Decursin Isolated from Angelica gigas Nakai Rescues PC12 Cells from Amyloid β-Protein-Induced Neurotoxicity through Nrf2-Mediated Upregulation of Heme Oxygenase-1: Potential Roles of MAPK.

PubMed

Li, Li; Du, Ji-Kun; Zou, Li-Yi; Wu, Tie; Lee, Yong-Woo; Kim, Yong-Ho

2013-01-01

Decursin (D), purified from Angelica gigas Nakai, has been proven to exert neuroprotective property. Previous study revealed that D reduced A β 25 ‒ 35-induced cytotoxicity in PC12 cells. Our study explored the underlying mechanisms by which D mediates its therapeutic effects in vitro. Pretreatment of cells with D diminished intracellular generation of ROS in response to A β 25 ‒ 35. Western blot revealed that D significantly increased the expression and activity of HO-1, which was correlated with its protection against A β 25 ‒ 35-induced injury. Addition of ZnPP, an HO-1 competitive inhibitor, significantly attenuated its protective effect in A β 25 ‒ 35-treated cells, indicating the vital role of HO-1 resistance to oxidative injury. Moreover, D induced Nrf2 nuclear translocation, the upstream of HO-1 expression. While investigating the signaling pathways responsible for HO-1 induction, D activated ERK and dephosphorylated p38 in PC12 cells. Addition of U0126, a selective inhibitor of ERK, blocked D-induced Nrf2 activation and HO-1 induction and meanwhile reversed the protection of D against A β 25 ‒ 35-induced cell death. These findings suggest D augments cellular antioxidant defense capacity through both intrinsic free radical scavenging activity and activation of MAPK signal pathways that leads to Nrf2 activation, and subsequently HO-1 induction, thereby protecting the PC12 cells from A β 25 ‒ 35-induced oxidative cytotoxicity.

Decursin Isolated from Angelica gigas Nakai Rescues PC12 Cells from Amyloid β-Protein-Induced Neurotoxicity through Nrf2-Mediated Upregulation of Heme Oxygenase-1: Potential Roles of MAPK

PubMed Central

Li, Li; Du, Ji-kun; Zou, Li-yi; Wu, Tie; Lee, Yong-woo; Kim, Yong-ho

2013-01-01

Decursin (D), purified from Angelica gigas Nakai, has been proven to exert neuroprotective property. Previous study revealed that D reduced Aβ 25‒35-induced cytotoxicity in PC12 cells. Our study explored the underlying mechanisms by which D mediates its therapeutic effects in vitro. Pretreatment of cells with D diminished intracellular generation of ROS in response to Aβ 25‒35. Western blot revealed that D significantly increased the expression and activity of HO-1, which was correlated with its protection against Aβ 25‒35-induced injury. Addition of ZnPP, an HO-1 competitive inhibitor, significantly attenuated its protective effect in Aβ 25‒35-treated cells, indicating the vital role of HO-1 resistance to oxidative injury. Moreover, D induced Nrf2 nuclear translocation, the upstream of HO-1 expression. While investigating the signaling pathways responsible for HO-1 induction, D activated ERK and dephosphorylated p38 in PC12 cells. Addition of U0126, a selective inhibitor of ERK, blocked D-induced Nrf2 activation and HO-1 induction and meanwhile reversed the protection of D against Aβ 25‒35-induced cell death. These findings suggest D augments cellular antioxidant defense capacity through both intrinsic free radical scavenging activity and activation of MAPK signal pathways that leads to Nrf2 activation, and subsequently HO-1 induction, thereby protecting the PC12 cells from Aβ 25‒35-induced oxidative cytotoxicity. PMID:23762139

The integrity of the RRGDL sequence of the proprotein convertase PC1 is critical for its zymogen and C-terminal processing and for its cellular trafficking.

PubMed Central

Lusson, J; Benjannet, S; Hamelin, J; Savaria, D; Chrétien, M; Seidah, N G

1997-01-01

In order to define the functional importance of the conserved RRGDL motif in the P-domain of the mammalian proprotein convertases(PCs) we generated and cellularly expressed three mutant PC1 vaccinia-virus (VV) recombinants: ARGDL-PC1, RAGDL-PC1 and RRGEL-PC1. Functionally, these mutants caused a decreased level of processing of pro-opiomelanocortin (POMC) into beta-lipotropic pituitary hormone (beta-LPH), especially in the constitutively secreting BSC40 cells. Pulse-chase analyses demonstrated that, in part, this effect was due to both an increased degradation of the mutant PC1s within the endoplasmic reticulum and to a diminished level of zymogen processing in the same compartment. In addition, within cells containing secretory granules such as PC12 and GH4C1 cells, such mutations prevented the C-terminal auto-processing of PC1 into the fully mature 66 kDa form stored in the secretory granules of regulated cells. Since the 66 kDa PC1 is the most active form of the enzyme, it is proposed that the RRGDL sequence is critical for the generation of maximal intracellular PC1 activity. In regulated cells, co-expression of POMC with PC1 or its mutants together with the general PC inhibitor alpha1-antitrypsin Portland (alpha1-PDX), which acts primarily within the constitutive secretory pathway, demonstrated that the latter completely inhibited the formation of beta-LPH by PC1 mutants, whereas it only partially inhibited the ability of wild-type PC1 to process POMC. This suggests that RRGDL mutations prevent PC1 from entering secretory granules and hence the formation of the 66 kDa PC1, and result in the mis-sorting of PC1 mutants towards the constitutive secretory pathway. This conclusion was further supported by immunocytochemical data demonstrating that RRGDL mutants exhibit an intracellular localization pattern different from that of the granule-associated wild-type PC1,but similar to that of the Golgi-localized convertase PC5-B. PMID:9307023

Synergistic effect of topography, surface chemistry and conductivity of the electrospun nanofibrous scaffold on cellular response of PC12 cells.

PubMed

Tian, Lingling; Prabhakaran, Molamma P; Hu, Jue; Chen, Menglin; Besenbacher, Flemming; Ramakrishna, Seeram

2016-09-01

Electrospun nanofibrous nerve implants is a promising therapy for peripheral nerve injury, and its performance can be tailored by chemical cues, topographical features as well as electrical properties. In this paper, a surface modified, electrically conductive, aligned nanofibrous scaffold composed of poly (lactic acid) (PLA) and polypyrrole (Ppy), referred to as o-PLAPpy_A, was fabricated for nerve regeneration. The morphology, surface chemistry and hydrophilicity of nanofibers were characterized by Scanning Electron Microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS) and water contact angle, respectively. The effects of these nanofibers on neuronal differentiation using PC12 cells were evaluated. A hydrophilic surface was created by Poly-ornithine coating, which was able to provide a better environment for cell attachment, and furthermore aligned fibers were proved to be able to guide PC12 cells grow along the fiber direction and be beneficial for neurite outgrowth. The cellular response of PC12 cells to pulsed electrical stimulation was evaluated by NF 200 and alpha tubulin expression, indicating that electrical stimulation with a voltage of 40mV could enhance the neurite outgrowth. The PC12 cells stimulated with electrical shock showed greater level of neurite outgrowth and smaller cell body size. Moreover, the PC12 cells under electrical stimulation showed better viability. In summary, the o-PLAPpy_A nanofibrous scaffold supported the attachment, proliferation and differentiation of PC12 cells in the absence of electrical stimulation, which could be potential candidate for nerve regeneration applications. Copyright © 2016 Elsevier B.V. All rights reserved.

Capns1, a new binding partner of RasGAP-SH3 domain in K-Ras(V12) oncogenic cells: modulation of cell survival and migration.

PubMed

Pamonsinlapatham, Perayot; Gril, Brunilde; Dufour, Sylvie; Hadj-Slimane, Réda; Gigoux, Véronique; Pethe, Stéphanie; L'hoste, Sébastien; Camonis, Jacques; Garbay, Christiane; Raynaud, Françoise; Vidal, Michel

2008-11-01

Ras GTPase-activating protein (RasGAP) is hypothesized to be an effector of oncogenic Ras stimulating numerous downstream cellular signaling cascades involved in survival, proliferation and motility. In this study, we identified calpain small subunit-1 (Capns1) as a new RasGAP-SH3 domain binding partner, using yeast two-hybrid screening. The interaction was confirmed by co-immunoprecipitation assay and was found specific to cells expressing oncogenic K-Ras. We used confocal microscopy to analyze our stably transfected cell model producing mutant Ras (PC3Ras(V12)). Staining for RasGAP-SH3/Capns1 co-localization was two-fold stronger in the protrusions of Ras(V12) cells than in PC3 cells. RasGAP or Capns1 knockdown in PC3Ras(V12) cells induced a two- to three-fold increase in apoptosis. Capns1 gene silencing reduced the speed and increased the persistence of movement in PC3Ras(V12) cells. In contrast, RasGAP knockdown in PC3Ras(V12) cells increased cell migration. Knockdown of both proteins altered the speed and directionality of cell motility. Our findings suggest that RasGAP and Capns1 interaction in oncogenic Ras cells is involved in regulating migration and cell survival.

The Neuroprotective Properties of Hericium erinaceus in Glutamate-Damaged Differentiated PC12 Cells and an Alzheimer's Disease Mouse Model.

PubMed

Zhang, Junrong; An, Shengshu; Hu, Wenji; Teng, Meiyu; Wang, Xue; Qu, Yidi; Liu, Yang; Yuan, Ye; Wang, Di

2016-11-01

Hericium erinaceus , an edible and medicinal mushroom, displays various pharmacological activities in the prevention of dementia in conditions such as Parkinson's and Alzheimer's disease. The present study explored the neuroprotective effects of H. erinaceus mycelium polysaccharide-enriched aqueous extract (HE) on an l-glutamic acid (l-Glu)-induced differentiated PC12 (DPC12) cellular apoptosis model and an AlCl₃ combined with d-galactose-induced Alzheimer's disease mouse model. The data revealed that HE successfully induced PC12 cell differentiation. A 3 h HE incubation at doses of 50 and 100 µg/mL before 25 mM of l-Glu effectively reversed the reduction of cell viability and the enhancement of the nuclear apoptosis rate in DPC12 cells. Compared with l-Glu-damaged cells, in PC12 cells, HE suppressed intracellular reactive oxygen species accumulation, blocked Ca 2+ overload and prevented mitochondrial membrane potential (MMP) depolarization. In the Alzheimer's disease mouse model, HE administration enhanced the horizontal and vertical movements in the autonomic activity test, improved the endurance time in the rotarod test, and decreased the escape latency time in the water maze test. It also improved the central cholinergic system function in the Alzheimer's mice, demonstrated by the fact that it dose-dependently enhanced the acetylcholine (Ach) and choline acetyltransferase (ChAT) concentrations in both the serum and the hypothalamus. Our findings provide experimental evidence that HE may provide neuroprotective candidates for treating or preventing neurodegenerative diseases.

Caspase cascade regulated mitochondria mediated apoptosis in monocrotophos exposed PC12 cells.

PubMed

Kashyap, M P; Singh, A K; Siddiqui, M A; Kumar, V; Tripathi, V K; Khanna, V K; Yadav, S; Jain, S K; Pant, A B

2010-11-15

Monocrotophos (MCP) is a commonly used organophosphorus (OP) pesticide. We studied apoptotic changes in PC12 cells exposed to MCP. A significant induction in reactive oxygen species (ROS), lipid peroxide (LPO), and the ratio of glutathione disulfide (GSSG)/reduced glutathione (GSH) was observed in cells exposed to selected doses of MCP. Following the exposure of PC12 cells to MCP, the levels of protein and mRNA expressions of Caspase-3, Caspase-9, Bax, p53, P(21), Puma, and cytochrome-c were significantly upregulated, whereas the levels of Bcl(2), Bcl(w), and Mcl1 were downregulated. TUNEL assay, DNA laddering, and micronuclei induction show that long-term exposure of PC12 cells to MCP at higher concentration (10(-5) M) decreases the number of apoptotic events due to an increase in the number of necrotic cells. MCP-induced translocation of Bax and cytochrome-c proteins between the cytoplasm and mitochondria confirmed the role of p53 and Puma in mitochondrial membrane permeability. Mitochondria mediated apoptosis induction was confirmed by the increased activity of caspase cascade. We believe that this is the first report showing MCP-induced apoptosis in PC12 cells, which is mitochondria mediated and regulated through the caspase cascade. Our data demonstrates that MCP induced the apoptotic cell death in neuronal cells and identifies the possible cellular and molecular mechanisms of organophosphate pesticide-induced apoptosis in neuronal cells.

Multi-walled carbon nanotubes suppress potassium channel activities in PC12 cells

NASA Astrophysics Data System (ADS)

Xu, Haifei; Bai, Juan; Meng, Jie; Hao, Wei; Xu, Haiyan; Cao, Ji-Min

2009-07-01

The advancement in nanotechnology has produced technological and conceptual breakthroughs but the effects nanomaterials have on organisms at the cellular level are poorly understood. Here we report that carboxyl-terminated multi-walled carbon nanotubes (MWCNTs) act as antagonists of three types of potassium channels as assessed by whole-cell patch clamp electrophysiology on undifferentiated pheochromocytoma (PC12) cells. Our results showed that carboxyl-terminated MWCNTs suppress the current densities of Ito, IK and IK1 in a time-dependent and irreversible manner. The suppressions were most distinct 24 h after incubation with MWCNTs. However, MWCNTs did not significantly change the expression levels of reactive oxygen species (ROS) or intracellular free calcium and also did not alter the mitochondrial membrane potential (ΔΨm) in PC12 cells. These results suggest that oxidative stress was not involved in the MWCNTs suppression of Ito, IK and IK1 current densities. Nonetheless, the suppression of potassium currents by MWCNTs will impact on electrical signaling of excitable cells such as neurons and muscles.

The polycystins are modulated by cellular oxygen-sensing pathways and regulate mitochondrial function

PubMed Central

Padovano, Valeria; Kuo, Ivana Y.; Stavola, Lindsey K.; Aerni, Hans R.; Flaherty, Benjamin J.; Chapin, Hannah C.; Ma, Ming; Somlo, Stefan; Boletta, Alessandra; Ehrlich, Barbara E.; Rinehart, Jesse; Caplan, Michael J.

2017-01-01

Autosomal dominant polycystic kidney disease is caused by mutations in the genes encoding polycystin-1 (PC1) and polycystin-2 (PC2), which form an ion channel complex that may mediate ciliary sensory processes and regulate endoplasmic reticulum (ER) Ca2+ release. Loss of PC1 expression profoundly alters cellular energy metabolism. The mechanisms that control the trafficking of PC1 and PC2, as well as their broader physiological roles, are poorly understood. We found that O2 levels regulate the subcellular localization and channel activity of the polycystin complex through its interaction with the O2-sensing prolyl hydroxylase domain containing protein EGLN3 (or PHD3), which hydroxylates PC1. Moreover, cells lacking PC1 expression use less O2 and show less mitochondrial Ca2+ uptake in response to bradykinin-induced ER Ca2+ release, indicating that PC1 can modulate mitochondrial function. These data suggest a novel role for the polycystins in sensing and responding to cellular O2 levels. PMID:27881662

Alpha-ketoglutarate and N-acetyl cysteine protect PC12 cells from cyanide-induced cytotoxicity and altered energy metabolism.

PubMed

Satpute, R M; Hariharakrishnan, J; Bhattacharya, R

2008-01-01

Cyanide is a rapidly acting neurotoxin that inhibits cellular respiration and energy metabolism leading to histotoxic hypoxia. This results in the dissipation of mitochondrial membrane potential (MMP) accompanied by decreased cellular ATP content which in turn is responsible for increased levels of intracellular calcium ions ([Ca(2+)](i)) and total lactic acid content of the cells. Rat pheochromocytoma (PC12) cells possess much of the biochemical machinery associated with synaptic neurons. In the present study, we evaluated the cytoprotective effects of alpha-ketoglutarate (A-KG) and N-acetylcysteine (NAC) against cyanide-induced cytotoxicity and altered energy metabolism in PC12 cells. Cyanide-antagonism by A-KG is attributed to cyanohydrin formation whereas NAC is known for its antioxidant properties. Data on leakage of intracellular lactate dehydrogenase and mitochondrial function (MTT assay) revealed that simultaneous treatment of A-KG (0.5 mM) and NAC (0.25 mM) significantly prevented the cytotoxicity of cyanide. Also, cellular ATP content was found to improve, followed by restoration of MMP, intracellular calcium [Ca(2+)](i) and lactic acid levels. Treatment with A-KG and NAC also attenuated the levels of peroxides generated by cyanide. The study indicates that combined administration of A-KG and NAC protected the cyanide-challenged PC12 cells by resolving the altered energy metabolism. The results have implications in the development of new treatment regimen for cyanide poisoning.

Ameliorating effects of aged garlic extracts against Aβ-induced neurotoxicity and cognitive impairment

PubMed Central

2013-01-01

Background In vitro antioxidant activities and neuron-like PC12 cell protective effects of solvent fractions from aged garlic extracts were investigated to evaluate their anti-amnesic functions. Ethyl acetate fractions of aged garlic had higher total phenolics than other fractions. Methods Antioxidant activities of ethyl acetate fractions from aged garlic were examined using 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt (ABTS) and malondialdehyde (MDA) inhibitory effect using mouse whole brain homogenates. Levels of cellular oxidative stress as reactive oxygen species (ROS) accumulation were measured using 2',7'-dichlorofluorescein diacetate (DCF-DA). PC12 cell viability was investigated by 3-[4,5-dimethythiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) and lactate dehydtrogenase (LDH) assay. The learning and memory impairment in institute of cancer research (ICR) mice was induced by neurotoxic amyloid beta protein (Aβ) to investigate in vivo anti-amnesic effects of aged garlic extracts by using Y-maze and passive avoidance tests. Results We discovered that ethyl acetate fractions showed the highest ABTS radical scavenging activity and MDA inhibitory effect. Intracellular ROS accumulation resulting from Aβ treatment in PC12 cells was significantly reduced when ethyl acetate fractions were presented in the medium compare to PC12 cells which was only treated with Aβ only. Ethyl acetate fractions from aged garlic extracts showed protection against Aβ-induced neurotoxicity. Pre-administration with aged garlic extracts attenuated Aβ-induced learning and memory deficits in both in vivo tests. Conclusions Our findings suggest that aged garlic extracts with antioxidant activities may improve cognitive impairment against Aβ-induced neuronal deficit, and possess a wide range of beneficial activities for neurodegenerative disorders, notably Alzheimer's disease (AD). PMID:24134394

The Neuroprotective Properties of Hericium erinaceus in Glutamate-Damaged Differentiated PC12 Cells and an Alzheimer’s Disease Mouse Model

PubMed Central

Zhang, Junrong; An, Shengshu; Hu, Wenji; Teng, Meiyu; Wang, Xue; Qu, Yidi; Liu, Yang; Yuan, Ye; Wang, Di

2016-01-01

Hericium erinaceus, an edible and medicinal mushroom, displays various pharmacological activities in the prevention of dementia in conditions such as Parkinson’s and Alzheimer’s disease. The present study explored the neuroprotective effects of H. erinaceus mycelium polysaccharide-enriched aqueous extract (HE) on an l-glutamic acid (l-Glu)-induced differentiated PC12 (DPC12) cellular apoptosis model and an AlCl3 combined with d-galactose-induced Alzheimer’s disease mouse model. The data revealed that HE successfully induced PC12 cell differentiation. A 3 h HE incubation at doses of 50 and 100 µg/mL before 25 mM of l-Glu effectively reversed the reduction of cell viability and the enhancement of the nuclear apoptosis rate in DPC12 cells. Compared with l-Glu-damaged cells, in PC12 cells, HE suppressed intracellular reactive oxygen species accumulation, blocked Ca2+ overload and prevented mitochondrial membrane potential (MMP) depolarization. In the Alzheimer’s disease mouse model, HE administration enhanced the horizontal and vertical movements in the autonomic activity test, improved the endurance time in the rotarod test, and decreased the escape latency time in the water maze test. It also improved the central cholinergic system function in the Alzheimer’s mice, demonstrated by the fact that it dose-dependently enhanced the acetylcholine (Ach) and choline acetyltransferase (ChAT) concentrations in both the serum and the hypothalamus. Our findings provide experimental evidence that HE may provide neuroprotective candidates for treating or preventing neurodegenerative diseases. PMID:27809277

Knockdown of NogoA prevents MPP+‑induced neurotoxicity in PC12 cells via the mTOR/STAT3 signaling pathway.

