent studies indicate that polymer-encapsulated PC12 cells release sufficient amounts of dopamine to significantly alter behavioural paradigms in animals with unilateral lesions of dopaminergic midbrain neurons. Because cell fine structure provides a useful measure for assessment of storage function, exocytosis, metabolism, cell activity and cell viability, we examined the ultrastructure of PC12 cells grown in semi-permeable polymer capsules maintained in vitro or implanted into the forebrain of rats or guinea pigs. Encapsulated PC12 cells remained viable and continued to divide for the entire evaluation period of six months. Overall morphologies of encapsulated PC12 cells were similar in both environments and they resembled PC12 cells grown in monolayer cultures. In short-term cultures, encapsulated PC12 cells typically contained abundant quantities of chromaffin cell-like granules. The encapsulated cells had initially abundant microvilli on their surfaces which decline in frequency over time. After long-term enclosure for ten weeks or more, fewer secretory granules were detected in the cytoplasm of cells in capsules cultured in vitro and in brain-implanted capsules. Some cells in implanted capsules had long slender filipodia that were not present on PC12 cells in cultured capsules. The morphological changes of PC12 cells may correlate with altered growth conditions such as serum and oxygen concentrations, the presence or absence of growth factors in different environments, and with changes of cell interactions related to cell densities and build up of debris within the capsules over time. Since dopaminergic PC12 pheochromocytoma cells remain viable in semi-permeable polymer capsules for at least six months, such 'cell-capsules' could provide an alternative to dopamine-secreting embryonic neural grafts in dopamine replacement therapies.