PubMed

Zhong, Jianbin; Li, Xie; Wan, Limei; Chen, Zhibang; Zhong, Simin; Xiao, Songhua; Yan, Zhengwen

2016-02-01

NogoA is a myelin‑associated protein, which is important in the inhibition of axonal fiber growth and in regeneration following injury of the mammalian central nervous system. A previous study suggested that NogoA may be key in the process of Parkinson's disease (PD), which is the second most common chronic neurodegenerative disorder worldwide. The regulatory mechanism underlying the effect of NogoA on the process of PD remains to be fully elucidated. The present study aimed to investigate the effect and underlying mechanism of NogoA on cellular viability, apoptosis and autophagy induced by 1-methyl-4-phenylpyridinium (MPP+) in PC12 cells, a commonly used in vitro PD model. PC12 cells were treated with 1 mM MPP+ for 24 h and the cells were harvested for western blotting. The results demonstrated that the protien expression levels of NogoA were increased in the PC12 cells treated with MPP+. Subsequently, NogoA small interfering RNA was synthesized and transfected into PC12 cells to silence the expression of NogoA. NogoA knockdown significantly reduced the MPP+‑induced decrease in cell viability and apoptosis, detected using a cell counting kit‑8 and flow cytometric analysis, respectively. Interference in the expression of NogoA increased the MPP+‑induced decrease in mitochondrial membrane potential, determined quantitatively by flow cytometry using JC-1 dye, and the protein levels of Beclin‑1. In addition, MPP+ treatment activated the mammalian target of rapamycin (mTOR)/signal transducer and activator of transcription 3 (STAT3) signaling pathway. Knockdown of NogoA significantly inhibited the expression levels of mTOR and STAT3. Furthermore, overexpression of NogoA had similar neurotoxic effects on the PC12 cells as MPP+ treatment. Treatment with rapamycin, an inhibitor of the mTOR/STAT3 signaling pathway had a similar effect to that of NogoA knockdown in the MPP+‑treated PC12 cells. Taken together, the results from the present study demonstrated that NogoA may regulate MPP+‑induced neurotoxicity in PC12 cells via the mTOR/STAT3 signaling pathway and provided an explanation regarding the regulatory mechanism of NogoA on the process of PD.

2, 3, 7, 8-Tetrachlorodibenzo-P-dioxin (TCDD) induces premature senescence in human and rodent neuronal cells via ROS-dependent mechanisms.

PubMed

Wan, Chunhua; Liu, Jiao; Nie, Xiaoke; Zhao, Jianya; Zhou, Songlin; Duan, Zhiqing; Tang, Cuiying; Liang, Lingwei; Xu, Guangfei

2014-01-01

The widespread environmental pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a potent toxicant that causes significant neurotoxicity. However, the biological events that participate in this process remain largely elusive. In the present study, we demonstrated that TCDD exposure triggered apparent premature senescence in rat pheochromocytoma (PC12) and human neuroblastoma SH-SY5Y cells. Senescence-associated β-galactosidase (SA-β-Gal) assay revealed that TCDD induced senescence in PC12 neuronal cells at doses as low as 10 nM. TCDD led to F-actin reorganization and the appearance of an alternative senescence marker, γ-H2AX foci, both of which are important features of cellular senescence. In addition, TCDD exposure altered the expression of senescence marker proteins, such as p16, p21 and p-Rb, in both dose- and time-dependent manners. Furthermore, we demonstrated that TCDD promotes mitochondrial dysfunction and the accumulation of cellular reactive oxygen species (ROS) in PC12 cells, leading to the activation of signaling pathways that are involved in ROS metabolism and senescence. TCDD-induced ROS generation promoted significant oxidative DNA damage and lipid peroxidation. Notably, treatment with the ROS scavenger N-acetylcysteine (NAC) markedly attenuated TCDD-induced ROS production, cellular oxidative damage and neuronal senescence. Moreover, we found that TCDD induced a similar ROS-mediated senescence response in human neuroblastoma SH-SY5Y cells. In sum, these results demonstrate for the first time that TCDD induces premature senescence in neuronal cells by promoting intracellular ROS production, supporting the idea that accelerating the onset of neuronal senescence may be an important mechanism underlying TCDD-induced neurotoxic effects.

Secretome of Differentiated PC12 Cells Restores the Monocrotophos-Induced Damages in Human Mesenchymal Stem Cells and SHSY-5Y Cells: Role of Autophagy and Mitochondrial Dynamics.

PubMed

Srivastava, A; Singh, S; Rajpurohit, C S; Srivastava, P; Pandey, A; Kumar, D; Khanna, V K; Pant, A B

2018-06-01

A perturbed cellular homeostasis is a key factor associated with xenobiotic exposure resulting in various ailments. The local cellular microenvironment enriched with secretory components aids in cell-cell communication that restores this homeostasis. Deciphering the underlying mechanism behind this restorative potential of secretome could serve as a possible solution to many health hazards. We, therefore, explored the protective efficacy of the secretome of differentiated PC12 cells with emphasis on induction of autophagy and mitochondrial biogenesis. Monocrotophos (MCP), a widely used neurotoxic organophosphate, was used as the test compound at sublethal concentration. The conditioned medium (CM) of differentiated PC12 cells comprising of their secretome restored the cell viability, oxidative stress and apoptotic cell death in MCP-challenged human mesenchymal stem cells and SHSY-5Y, a human neuroblastoma cell line. Delving further to identify the underlying mechanism of this restorative effect we observed a marked increase in the expression of autophagy markers LC3, Beclin-1, Atg5 and Atg7. Exposure to autophagy inhibitor, 3-methyladenine, led to a reduced expression of these markers with a concomitant increase in the expression of pro-apoptotic caspase-3. Besides that, the increased mitochondrial fission in MCP-exposed cells was balanced with increased fusion in the presence of CM facilitated by AMPK/SIRT1/PGC-1α signaling cascade. Mitochondrial dysfunctions are strongly associated with autophagy activation and as per our findings, cellular secretome too induces autophagy. Therefore, connecting these three potential apices can be a major breakthrough in repair and rescue of xenobiotic-damaged tissues and cells.

In vitro assessment of TAT — Ciliary Neurotrophic Factor therapeutic potential for peripheral nerve regeneration

DOE Office of Scientific and Technical Information (OSTI.GOV)

Barbon, Silvia, E-mail: silvia.barbon@yahoo.it

In regenerative neurobiology, Ciliary Neurotrophic Factor (CNTF) is raising high interest as a multifunctional neurocytokine, playing a key role in the regeneration of injured peripheral nerves. Despite its promising trophic and regulatory activity, its clinical application is limited by the onset of severe side effects, due to the lack of efficient intracellular trafficking after administration. In this study, recombinant CNTF linked to the transactivator transduction domain (TAT) was investigated in vitro and found to be an optimized fusion protein which preserves neurotrophic activity, besides enhancing cellular uptake for therapeutic advantage. Moreover, a compelling protein delivery method was defined, in themore » future perspective of improving nerve regeneration strategies. Following determination of TAT-CNTF molecular weight and concentration, its specific effect on neural SH-SY5Y and PC12 cultures was assessed. Cell proliferation assay demonstrated that the fusion protein triggers PC12 cell growth within 6 h of stimulation. At the same time, the activation of signal transduction pathway and enhancement of cellular trafficking were found to be accomplished in both neural cell lines after specific treatment with TAT-CNTF. Finally, the recombinant growth factor was successfully loaded on oxidized polyvinyl alcohol (PVA) scaffolds, and more efficiently released when polymer oxidation rate increased. Taken together, our results highlight that the TAT domain addiction to the protein sequence preserves CNTF specific neurotrophic activity in vitro, besides improving cellular uptake. Moreover, oxidized PVA could represent an ideal biomaterial for the development of nerve conduits loaded with the fusion protein to be delivered to the site of nerve injury. - Highlights: • TAT-CNTF is an optimized fusion protein that preserves neurotrophic activity. • In neural cell lines, TAT-CNTF triggers the activation of signal transduction. • Fast cellular uptake of TAT-CNTF was accomplished after cell treatment. • TAT-CNTF can be efficiently loaded on oxidized PVA cylinders for local delivery. • TAT-CNTF features make it ideal for peripheral nerve regeneration therapies.« less

Knockdown of cytosolic NADP(+) -dependent isocitrate dehydrogenase enhances MPP(+) -induced oxidative injury in PC12 cells.

PubMed

Yang, Eun Sun; Park, Jeen-Woo

2011-05-01

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its toxic metabolite 1-methyl-4-phenylpyridium ion (MPP(+)) have been shown to induce Parkinson's disease-like symptoms as well as neurotoxicity in humans and animal species. Recently, we reported that maintenance of redox balance and cellular defense against oxidative damage are primary functions of the novel antioxidant enzyme cytosolic NADP(+) -dependent isocitrate dehydrogenase (IDPc). In this study, we examined the role of IDPc in cellular defense against MPP(+) -induced oxidative injury using PC12 cells transfected with IDPc small interfering RNA (siRNA). Our results demonstrate that MPP(+) -mediated disruption of cellular redox status, oxidative damage to cells, and apoptotic cell death were significantly enhanced by knockdown of IDPc.

Conductive micropatterned polyurethane films as tissue engineering scaffolds for Schwann cells and PC12 cells.

PubMed

Wu, Yaobin; Wang, Ling; Hu, Tianli; Ma, Peter X; Guo, Baolin

2018-05-15

Controlling cellular alignment and elongation has been demonstrated as an important parameter for developing nerve tissue engineering scaffolds. Many approaches have been developed to guide cellular orientation for nerve regeneration such as micropatterning techniques. However, most of materials used for developing micropatterning scaffolds lack of bioactivity and biofunctionability. Here we present a functional conductive micropatterned scaffold based on bioactive conductive biodegradable polyurethane prepared using a micro-molding technique. These conductive micropatterned scaffolds are able to not only induce the Schwann cells (SCs) alignment and elongation by the micropatterned surface but also enhance the nerve growth factor (NGF) gene expression of SCs by the bioactivity of these materials. Additionally, the combined effect of the bioactivity of such conductive materials and the micropatterned structure also dramatically promotes the neurite extension and elongation of PC12 cells in a highly aligned direction. These data suggest that these conductive micropatterned scaffolds that easily control cellular orientation and organization, and dramatically enhance NGF gene expression and significantly induce the neurite extension of PC12 cells, have a great potential for nerve regeneration applications. Copyright © 2018 Elsevier Inc. All rights reserved.

Therapeutic Approaches Targeting MYC-Driven Prostate Cancer

PubMed Central

Rebello, Richard J.; Pearson, Richard B.; Hannan, Ross D.; Furic, Luc

2017-01-01

The transcript encoding the proto-oncogene MYC is commonly overexpressed in prostate cancer (PC). MYC protein abundance is also increased in the majority of cases of advanced and metastatic castrate-resistant PC (mCRPC). Accordingly, the MYC-directed transcriptional program directly contributes to PC by upregulating the expression of a number of pro-tumorigenic factors involved in cell growth and proliferation. A key cellular process downstream of MYC activity is the regulation of ribosome biogenesis which sustains tumor growth. MYC activity also cooperates with the dysregulation of the phosphoinositol-3-kinase (PI3K)/AKT/mTOR pathway to promote PC cell survival. Recent advances in the understanding of these interactions through the use of animal models have provided significant insight into the therapeutic efficacy of targeting MYC activity by interfering with its transcriptional program, and indirectly by targeting downstream cellular events linked to MYC transformation potential. PMID:28212321

Furanodictine A and B: amino sugar analogues produced by cellular slime mold Dictyostelium discoideum showing neuronal differentiation activity.

PubMed

Kikuchi, H; Saito, Y; Komiya, J; Takaya, Y; Honma, S; Nakahata, N; Ito, A; Oshima, Y

2001-10-19

We investigated the constituents of Dictyostelium discoideum to clarify the diversity of secondary metabolites of Dictyostelium cellular slime molds and to explore biologically active substances that could be useful in the development of novel drugs. From a methanol extract of the multicellular fruit body of D. discoideum, we isolated two novel amino sugar analogues, furanodictine A (1) and B (2). They are the first 3,6-anhydrosugars to be isolated from natural sources. Their relative structures were elucidated by spectral means, and the absolute configurations were confirmed by asymmetric syntheses of 1 and 2. These furanodictines potently induce neuronal differentiation of rat pheochromocytoma (PC-12) cells.

Reactive oxygen species regulated mitochondria-mediated apoptosis in PC12 cells exposed to chlorpyrifos

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Jeong Eun; Hanyang Biomedical Research Institute, Seoul; Park, Jae Hyeon

2012-09-01

Reactive oxidative species (ROS) generated by environmental toxicants including pesticides could be one of the factors underlying the neuronal cell damage in neurodegenerative diseases. In this study we found that chlorpyrifos (CPF) induced apoptosis in dopaminergic neuronal components of PC12 cells as demonstrated by the activation of caspases and nuclear condensation. Furthermore, CPF also reduced the tyrosine hydroxylase-positive immunoreactivity in substantia nigra of the rat. In addition, CPF induced inhibition of mitochondrial complex I activity. Importantly, N-acetyl cysteine (NAC) treatment effectively blocked apoptosis via the caspase-9 and caspase-3 pathways while NAC attenuated the inhibition of mitochondrial complex I activity asmore » well as the oxidative metabolism of dopamine (DA). These results demonstrated that CPF-induced apoptosis was involved in mitochondrial dysfunction through the production of ROS. In the response of cellular antioxidant systems to CPF, we found that CPF treatment increased HO-1 expression while the expression of CuZnSOD and MnSOD was reduced. In addition, we found that CPF treatment activated MAPK pathways, including ERK 1/2, the JNK, and the p38 MAP kinase in a time-dependent manner. NAC treatment abolished MAPK phosphorylation caused by CPF, indicating that ROS are upstream signals of MAPK. Interestingly, MAPK inhibitors abolished cytotoxicity and reduced ROS generation by CPF treatment. Our results demonstrate that CPF induced neuronal cell death in part through MAPK activation via ROS generation, suggesting its potential to generate oxidative stress via mitochondrial damage and its involvement in oxidative stress-related neurodegenerative disease. -- Highlights: ► Chlorpyrifos induces apoptosis. ► Chlorpyrifos inhibits mitochondrial complex I activity. ► ROS is involved in chlorpyrifos-induced apoptosis. ► Chlorpyrifos affects cellular antioxidant systems. ► Chlorpyrifos-induced apoptosis mediates activation of MAPK.« less

[Effect of mitogen activated protein kinase signal transduction on apoptosis of PC12 cells induced by electromagnetic exposure].

PubMed

Yang, Xue-Sen; Zhang, Wei; Gong, Qian-Fen

2008-06-01

To observe the effect of mitogen activated protein kinase (MAPK) signal transduction system on the apoptosis induced by electromagnetic exposure in PC12 cells. After pretreated by SB203580 alone or together with U0126, PC12 cells were exposed to 65 mW/cm(2) electromagnetic wave for 20 min. The phosphorylations of ERK1/2, JNK and P38 MAPK were tested by Western-blot at 3 h and 24 h after electromagnetic exposure. The apoptosis of PC12 cells were detected by Annexin-V-FITC flow cytometry. U0126, but not SB203580 could inhibit the activation of ERK1/2 induced by electromagnetic exposure. U0126 and SB203580 had no effects on the activation of JNK. SB203580 could inhibit the activation of P38 MAPK significantly. But U0126 had no such effect on the activation of P38 MAPK. After pretreated by SB203580 alone or together with U0126, the apoptosis of PC12 cells decreased. But the pretreatment by U0126 alone had no influence on the apoptosis of PC12 cells. The P38 MAPK signal transduction modulate the apoptosis of PC12 cells induced by electromagnetic exposure. ERK signal transduction has no effect on the apoptosis of PC12 cells. JNK signal transduction may promote the apoptosis of PC12 cells in the early stage after electromagnetic exposure.

Hypochlorite modification of sphingomyelin generates chlorinated lipid species that induce apoptosis and proteome alterations in dopaminergic PC12 neurons in vitro

PubMed Central

Nusshold, Christoph; Kollroser, Manfred; Köfeler, Harald; Rechberger, Gerald; Reicher, Helga; Üllen, Andreas; Bernhart, Eva; Waltl, Sabine; Kratzer, Ingrid; Hermetter, Albin; Hackl, Hubert; Trajanoski, Zlatko; Hrzenjak, Andelko; Malle, Ernst; Sattler, Wolfgang

2014-01-01

Recent observations link myeloperoxidase (MPO) activation to neurodegeneration. In multiple sclerosis MPO is present in areas of active demyelination where the potent oxidant hypochlorous acid (HOCl), formed by MPO from H2O2 and chloride ions, could oxidatively damage myelin-associated lipids. The purpose of this study was (i) to characterize reaction products of sphingomyelin (SM) formed in response to modification by HOCl, (ii) to define the impact of exogenously added SM and HOCl-modified SM (HOCl-SM) on viability parameters of a neuronal cell line (PC12), and (iii) to study alterations in the PC12 cell proteome in response to SM and HOCl-SM. MALDI-TOF-MS analyses revealed that HOCl, added as reagent or generated enzymatically, transforms SM into chlorinated species. On the cellular level HOCl-SM but not SM induced the formation of reactive oxygen species. HOCl-SM induced severely impaired cell viability, dissipation of the mitochondrial membrane potential, and activation of caspase-3 and DNA damage. Proteome analyses identified differential expression of specific subsets of proteins in response to SM and HOCl-SM. Our results demonstrate that HOCl modification of SM results in the generation of chlorinated lipid species with potent neurotoxic properties. Given the emerging connections between the MPO–H2O2–chloride axis and neurodegeneration, this chlorinating pathway might be implicated in neuropathogenesis. PMID:20226853

The α-Secretase-derived N-terminal Product of Cellular Prion, N1, Displays Neuroprotective Function in Vitro and in Vivo*

PubMed Central

Guillot-Sestier, Marie-Victoire; Sunyach, Claire; Druon, Charlotte; Scarzello, Sabine; Checler, Frédéric

2009-01-01

Cellular prion protein (PrPc) undergoes a disintegrin-mediated physiological cleavage, generating a soluble amino-terminal fragment (N1), the function of which remained unknown. Recombinant N1 inhibits staurosporine-induced caspase-3 activation by modulating p53 transcription and activity, whereas the PrPc-derived pathological fragment (N2) remains biologically inert. Furthermore, N1 protects retinal ganglion cells from hypoxia-induced apoptosis, reduces the number of terminal deoxynucleotidyltransferase-mediated biotinylated UTP nick end labeling-positive and p53-immunoreactive neurons in a pressure-induced ischemia model of the rat retina and triggers a partial recovery of b-waves but not a-waves of rat electroretinograms. Our work is the first demonstration that the α-secretase-derived PrPc fragment N1, but not N2, displays in vivo and in vitro neuroprotective function by modulating p53 pathway. It further demonstrates that distinct N-terminal cleavage products of PrPc harbor different biological activities underlying the various phenotypes linking PrPc to cell survival. PMID:19850936

A role for the tyrosine kinase ACK1 in neurotrophin signaling and neuronal extension and branching

PubMed Central

La Torre, A; del Mar Masdeu, M; Cotrufo, T; Moubarak, R S; del Río, J A; Comella, J X; Soriano, E; Ureña, J M

2013-01-01

Neurotrophins are involved in many crucial cellular functions, including neurite outgrowth, synapse formation, and plasticity. Although these events have long been known, the molecular determinants underlying neuritogenesis have not been fully characterized. Ack1 (activated Cdc42-associated tyrosine kinase) is a non-receptor tyrosine kinase that is highly expressed in the brain. Here, we demonstrate that Ack1 is a molecular constituent of neurotrophin signaling cascades in neurons and PC12 cells. We report that Ack1 interacts with Trk receptors and becomes tyrosine phosphorylated and its kinase activity is increased in response to neurotrophins. Moreover, our data indicate that Ack1 acts upstream of the Akt and MAPK pathways. We show that Ack1 overexpression induces neuritic outgrowth and promotes branching in neurotrophin-treated neuronal cells, whereas the expression of Ack1 dominant negatives or short-hairpin RNAs counteract neurotrophin-stimulated differentiation. Our results identify Ack1 as a novel regulator of neurotrophin-mediated events in primary neurons and in PC12 cells. PMID:23598414

[Carcinogenesis and its mechanism of mutant-type[12Asp]K-ras4B gene].

PubMed

Gui, Li-ming; Wei, Li-hui; Zhang, Ying-mei; Wang, Jian-liu; Wang, Ying; Chen, Ying; Ma, Da-long

2002-01-01

Ras gene plays an important role in the extra- and intra-cellular signal transduction pathway. It mediates series cascade reactions, and eventually actives transcriptional factors in nucleus. It is unknown on the mechanism of carcinogenesis of Ras gene in endometrial carcinoma, though K-ras mutant is very common in endometrial atypical hyperplasia and carcinoma. On basis of discovering the mutation in 12th codon of K-ras in endometrial carcinoma cell line, HEC-1A, we explored the carcinogenesis and molecular mechanism of mutant-type [12Asp] K-ras4B gene. (1) Full-length [12Asp]K-ras4B cDNA was amplified with RT-PCR, then inserted into pcDI eukaryotic expressive vector. (2) Morphological change, growth kinetics in vitro and tumorigencity in nude mice in vivo after-before transfection were observed. (3) To test the cell growth kinetics by methyl thiazolium tetrazolium (MTT) and [3H]thymidine incorporation method. (1) The authors have successfully constructed eukaryotic expression plasmid pcDI-[12Asp] K-ras4B; (2) To confirm that [12Asp] K-ras4B mutant can trigger the neoplastic transformation of NIH3T3 cells by test in vitro and in vivo. (3) After pMCV-RasN17 plasmid, a Ras mutant were transfected into pcDI-[12Asp] K-ras4B cells, the growth of this cell were restrained significantly in comparison with control group. (4) These findings indicate the expression of RafS621A resulted in remarkable inhibition in proliferation of pcDI-[12Asp]K-ras4B cell (P < 0.05). However, RafCAAX mutant can enhance pcDI-[12Asp]K-ras4B cell growth (P < 0.05). (1) [12Asp]K-ras4B gene alone is able to cause neoplastic transformation in NIH3T3 cells in vitro and in vivo. (2) [12Asp]K-ras4B-induced NIH3T3 cells neoplastic transformation required Raf signaling pathway.

Monocrotophos induced apoptosis in PC12 cells: role of xenobiotic metabolizing cytochrome P450s.

PubMed

Kashyap, Mahendra Pratap; Singh, Abhishek Kumar; Kumar, Vivek; Tripathi, Vinay Kumar; Srivastava, Ritesh Kumar; Agrawal, Megha; Khanna, Vinay Kumar; Yadav, Sanjay; Jain, Swatantra Kumar; Pant, Aditya Bhushan

2011-03-21

Monocrotophos (MCP) is a widely used organophosphate (OP) pesticide. We studied apoptotic changes and their correlation with expression of selected cytochrome P450s (CYPs) in PC12 cells exposed to MCP. A significant induction in reactive oxygen species (ROS) and decrease in glutathione (GSH) levels were observed in cells exposed to MCP. Following the exposure of PC12 cells to MCP (10(-5) M), the levels of protein and mRNA expressions of caspase-3/9, Bax, Bcl(2), P(53), P(21), GSTP1-1 were significantly upregulated, whereas the levels of Bclw, Mcl1 were downregulated. A significant induction in the expression of CYP1A1/1A2, 2B1/2B2, 2E1 was also observed in PC12 cells exposed to MCP (10(-5) M), whereas induction of CYPs was insignificant in cells exposed to 10(-6) M concentration of MCP. We believe that this is the first report showing altered expressions of selected CYPs in MCP-induced apoptosis in PC12 cells. These apoptotic changes were mitochondria mediated and regulated by caspase cascade. Our data confirm the involvement of specific CYPs in MCP-induced apoptosis in PC12 cells and also identifies possible cellular and molecular mechanisms of organophosphate pesticide-induced apoptosis in neuronal cells.

Identification of novel putative-binding proteins for cellular prion protein and a specific interaction with the STIP1 homology and U-Box-containing protein 1

PubMed Central

Gimenez, Ana Paula Lappas; Richter, Larissa Morato Luciani; Atherino, Mariana Campos; Beirão, Breno Castello Branco; Fávaro, Celso; Costa, Michele Dietrich Moura; Zanata, Silvio Marques; Malnic, Bettina; Mercadante, Adriana Frohlich

2015-01-01

ABSTRACT Prion diseases involve the conversion of the endogenous cellular prion protein, PrPC, into a misfolded infectious isoform, PrPSc. Several functions have been attributed to PrPC, and its role has also been investigated in the olfactory system. PrPC is expressed in both the olfactory bulb (OB) and olfactory epithelium (OE) and the nasal cavity is an important route of transmission of diseases caused by prions. Moreover, Prnp−/− mice showed impaired behavior in olfactory tests. Given the high PrPC expression in OE and its putative role in olfaction, we screened a mouse OE cDNA library to identify novel PrPC-binding partners. Ten different putative PrPC ligands were identified, which were involved in functions such as cellular proliferation and apoptosis, cytoskeleton and vesicle transport, ubiquitination of proteins, stress response, and other physiological processes. In vitro binding assays confirmed the interaction of PrPC with STIP1 homology and U-Box containing protein 1 (Stub1) and are reported here for the first time. Stub1 is a co-chaperone with ubiquitin E3-ligase activity, which is associated with neurodegenerative diseases characterized by protein misfolding and aggregation. Physiological and pathological implications of PrPC-Stub1 interaction are under investigation. The PrPC-binding proteins identified here are not exclusive to the OE, suggesting that these interactions may occur in other tissues and play general biological roles. These data corroborate the proposal that PrPC is part of a multiprotein complex that modulates several cellular functions and provide a platform for further studies on the physiological and pathological roles of prion protein. PMID:26237451

Ameliorative effects of selenium on arsenic-induced cytotoxicity in PC12 cells via modulating autophagy/apoptosis.

PubMed

Rahman, Md Mostafizur; Uson-Lopez, Rachael A; Sikder, Md Tajuddin; Tan, Gongxun; Hosokawa, Toshiyuki; Saito, Takeshi; Kurasaki, Masaaki

2018-04-01

Arsenic is well known toxicant responsible for human diseases including cancers. On the other hand, selenium is an essential trace element with significant chemopreventive effects, anticancer potentials and antioxidant properties. Although previous studies have reported antagonism/synergism between arsenic and selenium in biological systems, the biomolecular mechanism/s is still inconclusive. Therefore, to elucidate the molecular phenomena in cellular level, we hypothesized that co-exposure of selenium with arsenic may have suppressive effects on arsenic-induced cytotoxicity. We found that selenium in co-exposure with arsenic increases cell viability, and suppresses oxidative stress induced by arsenic in PC12 cells. Consequently, DNA fragmentation due to arsenic exposure was also reduced by arsenic and selenium co-exposure. Furthermore, western blot analyses revealed that simultaneous exposure of both metals significantly inhibited autophagy which further suppressed apoptosis through positively regulation of key proteins; p-mTOR, p-Akt, p-Foxo1A, p62, and expression of ubiquitin, Bax, Bcl2, NFкB, and caspases 3 and 9, although those are negatively regulated by arsenic. In addition, reverse transcriptase PCR analysis confirmed the involvement of caspase cascade in cell death process induced by arsenic and subsequent inhibition by co-exposure of selenium with arsenic. The cellular accumulation study of arsenic in presence/absence of selenium via inductively coupled plasma mass spectrometry confirmed that selenium effectively retarded the uptake of arsenic in PC12 cells. Finally, these findings imply that selenium is capable to modulate arsenic-induced intrinsic apoptosis pathway via enhancement of mTOR/Akt autophagy signaling pathway through employing antioxidant potentials and through inhibiting the cellular accumulation of arsenic in PC12 cells. Copyright © 2017 Elsevier Ltd. All rights reserved.

Caveolin-1 and glucose transporter 4 involved in the regulation of glucose-deprivation stress in PC12 cells.

PubMed

Zhang, Qi-Qi; Huang, Liang; Han, Chao; Guan, Xin; Wang, Ya-Jun; Liu, Jing; Wan, Jing-Hua; Zou, Wei

2015-08-25

Recent evidence suggests that caveolin-1 (Cav-1), the major protein constituent of caveolae, plays a prominent role in neuronal nutritional availability with cellular fate regulation besides in several cellular processes such as cholesterol homeostasis, regulation of signal transduction, integrin signaling and cell growth. Here, we aimed to investigate the function of Cav-1 and glucose transporter 4 (GLUT4) upon glucose deprivation (GD) in PC12 cells. The results demonstrated firstly that both Cav-1 and GLUT4 were up-regulated by glucose withdrawal in PC12 cells by using Western blot and laser confocal technology. Also, we found that the cell death rate, mitochondrial membrane potential (MMP) and intracellular free Ca(2+) concentration ([Ca(2+)]i) were also respectively changed followed the GD stress tested by CCK8 and flow cytometry. After knocking down of Cav-1 in the cells by siRNA, the level of [Ca(2+)]i was increased, and MMP was reduced further in GD-treated PC12 cells. Knockdown of Cav-1 or methylated-β-Cyclodextrin (M-β-CD) treatment inhibited the expression of GLUT4 protein upon GD. Additionally, we found that GLUT4 could translocate from cytoplasm to cell membrane upon GD. These findings might suggest a neuroprotective role for Cav-1, through coordination of GLUT4 in GD.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Raza, Haider; John, Annie

Tea polyphenols have been reported to be potent antioxidants and beneficial in oxidative stress related diseases. Prooxidant effects of tea polyphenols have also been reported in cell culture systems. In the present study, we have studied oxidative stress in the subcellular compartments of PC12 cells after treatment with different concentrations of the green tea polyphenol, epigallocatechin-3-gallate (EGCG). We have demonstrated that EGCG has differentially affected the production of reactive oxygen species (ROS), glutathione (GSH) metabolism and cytochrome P450 2E1 activity in the different subcellular compartments in PC12 cells. Our results have shown that although the cell survival was not inhibitedmore » by EGCG, there was, however, an increased DNA breakdown and activation of apoptotic markers, caspase 3 and poly- (ADP-ribose) polymerase (PARP) at higher concentrations of EGCG treatment. Our results suggest that the differential effects of EGCG might be related to the alterations in oxidative stress, GSH pools and CYP2E1 activity in different cellular compartments. These results may have implications in determining the chemopreventive therapeutic use of tea polyphenols in vivo.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kato, Haruo, E-mail: hal.kato@gunma-u.ac.jp; Sekine, Yoshitaka; Furuya, Yosuke

Metformin is a biguanide drug that is widely used for the treatment of type 2 diabetes. Recent studies have shown that metformin inhibits cancer cell proliferation and tumor growth both in vitro and in vivo. The anti-tumor mechanisms of metformin include activation of the AMP-activated protein kinase/mTOR pathway and direct inhibition of insulin/insulin-like growth factor (IGF)-mediated cellular proliferation. However, the anti-tumor mechanism in prostate cancer remains unclear. Because activation of the IGF-1 receptor (IGF-1R) is required for prostate cell proliferation, IGF-1R inhibitors may be of therapeutic value. Accordingly, we examined the effects of metformin on IGF-1R signaling in prostate cancer cells. Metforminmore » significantly inhibited PC-3 cell proliferation, migration, and invasion. IGF-1R mRNA expression decreased significantly after 48 h of treatment, and IGF-1R protein expression decreased in a similar manner. IGF-1R knockdown by siRNA transfection led to inhibited proliferation, migration and invasion of PC-3 cells. IGF-1 activated both ERK1/2 and Akt, but these effects were attenuated by metformin treatment. In addition, intraperitoneal treatment with metformin significantly reduced tumor growth and IGF-1R mRNA expression in PC-3 xenografts. Our results suggest that metformin is a potent inhibitor of the IGF-1/IGF-1R system and may be beneficial in prostate cancer treatment. - Highlights: • Metformin inhibited PC-3 cell proliferation, migration, and invasion. • Metformin decreased IGF-1R mRNA and protein expressions in PC-3 cells. • Metformin inhibited IGF-1 induced ERK and Akt phosphorylations in PC-3 cells. • Metformin treatment inhibited PC-3 cell growth and IGF-1R expression in vivo. • Metformin may be a potent inhibitor of the IGF-1/IGF-1R signaling.« less

Transfer of arachidonate from phosphatidylcholine to phosphatidylethanolamine and triacylglycerol in guinea pig alveolar macrophages

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nijssen, J.G.; Oosting, R.S.; Nkamp, F.Pv.

1986-10-01

Guinea pig alveolar macrophages were labeled by incubation with either arachidonate or linoleate. Arachidonate labeled phosphatidylcholine (PC), phosphatidylethanolamine (PE) and triglycerides (TG) equally well, with each lipid containing about 30% of total cellular radioactivity. In comparison to arachidonate, linoleate was recovered significantly less in PE (7%) and more in TG (47%). To investigate whether redistributions of acyl chains among lipid classes took place, the macrophages were incubated with 1-acyl-2-(1-/sup 14/C)arachidonoyl PC or 1-acyl-2-(1-/sup 14/C)linoleoyl PC. After harvesting, the cells incubated with 1-acyl-2-(1-/sup 14/C)linoleoyl PC contained 86% of the recovered cellular radioactivity in PC, with only small amounts of label beingmore » transferred to PE and TG (3 and 6%, respectively). More extensive redistributions were observed with arachidonate-labeled PC. In this case, only 60% of cellular radioactivity was still associated with PC, while 22 and 12%, respectively, had been transferred to PE and TG. Arachidonate transfer from PC to PE was unaffected by an excess of free arachidonate which inhibited this transfer to TG for over 90%, indicating that different mechanisms or arachidonoyl CoA pools were involved in the transfer of arachidonate from PC to PE and TG. Cells prelabeled with 1-acyl-2-(1-/sup 14/C)arachidonoyl PC released /sup 14/C-label into the medium upon further incubation. This release was slightly stimulated by zymosan and threefold higher in the presence of the Ca2+-ionophore A23187. Labeling of macrophages with intact phospholipid molecules appears to be a suitable method for studying acyl chain redistribution and release reactions.« less

The prion-ZIP connection: From cousins to partners in iron uptake

PubMed Central

Singh, Neena; Asthana, Abhishek; Baksi, Shounak; Desai, Vilok; Haldar, Swati; Hari, Sahi; Tripathi, Ajai K

2015-01-01

ABSTRACT Converging observations from disparate lines of inquiry are beginning to clarify the cause of brain iron dyshomeostasis in sporadic Creutzfeldt-Jakob disease (sCJD), a neurodegenerative condition associated with the conversion of prion protein (PrPC), a plasma membrane glycoprotein, from α-helical to a β-sheet rich PrP-scrapie (PrPSc) isoform. Biochemical evidence indicates that PrPC facilitates cellular iron uptake by functioning as a membrane-bound ferrireductase (FR), an activity necessary for the transport of iron across biological membranes through metal transporters. An entirely different experimental approach reveals an evolutionary link between PrPC and the Zrt, Irt-like protein (ZIP) family, a group of proteins involved in the transport of zinc, iron, and manganese across the plasma membrane. Close physical proximity of PrPC with certain members of the ZIP family on the plasma membrane and increased uptake of extracellular iron by cells that co-express PrPC and ZIP14 suggest that PrPC functions as a FR partner for certain members of this family. The connection between PrPC and ZIP proteins therefore extends beyond common ancestry to that of functional cooperation. Here, we summarize evidence supporting the facilitative role of PrPC in cellular iron uptake, and implications of this activity on iron metabolism in sCJD brains. PMID:26689487

The extraction of simple relationships in growth factor-specific multiple-input and multiple-output systems in cell-fate decisions by backward elimination PLS regression.

PubMed

Akimoto, Yuki; Yugi, Katsuyuki; Uda, Shinsuke; Kudo, Takamasa; Komori, Yasunori; Kubota, Hiroyuki; Kuroda, Shinya

2013-01-01

Cells use common signaling molecules for the selective control of downstream gene expression and cell-fate decisions. The relationship between signaling molecules and downstream gene expression and cellular phenotypes is a multiple-input and multiple-output (MIMO) system and is difficult to understand due to its complexity. For example, it has been reported that, in PC12 cells, different types of growth factors activate MAP kinases (MAPKs) including ERK, JNK, and p38, and CREB, for selective protein expression of immediate early genes (IEGs) such as c-FOS, c-JUN, EGR1, JUNB, and FOSB, leading to cell differentiation, proliferation and cell death; however, how multiple-inputs such as MAPKs and CREB regulate multiple-outputs such as expression of the IEGs and cellular phenotypes remains unclear. To address this issue, we employed a statistical method called partial least squares (PLS) regression, which involves a reduction of the dimensionality of the inputs and outputs into latent variables and a linear regression between these latent variables. We measured 1,200 data points for MAPKs and CREB as the inputs and 1,900 data points for IEGs and cellular phenotypes as the outputs, and we constructed the PLS model from these data. The PLS model highlighted the complexity of the MIMO system and growth factor-specific input-output relationships of cell-fate decisions in PC12 cells. Furthermore, to reduce the complexity, we applied a backward elimination method to the PLS regression, in which 60 input variables were reduced to 5 variables, including the phosphorylation of ERK at 10 min, CREB at 5 min and 60 min, AKT at 5 min and JNK at 30 min. The simple PLS model with only 5 input variables demonstrated a predictive ability comparable to that of the full PLS model. The 5 input variables effectively extracted the growth factor-specific simple relationships within the MIMO system in cell-fate decisions in PC12 cells.

Bioassay-Guided Isolation of Neuroprotective Compounds from Uncaria rhynchophylla against Beta-Amyloid-Induced Neurotoxicity

PubMed Central

Xian, Yan-Fang; Lin, Zhi-Xiu; Mao, Qing-Qiu; Hu, Zhen; Zhao, Ming; Che, Chun-Tao; Ip, Siu-Po

2012-01-01

Uncaria rhynchophylla is a component herb of many Chinese herbal formulae for the treatment of neurodegenerative diseases. Previous study in our laboratory has demonstrated that an ethanol extract of Uncaria rhynchophylla ameliorated cognitive deficits in a mouse model of Alzheimer's disease induced by D-galactose. However, the active ingredients of Uncaria rhynchophylla responsible for the anti-Alzheimer's disease activity have not been identified. This study aims to identify the active ingredients of Uncaria rhynchophylla by a bioassay-guided fractionation approach and explore the acting mechanism of these active ingredients by using a well-established cellular model of Alzheimer's disease, beta-amyloid- (Aβ-) induced neurotoxicity in PC12 cells. The results showed that six alkaloids, namely, corynoxine, corynoxine B, corynoxeine, isorhynchophylline, isocorynoxeine, and rhynchophylline were isolated from the extract of Uncaria rhynchophylla. Among them, rhynchophylline and isorhynchophylline significantly decreased Aβ-induced cell death, intracellular calcium overloading, and tau protein hyperphosphorylation in PC12 cells. These results suggest that rhynchophylline and isorhynchophylline are the major active ingredients responsible for the protective action of Uncaria rhynchophylla against Aβ-induced neuronal toxicity, and their neuroprotective effect may be mediated, at least in part, by inhibiting intracellular calcium overloading and tau protein hyperphosphorylation. PMID:22778778

Bioassay-Guided Isolation of Neuroprotective Compounds from Uncaria rhynchophylla against Beta-Amyloid-Induced Neurotoxicity.

PubMed

Xian, Yan-Fang; Lin, Zhi-Xiu; Mao, Qing-Qiu; Hu, Zhen; Zhao, Ming; Che, Chun-Tao; Ip, Siu-Po

2012-01-01

Uncaria rhynchophylla is a component herb of many Chinese herbal formulae for the treatment of neurodegenerative diseases. Previous study in our laboratory has demonstrated that an ethanol extract of Uncaria rhynchophylla ameliorated cognitive deficits in a mouse model of Alzheimer's disease induced by D-galactose. However, the active ingredients of Uncaria rhynchophylla responsible for the anti-Alzheimer's disease activity have not been identified. This study aims to identify the active ingredients of Uncaria rhynchophylla by a bioassay-guided fractionation approach and explore the acting mechanism of these active ingredients by using a well-established cellular model of Alzheimer's disease, beta-amyloid- (Aβ-) induced neurotoxicity in PC12 cells. The results showed that six alkaloids, namely, corynoxine, corynoxine B, corynoxeine, isorhynchophylline, isocorynoxeine, and rhynchophylline were isolated from the extract of Uncaria rhynchophylla. Among them, rhynchophylline and isorhynchophylline significantly decreased Aβ-induced cell death, intracellular calcium overloading, and tau protein hyperphosphorylation in PC12 cells. These results suggest that rhynchophylline and isorhynchophylline are the major active ingredients responsible for the protective action of Uncaria rhynchophylla against Aβ-induced neuronal toxicity, and their neuroprotective effect may be mediated, at least in part, by inhibiting intracellular calcium overloading and tau protein hyperphosphorylation.

Endothelial microparticles released by activated protein C protect beta cells through EPCR/PAR1 and annexin A1/FPR2 pathways in islets.

PubMed

Kreutter, Guillaume; Kassem, Mohamad; El Habhab, Ali; Baltzinger, Philippe; Abbas, Malak; Boisrame-Helms, Julie; Amoura, Lamia; Peluso, Jean; Yver, Blandine; Fatiha, Zobairi; Ubeaud-Sequier, Geneviève; Kessler, Laurence; Toti, Florence

2017-11-01

Islet transplantation is associated with early ischaemia/reperfusion, localized coagulation and redox-sensitive endothelial dysfunction. In animal models, islet cytoprotection by activated protein C (aPC) restores islet vascularization and protects graft function, suggesting that aPC triggers various lineages. aPC also prompts the release of endothelial MP that bear EPCR, its specific receptor. Microparticles (MP) are plasma membrane procoagulant vesicles, surrogate markers of stress and cellular effectors. We measured the cytoprotective effects of aPC on endothelial and insulin-secreting Rin-m5f β-cells and its role in autocrine and paracrine MP-mediated cell crosstalk under conditions of oxidative stress. MP from aPC-treated primary endothelial (EC) or β-cells were applied to H 2 O 2 -treated Rin-m5f. aPC activity was measured by enzymatic assay and ROS species by dihydroethidium. The capture of PKH26-stained MP and the expression of EPCR were probed by fluorescence microscopy and apoptosis by flow cytometry. aPC treatment enhanced both annexin A1 (ANXA1) and PAR-1 expression in EC and to a lesser extent in β-cells. MP from aPC-treated EC (eM aPC ) exhibited high EPCR and annexin A1 content, protected β-cells, restored insulin secretion and were captured by 80% of β cells in a phosphatidylserine and ANXA1-dependent mechanism. eMP activated EPCR/PAR-1 and ANXA1/FPR2-dependent pathways and up-regulated the expression of EPCR, and of FPR2/ALX, the ANXA1 receptor. Cytoprotection was confirmed in H 2 O 2 -treated rat islets with increased viability (62% versus 48% H 2 O 2 ), reduced apoptosis and preserved insulin secretion in response to glucose elevation (16 versus 5 ng/ml insulin per 10 islets). MP may prove a promising therapeutic tool in the protection of transplanted islets. © 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

The Human Polycomb Group Complex Associates with Pericentromeric Heterochromatin to Form a Novel Nuclear Domain

PubMed Central

Saurin, Andrew J.; Shiels, Carol; Williamson, Jill; Satijn, David P.E.; Otte, Arie P.; Sheer, Denise; Freemont, Paul S.

1998-01-01

The Polycomb group (PcG) complex is a chromatin-associated multiprotein complex, involved in the stable repression of homeotic gene activity in Drosophila. Recently, a mammalian PcG complex has been identified with several PcG proteins implicated in the regulation of Hox gene expression. Although the mammalian PcG complex appears analogous to the complex in Drosophila, the molecular mechanisms and functions for the mammalian PcG complex remain unknown. Here we describe a detailed characterization of the human PcG complex in terms of cellular localization and chromosomal association. By using antibodies that specifically recognize three human PcG proteins— RING1, BMI1, and hPc2—we demonstrate in a number of human cell lines that the PcG complex forms a unique discrete nuclear structure that we term PcG bodies. PcG bodies are prominent novel nuclear structures with the larger PcG foci generally localized near the centromeres, as visualized with a kinetochore antibody marker. In both normal fetal and adult fibroblasts, PcG bodies are not randomly dispersed, but appear clustered into defined areas within the nucleus. We show in three different human cell lines that the PcG complex can tightly associate with large pericentromeric heterochromatin regions (1q12) on chromosome 1, and with related pericentromeric sequences on different chromosomes, providing evidence for a mammalian PcG–heterochromatin association. Furthermore, these heterochromatin-bound PcG complexes remain stably associated throughout mitosis, thereby allowing the potential inheritance of the PcG complex through successive cell divisions. We discuss these results in terms of the known function of the PcG complex as a transcriptional repression complex. PMID:9722603

Phosphatidylserine metabolism modification precedes manganese-induced apoptosis and phosphatidylserine exposure in PC12 cells.

PubMed

Ferrara, G; Gambelunghe, A; Mozzi, R; Marchetti, M C; Migliorati, G; Muzi, G; Buratta, S

2013-12-01

Long-term exposure to high manganese (Mn) levels can lead to Parkinson-like neurological disorders. Molecular mechanisms underlying Mn cytotoxicity have been not defined. It is known that Mn induces apoptosis in PC12 cells and that this involves the activation of some signal transduction pathways. Although the role of phospholipids in apoptosis and signal transduction is well-known, the membrane phospholipid component in Mn-related damage has not yet been investigated. Phosphatidylserine (PS) facilitates protein translocation from cytosol to plasma membrane and PS exposure on the cell surface allows macrophage recognition of apoptotic cells. This study investigates the effects of MnCl2 on PS metabolism in PC12 cells, relating them to those on cell apoptosis. Apoptosis induction decreased PS radioactivity of PC12 cells incubated with radioactive serine. MnCl2 reduced PS radioactivity even under conditions that did not affect cell viability or PS exposure, suggesting that the effects on PS metabolism may represent an early event in cell apoptosis. Thus the latter conditions that also induced a greater PS decarboxylation were utilized for further investigating on the effects on PS synthesis, by measuring the activity and expression of PS-synthesizing enzymes, in cell lysates and in total cellular membranes (TM). Compared with corresponding controls, enzyme activity of MnCl2-treated cells was lower in cell lysates and greater in TM. Evaluating the expression of two isoforms of PS-synthesizing enzyme (PSS), PSSII was increased both in cell lysate and TM, while PSSI was unchanged. MnCl2 addition to control cell lysate reduced enzyme activity. These results suggest Mn plays a dual role on PS synthesis. Once inside the cell, Mn inhibits the enzyme/s, thus accounting for reduced PS synthesis in lysates and intact cells. On the other hand, it increases PSSII expression in cell membranes. The possibility that this occurs to counteract the direct effects of Mn ions on enzyme activity cannot be excluded. The effects on membrane enzyme activity and expression may also participate to PS exposure, observed at longer periods of treatment, by increasing membrane PS content. Copyright © 2013 Elsevier Inc. All rights reserved.

Proteomic characterization of an isolated fraction of synthetic proteasome inhibitor (PSI)-induced inclusions in PC12 cells might offer clues to aggresomes as a cellular defensive response against proteasome inhibition by PSI

PubMed Central

2010-01-01

Background Cooperation of constituents of the ubiquitin proteasome system (UPS) with chaperone proteins in degrading proteins mediate a wide range of cellular processes, such as synaptic function and neurotransmission, gene transcription, protein trafficking, mitochondrial function and metabolism, antioxidant defence mechanisms, and apoptotic signal transduction. It is supposed that constituents of the UPS and chaperone proteins are recruited into aggresomes where aberrant and potentially cytotoxic proteins may be sequestered in an inactive form. Results To determinate the proteomic pattern of synthetic proteasome inhibitor (PSI)-induced inclusions in PC12 cells after proteasome inhibition by PSI, we analyzed a fraction of PSI-induced inclusions. A proteomic feature of the isolated fraction was characterized by identification of fifty six proteins including twenty previously reported protein components of Lewy bodies, twenty eight newly identified proteins and eight unknown proteins. These proteins, most of which were recognized as a profile of proteins within cellular processes mediated by the UPS, a profile of constituents of the UPS and a profile of chaperone proteins, are classed into at least nine accepted categories. In addition, prolyl-4-hydroxylase beta polypeptide, an endoplasmic reticulum member of the protein disulfide isomerase family, was validated in the developmental process of PSI-induced inclusions in the cells. Conclusions It is speculated that proteomic characterization of an isolated fraction of PSI-induced inclusions in PC12 cells might offer clues to appearance of aggresomes serving as a cellular defensive response against proteasome inhibition. PMID:20704702

Monocrotophos Induced Apoptosis in PC12 Cells: Role of Xenobiotic Metabolizing Cytochrome P450s

PubMed Central

Kashyap, Mahendra Pratap; Singh, Abhishek Kumar; Kumar, Vivek; Tripathi, Vinay Kumar; Srivastava, Ritesh Kumar; Agrawal, Megha; Khanna, Vinay Kumar; Yadav, Sanjay; Jain, Swatantra Kumar; Pant, Aditya Bhushan

2011-01-01

Monocrotophos (MCP) is a widely used organophosphate (OP) pesticide. We studied apoptotic changes and their correlation with expression of selected cytochrome P450s (CYPs) in PC12 cells exposed to MCP. A significant induction in reactive oxygen species (ROS) and decrease in glutathione (GSH) levels were observed in cells exposed to MCP. Following the exposure of PC12 cells to MCP (10−5 M), the levels of protein and mRNA expressions of caspase-3/9, Bax, Bcl2, P53, P21, GSTP1-1 were significantly upregulated, whereas the levels of Bclw, Mcl1 were downregulated. A significant induction in the expression of CYP1A1/1A2, 2B1/2B2, 2E1 was also observed in PC12 cells exposed to MCP (10−5 M), whereas induction of CYPs was insignificant in cells exposed to 10−6 M concentration of MCP. We believe that this is the first report showing altered expressions of selected CYPs in MCP-induced apoptosis in PC12 cells. These apoptotic changes were mitochondria mediated and regulated by caspase cascade. Our data confirm the involvement of specific CYPs in MCP-induced apoptosis in PC12 cells and also identifies possible cellular and molecular mechanisms of organophosphate pesticide-induced apoptosis in neuronal cells. PMID:21445290

Sampling-based Bayesian approaches reveal the importance of quasi-bistable behavior in cellular decision processes on the example of the MAPK signaling pathway in PC-12 cell lines.

PubMed

Jensch, Antje; Thomaseth, Caterina; Radde, Nicole E

2017-01-25

Positive and negative feedback loops are ubiquitous motifs in biochemical signaling pathways. The mitogen-activated protein kinase (MAPK) pathway module is part of many distinct signaling networks and comprises several of these motifs, whose functioning depends on the cell line at hand and on the particular context. The maintainance of specificity of the response of the MAPK module to distinct stimuli has become a key paradigm especially in PC-12 cells, where the same module leads to different cell fates, depending on the stimulating growth factor. This cell fate is regulated by differences in the ERK (MAPK) activation profile, which shows a transient response upon stimulation with EGF, while the response is sustained in case of NGF. This behavior was explained by different effective network topologies. It is widely believed that this sustained response requires a bistable system. In this study we present a sampling-based Bayesian model analysis on a dataset, in which PC-12 cells have been stimulated with different growth factors. This is combined with novel analysis methods to investigate the role of feedback interconnections to shape ERK response. Results strongly suggest that, besides bistability, an additional effect called quasi-bistability can contribute to explain the observed responses of the system to different stimuli. Quasi-bistability is the ability of a monostable system to maintain two distinct states over a long time period upon a transient signal, which is also related to positive feedback, but cannot be detected by standard steady state analysis methods. Although applied on a specific example, our framework is generic enough to be also relevant for other regulatory network modeling studies that comprise positive feedback to explain cellular decision making processes. Overall, this study advices to focus not only on steady states, but also to take transient behavior into account in the analysis.

Polycomb Group (PcG) Proteins and Human Cancers: Multifaceted Functions and Therapeutic Implications

PubMed Central

Wang, Wei; Qin, Jiang-Jiang; Voruganti, Sukesh; Nag, Subhasree; Zhou, Jianwei; Zhang, Ruiwen

2016-01-01

Polycomb group (PcG) proteins are transcriptional repressors that regulate several crucial developmental and physiological processes in the cell. More recently, they have been found to play important roles in human carcinogenesis and cancer development and progression. The deregulation and dysfunction of PcG proteins often lead to blocking or inappropriate activation of developmental pathways, enhancing cellular proliferation, inhibiting apoptosis, and increasing the cancer stem cell population. Genetic and molecular investigations of PcG proteins have long been focused on their PcG functions. However, PcG proteins have recently been shown to exert non-polycomb functions, contributing to the regulation of diverse cellular functions. We and others have demonstrated that PcG proteins regulate the expression and function of several oncogenes and tumor suppressor genes in a PcG-independent manner, and PcG proteins are associated with the survival of patients with cancer. In this review, we summarize the recent advances in the research on PcG proteins, including both the polycomb-repressive and non-polycomb functions. We specifically focus on the mechanisms by which PcG proteins play roles in cancer initiation, development, and progression. Finally, we discuss the potential value of PcG proteins as molecular biomarkers for the diagnosis and prognosis of cancer, and as molecular targets for cancer therapy. PMID:26227500

Polycomb Group (PcG) Proteins and Human Cancers: Multifaceted Functions and Therapeutic Implications.

PubMed

Wang, Wei; Qin, Jiang-Jiang; Voruganti, Sukesh; Nag, Subhasree; Zhou, Jianwei; Zhang, Ruiwen

2015-11-01

Polycomb group (PcG) proteins are transcriptional repressors that regulate several crucial developmental and physiological processes in the cell. More recently, they have been found to play important roles in human carcinogenesis and cancer development and progression. The deregulation and dysfunction of PcG proteins often lead to blocking or inappropriate activation of developmental pathways, enhancing cellular proliferation, inhibiting apoptosis, and increasing the cancer stem cell population. Genetic and molecular investigations of PcG proteins have long been focused on their PcG functions. However, PcG proteins have recently been shown to exert non-classical-Pc-functions, contributing to the regulation of diverse cellular functions. We and others have demonstrated that PcG proteins regulate the expression and function of several oncogenes and tumor suppressor genes in a PcG-independent manner, and PcG proteins are associated with the survival of patients with cancer. In this review, we summarize the recent advances in the research on PcG proteins, including both the Pc-repressive and non-classical-Pc-functions. We specifically focus on the mechanisms by which PcG proteins play roles in cancer initiation, development, and progression. Finally, we discuss the potential value of PcG proteins as molecular biomarkers for the diagnosis and prognosis of cancer, and as molecular targets for cancer therapy. © 2015 Wiley Periodicals, Inc.

Pathogenic prions deviate PrPC signaling in neuronal cells and impair A-beta clearance

PubMed Central

Pradines, E; Hernandez-Rapp, J; Villa-Diaz, A; Dakowski, C; Ardila-Osorio, H; Haik, S; Schneider, B; Launay, J-M; Kellermann, O; Torres, J-M; Mouillet-Richard, S

2013-01-01

The subversion of the normal function exerted by the cellular prion protein (PrPC) in neurons by pathogenic prions is assumed to have a central role in the pathogenesis of transmissible spongiform encephalopathies. Using two murine models of prion infection, the 1C11 neuronal cell line and neurospheres, we document that prion infection is associated with the constitutive activation of signaling targets normally coupled with PrPC, including the Fyn kinase, the mitogen-associated protein kinases ERK1/2 and the CREB transcription factor. PrPC-dependent signaling overactivation in infected cells is associated with the recruitment of p38 and JNK stress-associated kinases. Downstream from CREB, prion-infected cells exhibit reduced activity of the matrix metalloprotease (MMP)-9. As MMP-9 catalyzes the degradation of the amyloid A-beta peptide, the decrease in MMP-9 activity in prion-infected cells causes a significant impairment of the clearance of A-beta, leading to its accumulation. By exploiting two 1C11-infected clones accumulating high or moderate levels of prions, we show that the prion-induced changes are correlated with the level of infectivity. Of note, a dose-dependent increase in A-beta levels was also found in the cerebrospinal fluid of mice inoculated with these infected clones. By demonstrating that pathogenic prions trigger increases in A-beta levels through the deviation of PrPC signaling, our data argue that A-beta may exacerbate prion-induced toxicity. PMID:23303130

Metabolic Regulation of Invadopodia and Invasion by Acetyl-CoA Carboxylase 1 and De novo Lipogenesis

PubMed Central

Scott, Kristen E. N.; Wheeler, Frances B.; Davis, Amanda L.; Thomas, Michael J.; Ntambi, James M.; Seals, Darren F.; Kridel, Steven J.

2012-01-01

Invadopodia are membrane protrusions that facilitate matrix degradation and cellular invasion. Although lipids have been implicated in several aspects of invadopodia formation, the contributions of de novo fatty acid synthesis and lipogenesis have not been defined. Inhibition of acetyl-CoA carboxylase 1 (ACC1), the committed step of fatty acid synthesis, reduced invadopodia formation in Src-transformed 3T3 (3T3-Src) cells, and also decreased the ability to degrade gelatin. Inhibition of fatty acid synthesis through AMP-activated kinase (AMPK) activation and ACC phosphorylation also decreased invadopodia incidence. The addition of exogenous 16∶0 and 18∶1 fatty acid, products of de novo fatty acid synthesis, restored invadopodia and gelatin degradation to cells with decreased ACC1 activity. Pharmacological inhibition of ACC also altered the phospholipid profile of 3T3-Src cells, with the majority of changes occurring in the phosphatidylcholine (PC) species. Exogenous supplementation with the most abundant PC species, 34∶1 PC, restored invadopodia incidence, the ability to degrade gelatin and the ability to invade through matrigel to cells deficient in ACC1 activity. On the other hand, 30∶0 PC did not restore invadopodia and 36∶2 PC only restored invadopodia incidence and gelatin degradation, but not cellular invasion through matrigel. Pharmacological inhibition of ACC also reduced the ability of MDA-MB-231 breast, Snb19 glioblastoma, and PC-3 prostate cancer cells to invade through matrigel. Invasion of PC-3 cells through matrigel was also restored by 34∶1 PC supplementation. Collectively, the data elucidate the novel metabolic regulation of invadopodia and the invasive process by de novo fatty acid synthesis and lipogenesis. PMID:22238651

Neuroprotective effect of novel cognitive enhancer noopept on AD-related cellular model involves the attenuation of apoptosis and tau hyperphosphorylation.

PubMed

Ostrovskaya, Rita U; Vakhitova, Yulia V; Kuzmina, Uliyana Sh; Salimgareeva, Milyausha Kh; Zainullina, Liana F; Gudasheva, Tatiana A; Vakhitov, Vener A; Seredenin, Sergey B

2014-08-06

Noopept (N-phenyl-acetyl-L-prolylglycine ethyl ester) was constructed as a dipeptide analog of the standard cognition enhancer, piracetam. Our previous experiments have demonstrated the cognition restoring effect of noopept in several animal models of Alzheimer disease (AD). Noopept was also shown to prevent ionic disbalance, excitotoxicity, free radicals and pro-inflammatory cytokines accumulation, and neurotrophine deficit typical for different kinds of brain damages, including AD. In this study, we investigated the neuroprotective action of noopept on cellular model of AD, Aβ 25-35-induced toxicity in PC12 cells and revealed the underlying mechanisms. The neuroprotective effect of noopept (added to the medium at 10 μM concentration, 72 hours before Аβ 25-35) was studied on Аβ 25-35-induced injury (5 μM for 24 h) in PC12 cells. The ability of drug to protect the impairments of cell viability, calcium homeostasis, ROS level, mitochondrial function, tau phosphorylation and neurite outgrowth caused by Аβ 25-35 were evaluated. Following the exposure of PC12 cells to Аβ 25-35 an increase of the level of ROS, intracellular calcium, and tau phosphorylation at Ser396 were observed; these changes were accompanied by a decrease in cell viability and an increase of apoptosis. Noopept treatment before the amyloid-beta exposure improved PC12 cells viability, reduced the number of early and late apoptotic cells, the levels of intracellular reactive oxygen species and calcium and enhanced the mitochondrial membrane potential. In addition, pretreatment of PC12 cell with noopept significantly attenuated tau hyperphosphorylation at Ser396 and ameliorated the alterations of neurite outgrowth evoked by Аβ25-35. Taken together, these data provide evidence that novel cognitive enhancer noopept protects PC12 cell against deleterious actions of Aβ through inhibiting the oxidative damage and calcium overload as well as suppressing the mitochondrial apoptotic pathway. Moreover, neuroprotective properties of noopept likely include its ability to decrease tau phosphorylation and to restore the altered morphology of PC12 cells. Therefore, this nootropic dipeptide is able to positively affect not only common pathogenic pathways but also disease-specific mechanisms underlying Aβ-related pathology.

Neuroprotective effect of novel cognitive enhancer noopept on AD-related cellular model involves the attenuation of apoptosis and tau hyperphosphorylation

PubMed Central

2014-01-01

Background Noopept (N-phenyl-acetyl-L-prolylglycine ethyl ester) was constructed as a dipeptide analog of the standard cognition enhancer, piracetam. Our previous experiments have demonstrated the cognition restoring effect of noopept in several animal models of Alzheimer disease (AD). Noopept was also shown to prevent ionic disbalance, excitotoxicity, free radicals and pro-inflammatory cytokines accumulation, and neurotrophine deficit typical for different kinds of brain damages, including AD. In this study, we investigated the neuroprotective action of noopept on cellular model of AD, Aβ25–35-induced toxicity in PC12 cells and revealed the underlying mechanisms. Results The neuroprotective effect of noopept (added to the medium at 10 μM concentration, 72 hours before Аβ25–35) was studied on Аβ25–35-induced injury (5 μM for 24 h) in PC12 cells. The ability of drug to protect the impairments of cell viability, calcium homeostasis, ROS level, mitochondrial function, tau phosphorylation and neurite outgrowth caused by Аβ25–35 were evaluated. Following the exposure of PC12 cells to Аβ25–35 an increase of the level of ROS, intracellular calcium, and tau phosphorylation at Ser396 were observed; these changes were accompanied by a decrease in cell viability and an increase of apoptosis. Noopept treatment before the amyloid-beta exposure improved PC12 cells viability, reduced the number of early and late apoptotic cells, the levels of intracellular reactive oxygen species and calcium and enhanced the mitochondrial membrane potential. In addition, pretreatment of PC12 cell with noopept significantly attenuated tau hyperphosphorylation at Ser396 and ameliorated the alterations of neurite outgrowth evoked by Аβ25–35. Conclusions Taken together, these data provide evidence that novel cognitive enhancer noopept protects PC12 cell against deleterious actions of Aβ through inhibiting the oxidative damage and calcium overload as well as suppressing the mitochondrial apoptotic pathway. Moreover, neuroprotective properties of noopept likely include its ability to decrease tau phosphorylation and to restore the altered morphology of PC12 cells. Therefore, this nootropic dipeptide is able to positively affect not only common pathogenic pathways but also disease-specific mechanisms underlying Aβ-related pathology. PMID:25096780

Humanin Derivatives Inhibit Necrotic Cell Death in Neurons

PubMed Central

Cohen, Aviv; Lerner-Yardeni, Jenny; Meridor, David; Kasher, Roni; Nathan, Ilana; Parola, Abraham H

2015-01-01

Humanin and its derivatives are peptides known for their protective antiapoptotic effects against Alzheimer’s disease. Herein, we identify a novel function of the humanin-derivative AGA(C8R)-HNG17 (namely, protection against cellular necrosis). Necrosis is one of the main modes of cell death, which was until recently considered an unmoderated process. However, recent findings suggest the opposite. We have found that AGA(C8R)-HNG17 confers protection against necrosis in the neuronal cell lines PC-12 and NSC-34, where necrosis is induced in a glucose-free medium by either chemohypoxia or by a shift from apoptosis to necrosis. Our studies in traumatic brain injury models in mice, where necrosis is the main mode of neuronal cell death, have shown that AGA(C8R)-HNG17 has a protective effect. This result is demonstrated by a decrease in a neuronal severity score and by a reduction in brain edema, as measured by magnetic resonance imaging (MRI). An insight into the peptide’s antinecrotic mechanism was attained through measurements of cellular ATP levels in PC-12 cells under necrotic conditions, showing that the peptide mitigates a necrosis-associated decrease in ATP levels. Further, we demonstrate the peptide’s direct enhancement of the activity of ATP synthase activity, isolated from rat-liver mitochondria, suggesting that AGA(C8R)-HNG17 targets the mitochondria and regulates cellular ATP levels. Thus, AGA(C8R)-HNG17 has potential use for the development of drug therapies for necrosis-related diseases, for example, traumatic brain injury, stroke, myocardial infarction, and other conditions for which no efficient drug-based treatment is currently available. Finally, this study provides new insight into the mechanisms underlying the antinecrotic mode of action of AGA(C8R)-HNG17. PMID:26062019

The effects of IL-1β, IL-8, G-CSF and TNF-α as molecular adjuvant on the immune response to an E. tarda subunit vaccine in flounder (Paralichthys olivaceus).

PubMed

Guo, Ming; Tang, Xiaoqian; Sheng, Xiuzhen; Xing, Jing; Zhan, Wenbin

2018-06-01

Cytokines play vital roles in mounting immune responses and activating host defense network. In this study, the expression plasmid pcDNA3.1 (pcN3) encoding four flounder (Paralichthys olivaceus) cytokines including IL-1β, TNF-α, IL-8 or G-CSF (pcIL-1β, pcTNF-α, pcIL-8 and pcG-CSF) were successfully constructed, and their adjuvant potential on an Edwardsiella tarda (E. tarda) subunit vaccine OmpV (rOmpV) were comparatively analyzed in vaccinated flounder model. Results revealed that flounder vaccinated with rOmpV plus pcIL-1β, pcIL-8 or pcG-CSF produced the relative percent survivals (RPS) of 71%, 65% and 49% respectively, which were higher than that in flounder vaccinated with rOmpV plus pcTNF-α (39%) or pcN3 (36%, the control group). Immunological analysis showed that: (1) except pcTNF-α, higher levels of anti-E. tarda serum antibodies and sIg + lymphocytes in spleen, head kidney and peripheral blood were significantly enhanced by pcIL-1β, pcIL-8 or pcG-CSF, however, pcIL-8 and pcIL-1β enhanced higher levels of sIg + lymphocytes and anti-E. tarda antibodies than pcG-CSF; (2) pcTNF-α could promote the up-regulation of genes participated in cellular immunity (MHCIα, IFN-γ, CD8α and CD8β), pcIL-1β could enhance the expression of genes related to humoral immunity (CD4-1, CD4-2, MHCIIα and IgM), and all the detected genes were augmented by pcIL-8 and pcG-CSF; Among the four cytokines, pcIL-8 and pcIL-1β could strengthen the highest levels of genes participated in cellular immunity and humoral immunity, respectively. These results demonstrated that pcIL-8 and pcIL-1β could enhance stronger cellular and/or humoral immunity induced by rOmpV than pcG-CSF and pcTNF-α, and evoked higher RPS against E. tarda challenge in flounder, which indicated that pcIL-8 and pcIL-1β are promising adjuvants of vaccines in controlling E. tarda infection. Copyright © 2018 Elsevier Ltd. All rights reserved.

Cerebrosides from Sea Cucumber Protect Against Oxidative Stress in SAMP8 Mice and PC12 Cells.

PubMed

Che, Hongxia; Du, Lei; Cong, Peixu; Tao, Suyuan; Ding, Ning; Wu, Fengjuan; Xue, Changhu; Xu, Jie; Wang, Yuming

2017-04-01

Alzheimer's disease (AD) is a neurodegenerative disorder. Emerging evidence implicates β-amyloid (Aβ) plays a critical role in the progression of AD. In this study, we investigated the protective effect of cerebrosides obtained from sea cucumber against senescence-accelerated mouse prone 8 (SAMP8) mice in vivo. We also studied the effect of cerebrosides on Aβ-induced cytotoxicity on the rat pheochromocytoma cell (PC12) and the underlying molecular mechanisms. Cerebrosides ameliorated learning and memory deficits and the Aβ accumulation in demented mice, decreased the content of malondialdehyde (MDA), 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-OHdG), 8-hydroxy-2'-deoxyguanosine (8-oxo-G), and nitric oxide (NO), and enhanced the superoxide dismutase (SOD) activity significantly. The neuroprotective effect of sea cucumber cerebrosides (SCC) was also verified in vitro: the cerebrosides increased the survival rate of PC12 cells, recovered the cellular morphology, downregulated the protein levels of Caspase-9, cleaved Caspase-3, total Caspase-3, and Bax, and upregulated the protein level of Bcl-2, revealing that cerebrosides could inhibit Aβ-induced cell apoptosis. The results showed the protective effect of SCC was regulated by the mitochondria-dependent apoptotic pathway. Our results provide a new approach to developing the marine organisms as functional foods for neuroprotection.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Aragon-Aguilar, Hector; Ramon-Gallegos, Eva; Arenas-Huertero, Francisco Jesus

The search of more specific and efficient photosensitizer in low oxygen tensions is a need in the Photodynamic Therapy (PDT). Phthalocyanines have demonstrated to have the above mentioned activity. The aim of this work was to determine the efficiency of PDT using two phthalocyanines synthesized in Mexico to eliminate melanoma cells. B16F0 melanoma mouse cells were exposed to concentrations from 8.95x10{sup -5} to 0.733 m/mL of F16VoPc and F16NbPcC13 during 24h, afterwards cellular mortality was measured. One kinetic was realized to determine the intracellular incorporation of phthalocyanines by confocal microscopy at 1, 2, 4, 8, 16 and 24 h ofmore » exposition. The PDT was applied exposing the cells to innocuous concentration (that does not provoke cellular death with out irradiation) and irradiating with an argon laser at 100 J/cm{sup 2}. For each phthalocyanine a control group was used; one group was not treated neither with light nor with phthalocyanine, the other group it was only irradiated. 24 h after treatment the citotoxicity was measured by Alamar blue assay. The innocuous concentration found for the phthalocyanines F16VoPc and F16NbPcC13 were 4.58x10-2 and 2.29xl0{sup -2} mg/mL, respectively. The time of maximum intracellular accumulation for both phthalocyanines was 24 h. Only the F16VoPc had anticancerous activity and induced 31.7% of cellular death. The PDT might offer a potential alternative to the treatment of this cancer when is used the phthalocyanine F16VoPc.« less

Discovery of small molecules binding to the normal conformation of prion by combining virtual screening and multiple biological activity evaluation methods

NASA Astrophysics Data System (ADS)

Li, Lanlan; Wei, Wei; Jia, Wen-Juan; Zhu, Yongchang; Zhang, Yan; Chen, Jiang-Huai; Tian, Jiaqi; Liu, Huanxiang; He, Yong-Xing; Yao, Xiaojun

2017-12-01

Conformational conversion of the normal cellular prion protein, PrPC, into the misfolded isoform, PrPSc, is considered to be a central event in the development of fatal neurodegenerative diseases. Stabilization of prion protein at the normal cellular form (PrPC) with small molecules is a rational and efficient strategy for treatment of prion related diseases. However, few compounds have been identified as potent prion inhibitors by binding to the normal conformation of prion. In this work, to rational screening of inhibitors capable of stabilizing cellular form of prion protein, multiple approaches combining docking-based virtual screening, steady-state fluorescence quenching, surface plasmon resonance and thioflavin T fluorescence assay were used to discover new compounds interrupting PrPC to PrPSc conversion. Compound 3253-0207 that can bind to PrPC with micromolar affinity and inhibit prion fibrillation was identified from small molecule databases. Molecular dynamics simulation indicated that compound 3253-0207 can bind to the hotspot residues in the binding pocket composed by β1, β2 and α2, which are significant structure moieties in conversion from PrPC to PrPSc.

Tat peptide and hexadecylphosphocholine introduction into pegylated liposomal doxorubicin: An in vitro and in vivo study on drug cellular delivery, release, biodistribution and antitumor activity.

PubMed

Teymouri, Manouchehr; Badiee, Ali; Golmohammadzadeh, Shiva; Sadri, Kayvan; Akhtari, Javad; Mellat, Mostafa; Nikpoor, Amin Reza; Jaafari, Mahmoud Reza

2016-09-10

We have investigated the co-addition of hexadecylphosphocholine (HePC) and a Tat derived peptide (Tat), coupled to Maleimide-PEG2000-DSPE pegylated liposomal doxorubicin (PLD) in many respects, including drug and liposome cellular delivery, drug release, biodistribution, in vivo cell delivery and antitumor activity. The liposomes were HePC-free and -containing liposomes, from which liposomes with 25, 50, 100 and 200 numbers of Tat/liposome were prepared. Similarly, DiI-C18 (3)-model liposomes (DiI-L and DiI-HePC-L) were prepared. HePC and Tat increased cellular delivery of Dox and cytotoxicity in B16F0 melanoma and C26 colon carcinoma cells. Tat enhanced liposome-cell interaction and caused Dox burst release. HePC and Tat reduced the serum retention time of liposomal Dox, slightly and dramatically, respectively. In comparison, Tat-liposomes enhanced Dox delivery to liver and spleen cells 3h post-injection. Likewise, Dox content of these tissues and tumor was lower at 24h. The naïve liposomes retarded tumor growth more effectively and their related median survival time of the treated C26 bearing BALB/c mice was longer than those of Tat-liposomes (MST>45days versus MST<38days). Overall liposomes exhibiting sustained drug release and negligible cell interaction were more suitable delivery systems in targeting cancerous tumors and suppressing their growth. Copyright © 2016 Elsevier B.V. All rights reserved.

Hydrogen Peroxide-Reducing Factor Released by PC12D Cells Increases Cell Tolerance against Oxidative Stress.

PubMed

Muraishi, Asami; Haneta, Emi; Saito, Yoshiro; Hitomi, Yutaka; Sano, Mamoru; Noguchi, Noriko

2018-01-01

PC12D cells, a subline of rat adrenal pheochromocytoma PC12 cells, extend neurites rapidly in response to differentiation stimuli and are used to investigate the molecular mechanisms of neurite extension. In the present study, we found significant tolerance of PC12D cells against Parkinson's disease-related stimuli such as dopamine and 6-hydroxydopamine; this tolerance was significantly decreased by a change in the medium. Conditioned medium from PC12D cells induced tolerance against oxidative stress, which suggests that cytoprotective factor may be released by PC12D cells into the culture medium. Conditioned medium-induced tolerance was not found for PC12 cells or human neuroblastoma SH-SY5Y cells. A cytoprotective factor generated by PC12D cells exhibited hydrogen peroxide-reducing activity. Chemical characterization showed that this cytoprotective factor is water soluble and has a molecular weight about 1000 Da, and that its activity is inhibited by sodium cyanide. Release of this cytoprotective factor was increased by differentiation stimuli and oxidative stress. Taken together, these results suggest that release of a hydrogen peroxide-reducing factor by PC12D cells increases cell tolerance against oxidative stress. This study provides new insights into the antioxidative properties of factors in extracellular fluid.

Loss of PTEN causes SHP2 activation, making lung cancer cells unresponsive to IFN-γ

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Chia-Ling; Chiang, Tzu-Hui; Tseng, Po-Chun

Src homology-2 domain-containing phosphatase (SHP) 2, an oncogenic phosphatase, inhibits type II immune interferon (IFN)-γ signaling by subverting signal transducers and activators of transcription 1 tyrosine phosphorylation and activation. For cancer immunoediting, this study aimed to investigate the decrease of phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a tumor suppressor protein, leading to cellular impairment of IFN-γ signaling. In comparison with human lung adenocarcinoma A549 cells, the natural PTEN loss in another human lung adenocarcinoma line, PC14PE6/AS2 cells, presents reduced responsiveness in IFN-γ-induced IFN regulatory factor 1 activation and CD54 expression. Artificially silencing PTEN expression in A549 cellsmore » also caused cells to be unresponsive to IFN-γ without affecting IFN-γ receptor expression. IFN-γ-induced inhibition of cell proliferation and cytotoxicity were demonstrated in A549 cells but were defective in PC14PE6/AS2 cells and in PTEN-deficient A549 cells. Aberrant activation of SHP2 by ROS was specifically shown in PC14PE6/AS2 cells and PTEN-deficient A549 cells. Inhibiting ROS and SHP2 rescued cellular responses to IFN-γ-induced cytotoxicity and inhibition of cell proliferation in PC14PE6/AS2 cells. These results demonstrate that a decrease in PTEN facilitates ROS/SHP2 signaling, causing lung cancer cells to become unresponsive to IFN-γ. - Highlights: • This study demonstrates that PTEN decrease causes cellular unresponsive to IFN-γ. • Lung cancer cells with PTEN deficiency show unresponsive to IFN-γ signaling. • PTEN decrease inhibits IFN-γ-induced CD54, cell proliferation inhibition, and cytotoxicity. • ROS-mediated SHP2 activation makes PTEN-deficient cells unresponsive to IFN-γ.« less

Phenolic Profiles, Antioxidant Activities, and Neuroprotective Properties of Mulberry (Morus atropurpurea Roxb.) Fruit Extracts from Different Ripening Stages.

PubMed

Yang, Jiufang; Liu, Xuanjun; Zhang, Xiaoxu; Jin, Qing; Li, Jingming

2016-10-01

The present work investigated the phenolic profiles (including nonanthocyanin and anthocyanin phenolics), antioxidant activities, and neuroprotective potential of mulberry fruit (MF) (Morus atropurpurea Roxb.) grown in China at different ripening stages. High-performance liquid chromatography-tandem mass spectrometry method (HPLC-MS/MS) was used to identify and quantify the phenolic compounds. The antioxidant capacity, total phenolic content (TPC), total flavonoid content (TFC), and total monomeric anthocyanin content (TAC) were determined using spectrophotometric methods. The neuroprotective effects of MFs at different ripening stages were investigated using Aβ 25-35 -treated PC12 cells as the cellular model of Alzheimer's disease. Of the 19 phenolic compounds characterized from the MF extracts, the contents of rutin and anthocyanins increased and that of chlorogenic acid decreased significantly with maturity. At the fully ripened stage, MF extracts showed the highest amounts of TPC (11.23 mg gallic acid equivalents/g fresh weight), TFC (15.1 mg rutin equivalents/g fresh weight), and TAC (1177 mg cyanidin 3-O-glucoside equivalents/100 g fresh weight). Meanwhile, antioxidant activity of MF extracts at this stage was highest according to ABTS (an IC50 value of 4.11 μg/mL) and DPPH (an IC50 value of 10.08 μg/mL) assays. Cellular assays revealed increased cell viability in cells treated with the ripe MF extracts; compared with the control groups, the ripening fruits also increased the antioxidant enzyme levels in PC12 cells. Together, these results suggest that the antioxidant activities and neuroprotective properties of ripening MFs are related to the contents and types of phenolic compounds that are present in the fruits. © 2016 Institute of Food Technologists®.

Lipotoxicity Mediated Cell Dysfunction and Death Involves Lysosomal Membrane Permeabilization and Cathepsin L Activity

PubMed Central

Almaguel, Frankis G.; Liu, Jo-Wen; Pacheco, Fabio J.; De Leon, Daisy; Casiano, Carlos A.; De Leon, Marino

2010-01-01

Lipotoxicity, which is triggered when cells are exposed to elevated levels of free fatty acids, involves cell dysfunction and apoptosis and is emerging as an underlying factor contributing to various pathological conditions including disorders of the central nervous system and diabetes. We have shown that palmitic acid (PA)-induced lipotoxicity (PA-LTx) in nerve growth factor-differentiated PC12 (NGFDPC12) cells is linked to an augmented state of cellular oxidative stress (ASCOS) and apoptosis, and that these events are inhibited by docosahexanoic acid (DHA). The mechanisms of PA-LTx in nerve cells are not well understood, but our previous findings indicate that it involves ROS generation, mitochondrial membrane permeabilization (MMP), and caspase activation. The present study used nerve growth factor differentiated PC12 cells (NGFDPC12 cells) and found that lysosomal membrane permeabilization (LMP) is an early event during PA-induced lipotoxicity that precedes MMP and apoptosis. Cathepsin L, but not cathepsin B, is an important contributor in this process since its pharmacological inhibition significantly attenuated LMP, MMP, and apoptosis. In addition, co-treatment of NGFDPC12 cells undergoing lipotoxicity with DHA significantly reduced LMP, suggesting that DHA acts by antagonizing upstream signals leading to lysosomal dysfunction. These results suggest that LMP is a key early mediator of lipotoxicity, and underscore the value of interventions targeting upstream signals leading to LMP for the treatment of pathological conditions associated with lipotoxicity. PMID:20043885

Kinetic of the Intracellular Incorporation of New Phthalocyanines Synthesized in mexico and Its Potential as Photosensibilizers in the Photodynamic Therapy

NASA Astrophysics Data System (ADS)

Aragón-Aguilar, Héctor; Ramón-Gallegos, Eva; Arenas-Huertero, Francisco Jesús; Contreras-Ramos, Alejandra; Cruz-Orea, Alfredo; Sosa-Sánchez, José Luís; Miranda, Maribel García

2008-08-01

The search of more specific and efficient photosensitizer in low oxygen tensions is a need in the Photodynamic Therapy (PDT). Phthalocyanines have demonstrated to have the above mentioned activity. The aim of this work was to determine the efficiency of PDT using two phthalocyanines synthesized in Mexico to eliminate melanoma cells. B16F0 melanoma mouse cells were exposed to concentrations from 8.95×10-5 to 0.733 m/mL of F16VoPc and F16NbPcC13 during 24h, afterwards cellular mortality was measured. One kinetic was realized to determine the intracellular incorporation of phthalocyanines by confocal microscopy at 1, 2, 4, 8, 16 and 24 h of exposition. The PDT was applied exposing the cells to innocuous concentration (that does not provoke cellular death with out irradiation) and irradiating with an argon laser at 100 J/cm2. For each phthalocyanine a control group was used; one group was not treated neither with light nor with phthalocyanine, the other group it was only irradiated. 24 h after treatment the citotoxicity was measured by Alamar blue assay. The innocuous concentration found for the phthalocyanines F16VoPc and F16NbPcC13 were 4.58×10-2 and 2.29×l0-2 mg/mL, respectively. The time of maximum intracellular accumulation for both phthalocyanines was 24 h. Only the F16VoPc had anticancerous activity and induced 31.7% of cellular death. The PDT might offer a potential alternative to the treatment of this cancer when is used the phthalocyanine F16VoPc.

Tumorigenesis of K-ras mutation in human endometrial carcinoma via upregulation of estrogen receptor.

PubMed

Tu, Zheng; Gui, Liming; Wang, Jianliu; Li, Xiaoping; Sun, Pengming; Wei, Lihui

2006-05-01

To investigate the tumorigenesis of mutant [12Asp]-K-ras in endometrial carcinoma and its relationship with ER. We constructed pcDI-[12Asp]K-ras4B by inserting full-length [12Asp]K-ras4B from human endometrial carcinoma Hec-1A cells, into pcDI vector. Cell proliferation of NIH3T3 after transfection with pcDI-[12Asp]K-ras4B was measured by MTT assay. The cell transformation was determined by colony formation and tumor nodule development. [12Asp]-K-ras4B-NIH3T3 cells were transfected with constitutively active pCMV-RafCAAX and dominant-negative pCMV-RafS621A. Cell growth was measured by MTT assay and [3H]thymidine incorporation. After transfected with pcDI-[12Asp]K-ras4B or pCMV-RafS621A, the cells were harvested for Western blot and reporter assay to determine the expression and transcriptional activity of ERalpha and ERbeta, respectively. [12Asp]-K-ras4B enhanced NIH3T3 cells proliferation after 48 h post-transfection (P < 0.05). More colonies were grown 10 days after incubating pcDI-[12Asp]-K-ras4B-NIH3T3 cells (13.48%) than pcDI-NIH3T3 (4.26%) or untreated NIH3T3 (2.33%). The pcDI-[12Asp]-K-ras4B-NIH3T3 cells injected to the nude mice Balb/C developed tumor nodules with poor-differentiated cells after 12 days. An increase of ERalpha and ERbeta was observed in pcDI-[12Asp]-K-ras4B-NIH3T3 cells. RafS621A downregulated ERalpha and ERbeta expression. Estrogen induced the ER transcriptional activity by 5-fold in pcDI-NIH3T3 cells, 13-fold in pcDI-[12Asp]K-ras4B-NIH3T3 and 19-fold in HEC-1A. RafS621A suppressed the ER transcriptional activity. K-ras mutation induces tumorigenesis in endometrium, and this malignant transformation involves Raf signaling pathway and ER.

Oxidative stress and dysfunctional NRF2 underlie pachyonychia congenita phenotypes

PubMed Central

Kerns, Michelle L.; Hakim, Jill M.C.; Lu, Rosemary G.; Guo, Yajuan; Berroth, Andreas; Kaspar, Roger L.

2016-01-01

Palmoplantar keratoderma (PPK) are debilitating lesions that arise in individuals with pachyonychia congenita (PC) and feature upregulation of danger-associated molecular patterns and skin barrier regulators. The defining features of PC-associated PPK are reproduced in mice null for keratin 16 (Krt16), which is commonly mutated in PC patients. Here, we have shown that PPK onset is preceded by oxidative stress in footpad skin of Krt16–/– mice and correlates with an inability of keratinocytes to sustain nuclear factor erythroid–derived 2 related factor 2–dependent (NRF2-dependent) synthesis of the cellular antioxidant glutathione (GSH). Additionally, examination of plantar skin biopsies from individuals with PC confirmed the presence of high levels of hypophosphorylated NRF2 in lesional tissue. In Krt16–/– mice, genetic ablation of Nrf2 worsened spontaneous skin lesions and accelerated PPK development in footpad skin. Hypoactivity of NRF2 in Krt16–/– footpad skin correlated with decreased levels or activity of upstream NRF2 activators, including PKCδ, receptor for activated C kinase 1 (RACK1), and p21. Topical application of the NRF2 activator sulforaphane to the footpad of Krt16–/– mice prevented the development of PPK and normalized redox balance via regeneration of GSH from existing cellular pools. Together, these findings point to oxidative stress and dysfunctional NRF2 as contributors to PPK pathogenesis, identify K16 as a regulator of NRF2 activation, and suggest that pharmacological activation of NRF2 should be further explored for PC treatment. PMID:27183391

Opposite Roles of Furin and PC5A in N-Cadherin Processing12

PubMed Central

Maret, Deborah; Sadr, Mohamad Seyed; Sadr, Emad Seyed; Colman, David R; Del Maestro, Rolando F; Seidah, Nabil G

2012-01-01

We recently demonstrated that lack of Furin-processing of the N-cadherin precursor (proNCAD) in highly invasive melanoma and brain tumor cells results in the cell-surface expression of a nonadhesive protein favoring cell migration and invasion in vitro. Quantitative polymerase chain reaction analysis of malignant human brain tumor cells revealed that of all proprotein convertases (PCs) only the levels of Furin and PC5A are modulated, being inversely (Furin) or directly (PC5A) correlated with brain tumor invasive capacity. Intriguingly, the N-terminal sequence following the Furin-activated NCAD site (RQKR↓DW161, mouse nomenclature) reveals a second putative PC-processing site (RIRSDR↓DK189) located in the first extracellular domain. Cleavage at this site would abolish the adhesive functions of NCAD because of the loss of the critical Trp161. This was confirmed upon analysis of the fate of the endogenous prosegment of proNCAD in human malignant glioma cells expressing high levels of Furin and low levels of PC5A (U343) or high levels of PC5A and negligible Furin levels (U251). Cellular analyses revealed that Furin is the best activating convertase releasing an ∼17-kDa prosegment, whereas PC5A is the major inactivating enzyme resulting in the secretion of an ∼20-kDa product. Like expression of proNCAD at the cell surface, cleavage of the NCAD molecule at RIRSDR↓DK189 renders the U251 cancer cells less adhesive to one another and more migratory. Our work modifies the present view on posttranslational processing and surface expression of classic cadherins and clarifies how NCAD possesses a range of adhesive potentials and plays a critical role in tumor progression. PMID:23097623

Activated Microglia Desialylate and Phagocytose Cells via Neuraminidase, Galectin-3, and Mer Tyrosine Kinase

PubMed Central

Nomura, Koji; Vilalta, Anna; Allendorf, David H.; Hornik, Tamara C.

2017-01-01

Activated microglia can phagocytose dying, stressed, or excess neurons and synapses via the phagocytic receptor Mer tyrosine kinase (MerTK). Galectin-3 (Gal-3) can cross-link surface glycoproteins by binding galactose residues that are normally hidden below terminal sialic acid residues. Gal-3 was recently reported to opsonize cells via activating MerTK. We found that LPS-activated BV-2 microglia rapidly released Gal-3, which was blocked by calcineurin inhibitors. Gal-3 bound to MerTK on microglia and to stressed PC12 (neuron-like) cells, and it increased microglial phagocytosis of PC12 cells or primary neurons, which was blocked by inhibition of MerTK. LPS-activated microglia exhibited a sialidase activity that desialylated PC12 cells and could be inhibited by Tamiflu, a neuraminidase (sialidase) inhibitor. Sialidase treatment of PC12 cells enabled Gal-3 to bind and opsonize the live cells for phagocytosis by microglia. LPS-induced microglial phagocytosis of PC12 was prevented by small interfering RNA knockdown of Gal-3 in microglia, lactose inhibition of Gal-3 binding, inhibition of neuraminidase with Tamiflu, or inhibition of MerTK by UNC569. LPS-induced phagocytosis of primary neurons by primary microglia was also blocked by inhibition of MerTK. We conclude that activated microglia release Gal-3 and a neuraminidase that desialylates microglial and PC12 surfaces, enabling Gal-3 binding to PC12 cells and their phagocytosis via MerTK. Thus, Gal-3 acts as an opsonin of desialylated surfaces, and inflammatory loss of neurons or synapses may potentially be blocked by inhibiting neuraminidases, Gal-3, or MerTK. PMID:28500071

Convergent RANK- and c-Met-mediated signaling components predict survival of patients with prostate cancer: an interracial comparative study.

PubMed

Hu, Peizhen; Chung, Leland W K; Berel, Dror; Frierson, Henry F; Yang, Hua; Liu, Chunyan; Wang, Ruoxiang; Li, Qinlong; Rogatko, Andre; Zhau, Haiyen E

2013-01-01

We reported (PLoS One 6 (12):e28670, 2011) that the activation of c-Met signaling in RANKL-overexpressing bone metastatic LNCaP cell and xenograft models increased expression of RANK, RANKL, c-Met, and phosphorylated c-Met, and mediated downstream signaling. We confirmed the significance of the RANK-mediated signaling network in castration resistant clinical human prostate cancer (PC) tissues. In this report, we used a multispectral quantum dot labeling technique to label six RANK and c-Met convergent signaling pathway mediators simultaneously in formalin fixed paraffin embedded (FFPE) tissue specimens, quantify the intensity of each expression at the sub-cellular level, and investigated their potential utility as predictors of patient survival in Caucasian-American, African-American and Chinese men. We found that RANKL and neuropilin-1 (NRP-1) expression predicts survival of Caucasian-Americans with PC. A Gleason score ≥ 8 combined with nuclear p-c-Met expression predicts survival in African-American PC patients. Neuropilin-1, p-NF-κB p65 and VEGF are predictors for the overall survival of Chinese men with PC. These results collectively support interracial differences in cell signaling networks that can predict the survival of PC patients.

Signal transduction in neurons: effects of cellular prion protein on fyn kinase and ERK1/2 kinase.

PubMed

Tomasi, Vittorio

2010-12-16

It has been reported that cellular prion protein (PrPc) co-localizes with caveolin-1 and participates to signal transduction events by recruiting Fyn kinase. As PrPc is a secreted protein anchored to the outer surface membrane through a glycosylphosphatidylinositol (GPI) anchor (secPrP) and caveolin-1 is located in the inner leaflet of plasma membrane, there is a problem of how the two proteins can physically interact each other and transduce signals. By using the GST-fusion proteins system we observed that PrPc strongly interacts with caveolin-1 scaffolding domain and with a caveolin-1 hydrophilic C-terminal region, but not with the caveolin-1 N-terminal region. In vitro binding experiments were also performed to define the site(s) of PrPc interacting with cav-1. The results are consistent with a participation of PrPc octapeptide repeats motif in the binding to caveolin-1 scaffolding domain. The caveolar localization of PrPc was ascertained by co-immunoprecipitation, by co-localization after flotation in density gradients and by confocal microscopy analysis of PrPc and caveolin-1 distributions in a neuronal cell line (GN11) expressing caveolin-1 at high levels. We observed that, after antibody-mediated cross-linking or copper treatment, PrPc was internalized probably into caveolae. We propose that following translocation from rafts to caveolae or caveolae-like domains, secPrP could interact with caveolin-1 and induce signal transduction events.

Conditional Deletion of Prnp Rescues Behavioral and Synaptic Deficits after Disease Onset in Transgenic Alzheimer's Disease

PubMed Central

Kaufman, Adam C.; Herber, Charlotte S.; Haas, Laura T.; Robinson, Sophie; Lee, Michael K.

2017-01-01

Biochemical and genetic evidence implicate soluble oligomeric amyloid-β (Aβo) in triggering Alzheimer's disease (AD) pathophysiology. Moreover, constitutive deletion of the Aβo-binding cellular prion protein (PrPC) prevents development of memory deficits in APPswe/PS1ΔE9 mice, a model of familial AD. Here, we define the role of PrPC to rescue or halt established AD endophenotypes in a therapeutic disease-modifying time window after symptom onset. Deletion of Prnp at either 12 or 16 months of age fully reverses hippocampal synapse loss and completely rescues preexisting behavioral deficits by 17 months. In contrast, but consistent with a neuronal function for Aβo/PrPC signaling, plaque density, microgliosis, and astrocytosis are not altered. Degeneration of catecholaminergic neurons remains unchanged by PrPC reduction after disease onset. These results define the potential of targeting PrPC as a disease-modifying therapy for certain AD-related phenotypes after disease onset. SIGNIFICANCE STATEMENT The study presented here further elucidates our understanding of the soluble oligomeric amyloid-β–Aβo-binding cellular prion protein (PrPC) signaling pathway in a familial form of Alzheimer's disease (AD) by implicating PrPC as a potential therapeutic target for AD. In particular, genetic deletion of Prnp rescued several familial AD (FAD)-associated phenotypes after disease onset in a mouse model of FAD. This study underscores the therapeutic potential of PrPC deletion given that patients already present symptoms at the time of diagnosis. PMID:28842420

Neuroprotective and anti-apoptotic propensity of Bacopa monniera extract against sodium nitroprusside induced activation of iNOS, heat shock proteins and apoptotic markers in PC12 cells.

PubMed

Pandareesh, M D; Anand, T

2014-05-01

Sodium nitroprusside (SNP) is a widely used nitric oxide (NO) donor, known to exert nitrative stress by up-regulation of inducible nitric oxide synthase (iNOS). Nω-nitro-L-arginine-methyl esther (L-NAME) is a NO inhibitor, which inhibits iNOS expression, is used as positive control. The present study was designed to assess neuroprotective propensity of Bacopa monniera extract (BME) in SNP-induced neuronal damage and oxido-nitrative stress in PC12 cells via modulation of iNOS, heat shock proteins and apoptotic markers. Our results elucidate that pre-treatment of PC12 cells with BME ameliorates the mitochondrial and plasma membrane damage induced by SNP (200 μM) as evidenced by MTT and LDH assays. BME pre-treatment inhibited NO generation by down regulating iNOS expression. BME replenished the depleted antioxidant status induced by SNP treatment. SNP-induced damage to cellular, nuclear and mitochondrial integrity was also restored by BME, which was confirmed by ROS estimation, comet assay and mitochondrial membrane potential assays respectively. BME pre-treatment efficiently attenuated the SNP-induced apoptotic protein biomarkers such as Bax, Bcl-2, cytochrome-c and caspase-3, which orchestrate the proteolytic damage of the cell. Q-PCR results further elucidated up-regulation of neuronal cell stress markers like HO-1 and iNOS and down-regulation of BDNF upon SNP exposure was attenuated by BME pre-treatment. By considering all these findings, we report that BME protects PC12 cells against SNP-induced toxicity via its free radical scavenging and neuroprotective mechanism.

The CNS glycoprotein Shadoo has PrPC-like protective properties and displays reduced levels in prion infections

PubMed Central

Watts, Joel C; Drisaldi, Bettina; Ng, Vivian; Yang, Jing; Strome, Bob; Horne, Patrick; Sy, Man-Sun; Yoong, Larry; Young, Rebecca; Mastrangelo, Peter; Bergeron, Catherine; Fraser, Paul E; Carlson, George A; Mount, Howard T J; Schmitt-Ulms, Gerold; Westaway, David

2007-01-01

The cellular prion protein, PrPC, is neuroprotective in a number of settings and in particular prevents cerebellar degeneration mediated by CNS-expressed Doppel or internally deleted PrP (‘ΔPrP'). This paradigm has facilitated mapping of activity determinants in PrPC and implicated a cryptic PrPC-like protein, ‘π'. Shadoo (Sho) is a hypothetical GPI-anchored protein encoded by the Sprn gene, exhibiting homology and domain organization similar to the N-terminus of PrP. Here we demonstrate Sprn expression and Sho protein in the adult CNS. Sho expression overlaps PrPC, but is low in cerebellar granular neurons (CGNs) containing PrPC and high in PrPC-deficient dendritic processes. In Prnp0/0 CGNs, Sho transgenes were PrPC-like in their ability to counteract neurotoxic effects of either Doppel or ΔPrP. Additionally, prion-infected mice exhibit a dramatic reduction in endogenous Sho protein. Sho is a candidate for π, and since it engenders a PrPC-like neuroprotective activity, compromised neuroprotective activity resulting from reduced levels may exacerbate damage in prion infections. Sho may prove useful in deciphering several unresolved facets of prion biology. PMID:17703189

Chimeras of the native form or achondroplasia mutant (G375C) of human fibroblast growth factor receptor 3 induce ligand-dependent differentiation of PC12 cells.

PubMed Central

Thompson, L M; Raffioni, S; Wasmuth, J J; Bradshaw, R A

1997-01-01

Mutations in the gene for human fibroblast growth factor receptor 3 (hFGFR3) cause a variety of skeletal dysplasias, including the most common genetic form of dwarfism, achondroplasia (ACH). Evidence indicates that these phenotypes are not due to simple haploinsufficiency of FGFR3 but are more likely related to a role in negatively regulating skeletal growth. The effects of one of these mutations on FGFR3 signaling were examined by constructing chimeric receptors composed of the extracellular domain of human platelet-derived growth factor receptor beta (hPDGFR beta) and the transmembrane and intracellular domains of hFGFR3 or of an ACH (G375C) mutant. Following stable transfection in PC12 cells, which lack platelet-derived growth factor (PDGF) receptors, all clonal cell lines, with either type of chimera, showed strong neurite outgrowth in the presence of PDGF but not in its absence. Antiphosphotyrosine immunoblots showed ligand-dependent autophosphorylation, and both receptor types stimulated strong phosphorylation of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase, an event associated with the differentiative response of these cells. In addition, ligand-dependent phosphorylation of phospholipase Cgamma and Shc was also observed. All of these responses were comparable to those observed from ligand activation, such as by nerve growth factor, of the native PC12 cells used to prepare the stable transfectants. The cells with the chimera bearing the ACH mutation were more rapidly responsive to ligand with less sustained MAPK activation, indicative of a preactivated or primed condition and consistent with the view that these mutations weaken ligand control of FGFR3 function. However, the full effect of the mutation likely depends in part on structural features of the extracellular domain. Although FGFR3 has been suggested to act as a negative regulator of long-bone growth in chrondrocytes, it produces differentiative signals similar to those of FGFR1, to which only positive effects have been ascribed, in PC12 cells. Therefore, its regulatory effects on bone growth likely result from cellular contexts and not the induction of a unique FGFR3 signaling pathway. PMID:9199352

Structure-activity relationships of neoechinulin A analogues with cytoprotection against peroxynitrite-induced PC12 cell death.

PubMed

Kimoto, Kuniaki; Aoki, Toshiaki; Shibata, Yasushi; Kamisuki, Shinji; Sugawara, Fumio; Kuramochi, Kouji; Nakazaki, Atsuo; Kobayashi, Susumu; Kuroiwa, Kenji; Watanabe, Nobuo; Arai, Takao

2007-10-01

Neoechinulin A, an alkaloid from Eurotium rubrum Hiji025, protected neuronal PC12 cells against cell death induced by peroxynitrite derived from SIN-1 (3-(4-morpholinyl)sydnonimine hydrochloride). In this study, we investigated the structure-activity relationships of neoechinulin A and a set of its analogues by using assays to measure anti-nitration and antioxidant activities and cytoprotection against SIN-1-induced PC12 cell death. The presence of the diketopiperazine ring was essential for both the antioxidant and anti-nitration activities of neoechinulin A derivatives. Nevertheless, a derivative lacking the diketopiperazine ring could still protect PC12 cells against SIN-1 cytotoxicity. An acyclic analogue completely lost the cytoprotective effect while retaining its antioxidant/anti-nitration activities. Pre-incubation of the cells with neoechinulin A for at least 12 hours was essential for the cells to gain SIN-1 resistance. These results suggest that neoechinulin A endows the cells with cytoprotection through a biological effect different from the apparent antioxidant/anti-nitration activities.

Mic60/Mitofilin Overexpression Alters Mitochondrial Dynamics and Attenuates Vulnerability of Dopaminergic Cells to Dopamine and Rotenone

PubMed Central

Van Laar, Victor S.; Berman, Sarah B.; Hastings, Teresa G.

2017-01-01

Mitochondrial dysfunction has been implicated in Parkinson’s disease (PD) neuropathology. Mic60, also known as mitofilin, is a protein of the inner mitochondrial membrane and a key component of the mitochondrial contact site and cristae junction organizing system (MICOS). Mic60 is critical for maintaining mitochondrial membrane structure and function. We previously demonstrated that mitochondrial Mic60 protein is susceptible to both covalent modification and loss in abundance following exposure to dopamine quinone. In this study, we utilized neuronally-differentiated SH-SY5Y and PC12 dopaminergic cell lines to examine the effects of altered Mic60 levels on mitochondrial function and cellular vulnerability in response to PD-relevant stressors. Short hairpin RNA (shRNA)-mediated knockdown of endogenous Mic60 protein in neuronal SH-SY5Y cells significantly potentiated dopamine-induced cell death, which was rescued by co-expressing shRNA-insensitive Mic60. Conversely, in PC12 and SH-SY5Y cells, Mic60 overexpression significantly attenuated both dopamine- and rotenone-induced cell death as compared to controls. Mic60 overexpression in SH-SY5Y cells was also associated with increased mitochondrial respiration, and, following rotenone exposure, increased spare respiratory capacity. Mic60 knockdown cells exhibited suppressed respiration and, following rotenone treatment, decreased spare respiratory capacity. Mic60 overexpression also affected mitochondrial fission/fusion dynamics. PC12 cells overexpressing Mic60 exhibited increased mitochondrial interconnectivity. Further, both PC12 cells and primary rat cortical neurons overexpressing Mic60 displayed suppressed mitochondrial fission and increased mitochondrial length in neurites. These results suggest that altering levels of Mic60 in dopaminergic neuronal cells significantly affects both mitochondrial homeostasis and cellular vulnerability to the PD-relevant stressors dopamine and rotenone, carrying implications for PD pathogenesis. PMID:27001148

The protective effect of sodium benzoate on aluminum toxicity in PC12 cell line.

PubMed

Arabsolghar, Rita; Saberzadeh, Jamileh; Khodaei, Forouzan; Borojeni, Rozhin Abbasi; Khorsand, Marjan; Rashedinia, Marzieh

2017-10-01

Sodium benzoate (SB) is one of the food additives and preservatives that prevent the growth of fungi and bacteria. SB has been shown to improve the symptoms of neurodegenerative disease such as Alzheimer's disease. The aim of this study was to evaluate the effect of SB on the cell survival and cellular antioxidant indices after exposure to aluminum maltolate (Almal) in PC12 cell line as a model of neurotoxicity. The cells exposed to different concentrations of SB (0.125 to 3 mg/mL) in the presence of Almal (500 µM) and cell viability, the level of reactive oxygen species (ROS), glutathione content and catalase activity were measured. The results showed that low concentrations of SB caused an increase in the cell survival, but cell viability was reduced in high concentrations. SB could neither prevent the level of ROS production nor change glutathione content. SB (0.5 mg/mL) significantly increased the catalase enzyme activity as compared to the Almal. This study suggested that SB did not completely protect the cell to aluminum-induced free radicals toxicity. Possibly SB improves the symptoms of neurodegenerative disease by other mechanisms.

Attenuation of Cisplatin-Induced Neurotoxicity by Cyanidin, a Natural Inhibitor of ROS-Mediated Apoptosis in PC12 Cells.

PubMed

Li, Da-wei; Sun, Jing-yi; Wang, Kun; Zhang, Shuai; Hou, Ya-jun; Yang, Ming-feng; Fu, Xiao-yan; Zhang, Zong-yong; Mao, Lei-lei; Yuan, Hui; Fang, Jie; Fan, Cun-dong; Zhu, Mei-jia; Sun, Bao-liang

2015-10-01

Cisplatin-based chemotherapy in clinic is severely limited by its adverse effect, including neurotoxicity. Oxidative damage contributes to cisplatin-induced neurotoxicity, but the mechanism remains unclearly. Cyanidin, a natural flavonoid compound, exhibits powerful antioxidant activity. Hence, we investigated the protective effects of cyanidin on PC12 cells against cisplatin-induced neurotoxicity and explored the underlying mechanisms. The results showed that cisplatin-induced cytotoxicity was completely reversed by cyanidin through inhibition of PC12 cell apoptosis, as proved by the attenuation of Sub-G1 peak, PARP cleavage, and caspases-3 activation. Mechanistically, cyanidin significantly inhibited reactive oxygen species (ROS)-induced DNA damage in cisplatin-treated PC12 cells. Our findings revealed that cyanidin as an apoptotic inhibitor effectively blocked cisplatin-induced neurotoxicity through inhibition of ROS-mediated DNA damage and apoptosis, predicating its therapeutic potential in prevention of chemotherapy-induced neurotoxicity. Cisplatin caused DNA damage, activated p53, and subsequently induced PC12 cells apoptosis by triggering ROS overproduction. However, cyanidin administration effectively inhibited DNA damage, attenuated p53 phosphorylation, and eventually reversed cisplatin-induced PC12 cell apoptosis through inhibition ROS accumulation.

Phosphorylation of serine 779 in fibroblast growth factor receptor 1 and 2 by protein kinase C(epsilon) regulates Ras/mitogen-activated protein kinase signaling and neuronal differentiation.

PubMed

Lonic, Ana; Powell, Jason A; Kong, Yang; Thomas, Daniel; Holien, Jessica K; Truong, Nhan; Parker, Michael W; Guthridge, Mark A

2013-05-24

The FGF receptors (FGFRs) control a multitude of cellular processes both during development and in the adult through the initiation of signaling cascades that regulate proliferation, survival, and differentiation. Although FGFR tyrosine phosphorylation and the recruitment of Src homology 2 domain proteins have been widely described, we have previously shown that FGFR is also phosphorylated on Ser(779) in response to ligand and binds the 14-3-3 family of phosphoserine/threonine-binding adaptor/scaffold proteins. However, whether this receptor phosphoserine mode of signaling is able to regulate specific signaling pathways and biological responses is unclear. Using PC12 pheochromocytoma cells and primary mouse bone marrow stromal cells as models for growth factor-regulated neuronal differentiation, we show that Ser(779) in the cytoplasmic domains of FGFR1 and FGFR2 is required for the sustained activation of Ras and ERK but not for other FGFR phosphotyrosine pathways. The regulation of Ras and ERK signaling by Ser(779) was critical not only for neuronal differentiation but also for cell survival under limiting growth factor concentrations. PKCε can phosphorylate Ser(779) in vitro, whereas overexpression of PKCε results in constitutive Ser(779) phosphorylation and enhanced PC12 cell differentiation. Furthermore, siRNA knockdown of PKCε reduces both growth factor-induced Ser(779) phosphorylation and neuronal differentiation. Our findings show that in addition to FGFR tyrosine phosphorylation, the phosphorylation of a conserved serine residue, Ser(779), can quantitatively control Ras/MAPK signaling to promote specific cellular responses.

Inhibition of 6-hydroxydopamine-induced PC12 cell apoptosis by olive (Olea europaea L.) leaf extract is performed by its main component oleuropein.

PubMed

Pasban-Aliabadi, Hamzeh; Esmaeili-Mahani, Saeed; Sheibani, Vahid; Abbasnejad, Mehdi; Mehdizadeh, Anahita; Yaghoobi, Mohammad Mehdi

2013-04-01

Parkinson disease (PD) is the most common progressive neurodegenerative disorder characterized by progressive death of midbrain dopaminergic neurons. Most neurodegenerative disease treatments are, at present, palliative. However, some natural herbal products have been shown to rescue neurons from death and apoptosis in some of neurodegenerative diseases. Not only Olea europaea L. olive oil, but also the leaves of this plant have been used for medical purposes. Olive leaf extract (OLE) is being used by people as a drink across the world and as an integral ingredient in their desire to maintain and improve their health. Here, we investigated the effects of OLE and its main phenolic component oleuropein on 6-hydroxydopamine (6-OHDA)-induced toxicity in rat adrenal pheochromocytoma (PC12) cells as an in vitro model of PD. Cell damage was induced by 150 μM 6-OHDA. The cell survival rate was examined by MTT assay. Generation of intra-cellular reactive oxygen species (ROS) was studied using fluorescence spectrophotometry. Immunoblotting and DNA analysis were also employed to determine the levels of biochemical markers of apoptosis in the cells. The data showed that 6-OHDA could decrease the viability of the cells. In addition, intra-cellular ROS, activated caspase 3, Bax/Bcl-2 ratio, as well as DNA fragmentation were significantly increased in 6-OHDA-treated cells. Incubation of cells with OLE (400 and 600 μg/mL) and oleuropein (20 and 25 μg/mL) could decrease cell damage and reduce biochemical markers of cell death. The results suggest that OLE and oleuropein have anti-oxidant protective effects against 6-OHDA-induced PC12 cell damage. The protective effects of OLE and oleuropein are correlative with their anti-oxidative and anti-apoptotic properties and suggest their therapeutic potential in the treatment of PD.

Targeting DNA repair with PNKP inhibition sensitizes radioresistant prostate cancer cells to high LET radiation

PubMed Central

Srivastava, Pallavi; Sarma, Asitikantha

2018-01-01

High linear energy transfer (LET) radiation or heavy ion such as carbon ion radiation is used as a method for advanced radiotherapy in the treatment of cancer. It has many advantages over the conventional photon based radiotherapy using Co-60 gamma or high energy X-rays from a Linear Accelerator. However, charged particle therapy is very costly. One way to reduce the cost as well as irradiation effects on normal cells is to reduce the dose of radiation by enhancing the radiation sensitivity through the use of a radiomodulator. PNKP (polynucleotide kinase/phosphatase) is an enzyme which plays important role in the non-homologous end joining (NHEJ) DNA repair pathway. It is expected that inhibition of PNKP activity may enhance the efficacy of the charged particle irradiation in the radioresistant prostate cancer cell line PC-3. To test this hypothesis, we investigated cellular radiosensitivity by clonogenic cell survival assay in PC-3 cells.12Carbon ion beam of62 MeVenergy (equivalent 5.16 MeV/nucleon) and with an entrance LET of 287 kev/μm was used for the present study. Apoptotic parameters such as nuclear fragmentation and caspase-3 activity were measured by DAPI staining, nuclear ladder assay and colorimetric caspase-3method. Cell cycle arrest was determined by FACS analysis. Cell death was enhanced when carbon ion irradiation is combined with PNKPi (PNKP inhibitor) to treat cells as compared to that seen for PNKPi untreated cells. A low concentration (10μM) of PNKPi effectively radiosensitized the PC-3 cells in terms of reduction of dose in achieving the same survival fraction. PC-3 cells underwent significant apoptosis and cell cycle arrest too was enhanced at G2/M phase when carbon ion irradiation was combined with PNKPi treatment. Our findings suggest that combined treatment of carbon ion irradiation and PNKP inhibition could enhance cellular radiosensitivity in a radioresistant prostate cancer cell line PC-3. The synergistic effect of PNKPi and carbon ion irradiation could be used as a promising method for carbon-ion therapy in radioresistant cells. PMID:29320576

Effects and mechanisms of melatonin on the proliferation and neural differentiation of PC12 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Yumei; Zhang, Ziqiang; Lv, Qiongxia

Melatonin, a lipophilic molecule that is mainly synthesized in the pineal gland, performs various neuroprotective functions. However, the detailed role and mechanisms of promoting neuronal differentiation remains limited. This study demonstrated that 10 μM melatonin led to significant increases in the proliferation and neurite outgrowth of PC12 cells. Increased expression of microtubule-associated protein 2 (MAP2, a neuron-specific protein) was also observed. However, luzindole (melatonin receptor antagonist) and PD98059 (MEK inhibitor) attenuated these increases. LY294002 (AKT inhibitor) inhibited melatonin-mediated proliferation in PC12 cells and did not affect melatonin-induced neural differentiation. The expression of p-ERK1/2/ERK1/2 was increased by melatonin treatment for 14 days in PC12 cells,more » whereas luzindole or PD98059 reduced the melatonin-induced increase. These results suggest that the activation of both the MEK/ERK and PI3K/AKT signaling pathways could potentially contribute to melatonin-mediated proliferation, but that only the MEK/ERK pathway participates in the melatonin-induced neural differentiation of PC12 cells. Altogether, our study demonstrates for the first time that melatonin may exert a positive effect on neural differentiation via melatonin receptor signalling and that the MEK/ERK1/2 signalling may act down stream from the melatonin pathway. - Highlights: • Melatonin improves the proliferation of PC12 cells. • Melatonin induces neural differentiation of PC12 cells. • Melatonin-mediated proliferation in PC12 cells relies on the ERK and AKT pathways. • Activation of ERK is essential for melatonin-induced neural differentiation of PC12.« less

Epidermal fatty acid-binding protein protects nerve growth factor-differentiated PC12 cells from lipotoxic injury

PubMed Central

Liu, Jo-Wen; Montero, Manuel; Bu, Liming; De Leon, Marino

2015-01-01

Epidermal fatty acid-binding protein (E-FABP/FABP5/DA11) binds and transport long-chain fatty acids in the cytoplasm and may play a protecting role during neuronal injury. We examined whether E-FABP protects nerve growth factor-differentiated PC12 cells (NGFDPC12 cells) from lipotoxic injury observed after palmitic acid (C16:0; PAM) overload. NGFDPC12 cells cultures treated with PAM/bovine serum albumin at 0.3 mM/0.15 mM show PAM-induced lipotoxicity (PAM-LTx) and apoptosis. The apoptosis was preceded by a cellular accumulation of reactive oxygen species (ROS) and higher levels of E-FABP. Antioxidants MCI-186 and N-acetyl cysteine prevented E-FABP's induction in expression by PAM-LTx, while tert-butyl hydroperoxide increased ROS and E-FABP expression. Non-metabolized methyl ester of PAM, methyl palmitic acid (mPAM), failed to increase cellular ROS, E-FABP gene expression, or trigger apoptosis. Treatment of NGFDPC12 cultures with siE-FABP showed reduced E-FABP levels correlating with higher accumulation of ROS and cell death after exposure to PAM. In contrast, increasing E-FABP cellular levels by pre-loading the cells with recombinant E-FABP diminished the PAM-induced ROS and cell death. Finally, agonists for PPARβ (GW0742) or PPARγ (GW1929) increased E-FABP expression and enhanced the resistance of NGFDPC12 cells to PAM-LTx. We conclude that E-FABP protects NGFDPC12 cells from lipotoxic injury through mechanisms that involve reduction of ROS. Epidermal fatty acid-binding protein (E-FABP) may protect nerve cells from the damaging exposure to high levels of free fatty acids (FA). We show that E-FABP can neutralize the effects of reactive oxygen species (ROS) generated by the high levels of FA in the cell and protect PC12 cells from lipotoxic injuries common in Type 2 diabetes neuropathy. Potentially, E-FABP gene up-regulation may be mediated through the NFkB pathway and future studies are needed to further evaluate this proposition. PMID:25147052

Prion Protein Regulates Iron Transport by Functioning as a Ferrireductase

PubMed Central

Singh, Ajay; Haldar, Swati; Horback, Katharine; Tom, Cynthia; Zhou, Lan; Meyerson, Howard; Singh, Neena

2017-01-01

Prion protein (PrPC) is implicated in the pathogenesis of prion disorders, but its normal function is unclear. We demonstrate that PrPC is a ferrireductase (FR), and its absence causes systemic iron deficiency in PrP knock-out mice (PrP−/−). When exposed to non-transferrin-bound (NTB) radioactive-iron (59FeCl3) by gastric-gavage, PrP−/− mice absorb significantly more 59Fe from the intestinal lumen relative to controls, indicating appropriate systemic response to the iron deficiency. Chronic exposure to excess dietary iron corrects this deficiency, but unlike wild-type (PrP+/+) controls that remain iron over-loaded, PrP−/− mice revert back to the iron deficient phenotype after 5 months of chase on normal diet. Bone marrow (BM) preparations of PrP−/− mice on normal diet show relatively less stainable iron, and this phenotype is only partially corrected by intraperitoneal administration of excess iron-dextran. Cultured PrP−/− BM-macrophages incorporate significantly less NTB-59Fe in the absence or presence of excess extracellular iron, indicating reduced uptake and/or storage of available iron in the absence of PrPC. When expressed in neuroblastoma cells, PrPC exhibits NAD(P)H-dependent cell-surface and intracellular FR activity that requires the copper-binding octa-peptide-repeat region and linkage to the plasma membrane for optimal function. Incorporation of NTB-59Fe by neuroblastoma cells correlates with FR activity of PrPC, implicating PrPC in cellular iron uptake and metabolism. These observations explain the correlation between PrPC expression and cellular iron levels, and the cause of iron imbalance in sporadic-Creutzfeldt-Jakob-disease brains where PrPC accumulates as insoluble aggregates. PMID:23478311

Post-Translational Regulation of Polycystin-2 Protein Expression as a Novel Mechanism of Cholangiocyte Reaction and Repair from Biliary Damage

PubMed Central

Spirli, Carlo; Villani, Ambra; Mariotti, Valeria; Fabris, Luca; Fiorotto, Romina; Strazzabosco, Mario

2015-01-01

Polycystin-2 (PC2 /TRPP2), a member of the transient receptor potential channels (TRP) family, is a non-selective calcium channel. Mutations in PC2/TRPP2 are associated with Polycystic Liver Diseases. PC2-defective cholangiocytes shows increased production of cAMP, PKA-dependent activation of the ERK1/2 pathway, HIF1α-mediated VEGF production, and stimulation of cyst growth and progression. Activation of the ERK/HIF1α/VEGF pathway in cholangiocytes plays a key role during repair from biliary damage. We hypothesized that PC2 levels are modulated during biliary damage/repair, resulting in activation of the ERK/HIF1α/VEGF pathway. Results PC2 protein expression, but not its gene expression, was significantly reduced in mouse livers with biliary damage (Mdr2−/−-KO, bile duct ligation, DDC-treatment). Treatment of colangiocytes with pro-inflammatory cytokines, nitric oxide (NO) donors and ER stressors), increased ERK1/2 phosphorylation, HIF1α transcriptional activity, secretion of VEGF, VEGFR2 phosphorylation and downregulated PC2 protein expression without affecting PC2 gene expression. Expression of Herp and NEK, ubiquitin-like proteins that promote proteosomal PC2 degradation was increased. Pre-treatment with the proteasome inhibitor MG-132 restored the expression of PC2 in cells treated with cytokines but not in cells treated with NO donors or with ER stressors. In these conditions, PC2 degradation was instead inhibited by interfering with the autophagy pathway. Treatment of DDC-mice and of Mdr2−/−-mice with the proteasome inhibitor bortezomib, restored PC2 expression and significantly reduced the ductular reaction, fibrosis and p-ERK1/2. In conclusion, in response to biliary damage, PC2 expression is modulated post-translationally by the proteasome or the autophagy pathways. PC2-dowregulation is associated with activation of ERK1/2 and increase of HIF1α-mediated VEGF secretion. Treatments able to restore PC2 expression and to reduce ductular reaction and fibrosis may represent a new therapeutic approach in biliary diseases. PMID:26313562

Protective effects of decursin and decursinol angelate against amyloid β-protein-induced oxidative stress in the PC12 cell line: the role of Nrf2 and antioxidant enzymes.

PubMed

Li, Li; Li, Wei; Jung, Sang-Won; Lee, Yong-Woo; Kim, Yong-Ho

2011-01-01

The protective effects of decursin (D) and decursinol angelate (DA) purified from Angelica gigas Nakai on amyloid β-protein (Aβ)-induced neurotoxicity and the underlying mechanisms were investigated. Aβ plays a major role in the pathogenesis of Alzheimer's disease (AD) by eliciting oxidative stress. It significantly increased cytotoxicity and lipid peroxidation, but decreased glutathione contents and antioxidant enzyme activities. All of these results were markedly reversed by pretreatment with D or DA. Nuclear transcription factor Nrf2, which regulates the expression of antioxidant enzymes, was significantly increased by D or DA pretreatment. Furthermore, D and DA suppressed Aβ aggregation. These results suggest that D and DA increase cellular resistance to Aβ-induced oxidative injury in the rat pheochromocytoma (PC12) cells, presumably through not only the induction of Nrf2 and related antioxidant enzymes, but also the anti-aggregation of Aβ. Thus D and DA have therapeutic potential in treating AD and other oxidative stress-related diseases.

Overexpression of the human ubiquitin E3 ligase CUL4A alleviates hypoxia-reoxygenation injury in pheochromocytoma (PC12) cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tan, Can; Zhang, Li-Yang; Chen, Hong

2011-12-16

Highlights: Black-Right-Pointing-Pointer Overexpression of human CUL4A (hCUL4A) in PC12 cells. Black-Right-Pointing-Pointer The effects of hCUL4A on hypoxia-reoxygenation injury were investigated. Black-Right-Pointing-Pointer hCUL4A suppresses apoptosis and DNA damage and thus promotes cell survival. Black-Right-Pointing-Pointer hCUL4A regulates apoptosis-related proteins and cell cycle regulators. -- Abstract: The ubiquitin E3 ligase CUL4A plays important roles in diverse cellular processes including carcinogenesis and proliferation. It has been reported that the expression of CUL4A can be induced by hypoxic-ischemic injury. However, the effect of elevated expression of CUL4A on hypoxia-reoxygenation injury is currently unclear. In this study, human CUL4A (hCUL4A) was expressed in rat pheochromocytoma (PC12)more » cells using adenoviral vector-mediated gene transfer, and the effects of hCUL4A expression on hypoxia-reoxygenation injury were investigated. In PC12 cells subjected to hypoxia and reoxygenation, we found that hCUL4A suppresses apoptosis and DNA damage by regulating apoptosis-related proteins and cell cycle regulators (Bcl-2, caspase-3, p53 and p27); consequently, hCUL4A promotes cell survival. Taken together, our results reveal the beneficial effects of hCUL4A in PC12 cells upon hypoxia-reoxygenation injury.« less

Design and synthesis of formononetin-dithiocarbamate hybrids that inhibit growth and migration of PC-3 cells via MAPK/Wnt signaling pathways

PubMed Central

Fu, Dong-Jun; Zhang, Li; Song, Jian; Mao, Ruo-Wang; Zhao, Ruo-Han; Liu, Ying-Chao; Hou, Yu-Hui; Li, Jia-Huan; Yang, Jia-Jia; Jin, Cheng-Yun; Li, Ping; Zi, Xiao-Lin; Liu, Hong-Min; Zhang, Sai-Yang; Zhang, Yan-Bing

2017-01-01

A series of novel formononetin-dithiocarbamate derivatives were designed, synthesized and evaluated for antiproliferative activity against three selected cancer cell line (MGC-803, EC-109, PC-3). The first structure-activity relationship (SAR) for this formononetin-dithiocarbamate scaffold is explored in this report with evaluation of 14 variants of the structural class. Among these analogues, tert-butyl 4-(((3-((3-(4-methoxyphenyl)-4-oxo-4H–chromen-7-yl)oxy)propyl)thio)carbonothioyl)piperazine-1-carboxylate (8i) showed the best inhibitory activity against PC-3 cells (IC50 = 1. 97 µM). Cellular mechanism studies elucidated 8i arrests cell cycle at G1 phase and regulates the expression of G1 checkpoint-related proteins in concentration-dependent manners. Furthermore, 8i could inhibit cell growth via MAPK signaling pathway and inhibit migration via Wnt pathway in PC-3 cells. PMID:28038329

Hypothermic inhibition of apoptotic pathways for combined neurotoxicity of iron and ascorbic acid in differentiated PC12 cells: reduction of oxidative stress and maintenance of the glutathione redox state.

PubMed

Hasegawa, Masashi; Ogihara, Tohru; Tamai, Hiroshi; Hiroi, Mayo

2009-08-04

Recent clinical trials have demonstrated the efficacy and safety of therapeutic hypothermia for neonatal hypoxic ischemic encephalopathy (HIE). We previously reported that the levels of non-protein-bound iron and ascorbic acid (AA) are increased in the CSF of infants with HIE. In this study, we investigated the effect of hypothermia on the combined cytotoxicity of Fe and AA for differentiated PC12 cells. The optimal settings for hypothermic treatment were a temperature of 30-32 degrees C, rescue time window of less than 6 h, and minimum duration of at least 24 h. Hypothermia effectively prevented the loss of the mitochondrial transmembrane potential from 6 h to 72 h (end of the study period) and attenuated the release of apoptotic proteins (cytochrome c and apoptosis-inducing factor) at 6 h of exposure to Fe-AA. Activation of caspase-3 was also delayed until 24 h. Akt was transiently activated, although no influence of temperature was observed. Elevation of oxidative stress markers, including ortho-, meta-, and di-tyrosine (markers of protein oxidation) and 4-hydroxynonenal (lipid peroxidation) was significantly attenuated when the temperature was reduced by 5 degrees C. The half-cell reduction potential (Ehc) of GSSG/2GSH redox couple ranged from -220 to -180 mV in unstressed differentiated PC12 cells, and apoptosis was triggered when Ehc exceeded -180 mV. Hypothermia prevented Ehc from rising above -180 mV within 24 h of exposure to Fe-AA. In conclusion, hypothermia prevented cell death due to Fe-AA toxicity by inhibiting apoptotic pathways through maintenance of a reduced cellular environment, as well as by alleviating oxidative stress.

Altered Expression of Urea Cycle Enzymes in Amyloid-β Protein Precursor Overexpressing PC12 Cells and in Sporadic Alzheimer's Disease Brain.

PubMed

Jęśko, Henryk; Lukiw, Walter J; Wilkaniec, Anna; Cieślik, Magdalena; Gąssowska-Dobrowolska, Magdalena; Murawska, Emilia; Hilgier, Wojciech; Adamczyk, Agata

2018-01-01

Urea cycle enzymes may play important yet poorly characterized roles in Alzheimer's disease (AD). Our previous results showed that amyloid-β (Aβ) affects urea cycle enzymes in rat pheochromocytoma (PC12) cells. The aim of the present study was to investigate the changes in arginases, other urea cycle enzymes, and nitric oxide synthases (NOSs) in PC12 cells transfected with AβPP bearing the double 'Swedish' mutation (APPsw, K670M/N671L) and in postmortem sporadic AD brain hippocampus; the mutation intensifies Aβ production and strongly associates with AD neuropathology. mRNA expression was analyzed using real-time PCR in cell cultures and DNA microarrays in hippocampal CA1 area of human AD brains. Arginase activity was measured spectrophotometrically, and arginine, ornithine, and citrulline levels by high-performance liquid chromatography. Our data demonstrated that the expression and activity of arginases (Arg1 and Arg2), as well as the expression of argininosuccinate synthase (Ass) were significantly reduced in APPsw cells compared to control. However, argininosuccinate lyase (Asl) was upregulated in APPsw cells. Real-time PCR analysis revealed significant elevation of neuronal nitric oxide synthase (Nnos) mRNA in APPsw cells, without changes in the endothelial Enos, whereas inducible Inos was undetectable. The changes were found to follow closely those observed in the human hippocampal CA1 region of sporadic AD brains. The changes in enzyme expression were accompanied in APPsw cells by significantly elevated citrulline, ornithine, and arginine. Our findings demonstrate that AβPP/Aβ alters arginine metabolism and induces a shift of cellular homeostasis that may support the oxidative/nitrosative stress observed in AD.

Preparation and sonodynamic activities of water-soluble tetra-α-(3-carboxyphenoxyl) zinc(II) phthalocyanine and its bovine serum albumin conjugate.

PubMed

Xu, He-Nan; Chen, Hai-Jun; Zheng, Bi-Yuan; Zheng, Yun-Quan; Ke, Mei-Rong; Huang, Jian-Dong

2015-01-01

Sonodynamic therapy (SDT) is a new approach for cancer treatment, involving the synergistic effect of ultrasound and certain chemical compounds termed as sonosensitizers. A water-soluble phthalocyanine, namely tetra-α-(3-carboxyphenoxyl) zinc(II) phthalocyanine (ZnPcC4), has been prepared and characterized. The interactions between ZnPcC4 and bovine serum albumin (BSA) were also investigated by absorption and fluorescence spectroscopy. It was found that there were strong interactions between ZnPcC4 and BSA with a binding constant of 6.83×10(7)M(-1). A non-covalent BSA conjugate of ZnPcC4 (ZnPcC4-BSA) was prepared. Both ZnPcC4 and ZnPcC4-BSA exhibited efficient sonodynamic activities against HepG2 human hepatocarcinoma cells. Compared with ZnPcC4, conjugate ZnPcC4-BSA showed a higher sonodynamic activity with an IC50 value of 7.5μM. Upon illumination with ultrasound, ZnPcC4-BSA can induce an increase of intracellular reactive oxygen species (ROS) level, resulting in cellular apoptosis. The results suggest that the albumin conjugates of zinc(II) phthalocyanines functionalized with carboxyls can serve as promising sonosensitizers for sonodynamic therapy. Copyright © 2014 Elsevier B.V. All rights reserved.

Photodynamic effect and mechanism study of selenium-enriched phycocyanin from Spirulina platensis against liver tumours.

PubMed

Liu, Zijian; Fu, Xiang; Huang, Wei; Li, Chunxia; Wang, Xinyan; Huang, Bei

2018-03-01

Selenium-containing phycocyanin (Se-PC) has been proved to have many biological effects, including anti-inflammatory and antioxidant. In this study, we investigated the photodynamic therapy (PDT) effects of Se-PC against liver tumour in vitro and in vivo experiment. Our results demonstrated that the half lethal dose of Se-PC PDT on HepG2 cells was 100μg/ml PC containing 20% selenium. Se-PC location migration from lysosomes to mitochondria was time dependent. In in vivo experiments, the tumour inhibition rate was 75.4% in the Se-PC PDT group, compared to 52.6% in PC PDT group. Histological observations revealed that the tumour cells outside the tissue showed cellular necrosis, and those inside the tissue exhibited apoptotic nuclei and digested vacuoles in the cytoplasm after Se-PC PDT treatment. Antioxidant enzyme analysis indicated that GSH-Px activity was linked to the selenium content of Se-PC, and SOD activity was affected by PC PDT. Therefore, Se-PC PDT could induce cell death through free radical production of PDT in tumours and enhance the activity of antioxidant enzymes with selenium in vivo. The mechanism of Se-PC PDT against liver tumour involves hematocyte damage and mitochondria-mediated apoptosis accompanied with autophagy inhibition during early stage of tumour development, which displayed new prospect and offered relatively safe way for cancer therapy. Copyright © 2017 Elsevier B.V. All rights reserved.

Shikonin Exerts Antitumor Activity via Proteasome Inhibition and Cell Death Induction in vitro and in vivo

PubMed Central

Yang, Huanjie; Zhou, Ping; Huang, Hongbiao; Chen, Di; Ma, Ningfang; Cui, Cindy Qiuzhi; Shen, Shouxing; Dong, Weihua; Zhang, Xiaoyan; Lian, Wen; Wang, Xuejun; Dou, Q. Ping; Liu, Jinbao

2009-01-01

Dysregulation of the ubiquitin-proteasome pathway plays an essential role in tumor growth and development. Shikonin, a natural naphthoquinone isolated from the traditional Chinese medicine Zi Cao (gromwell), has been reported to possess tumor cell-killing activity, and results from a clinical study using a shikonin-containing mixture demonstrated its safety and efficacy for the treatment of late-stage lung cancer. In the present study, we reported that shikonin is an inhibitor of tumor proteasome activity in vitro and in vivo. Our computational modeling predicts that the carbonyl carbons C1 and C4 of shikonin potentially interact with the catalytic site of β5 chymotryptic subunit of the proteasome. Indeed, shikonin potently inhibits the chymotrypsin-like activity of purified 20S proteasome (IC50 12.5 μmol/L) and tumor cellular 26S proteasome (IC50 between 2-16 μmol/L). Inhibition of the proteasome by shikonin in murine hepatoma H22, leukemia P388 and human prostate cancer PC-3 cultures resulted in accumulation of ubiquitinated proteins and several proteasome target proapoptotic proteins (IκB-α, Bax and p27), followed by induction of cell death. Shikonin treatment resulted in tumor growth inhibition in both H22 allografts and PC-3 xenografts, associated with suppression of the proteasomal activity and induction of cell death in vivo. Finally, shikonin treatment significantly prolonged the survival period of mice bearing P388 leukemia. Our results indicate that the tumor proteasome is one of the cellular targets of shikonin, and inhibition of the proteasome activity by shikonin contributes to its anti-tumor property. PMID:19165859

Cell cycle activation in p21 dependent pathway: An alternative mechanism of organophosphate induced dopaminergic neurodegeneration.

PubMed

Wani, Willayat Yousuf; Kandimalla, Ramesh J L; Sharma, Deep Raj; Kaushal, Alka; Ruban, Anand; Sunkaria, Aditya; Vallamkondu, Jayalakshmi; Chiarugi, Alberto; Reddy, P Hemachandra; Gill, Kiran Dip

2017-07-01

In the previous study, we demonstrated that dichlorvos induces oxidative stress in dopaminergic neuronal cells and subsequent caspase activation mediates apoptosis. In the present study, we evaluated the effect and mechanism of dichlorvos induced oxidative stress on cell cycle activation in NGF-differentiated PC12 cells. Dichlorvos exposure resulted in oxidative DNA damage along with activation of cell cycle machinery in differentiated PC12 cells. Dichlorvos exposed cells exhibited an increased expression of p53, cyclin-D1, pRb and decreased expression of p21suggesting a re-entry of differentiated cells into the cell cycle. Cell cycle analysis of dichlorvos exposed cells revealed a reduction of cells in the G 0 /G 1 phase of the cell cycle (25%), and a concomitant increase of cells in S phase (30%) and G2/M phase (43.3%) compared to control PC12 cells. Further, immunoblotting of cytochrome c, Bax, Bcl-2 and cleaved caspase-3 revealed that dichlorvos induces a caspase-dependent cell death in PC12 cells. These results suggest that Dichlorvos exposure has the potential to generate oxidative stress which evokes activation of cell cycle machinery leading to apoptotic cell death via cytochrome c release from mitochondria and subsequent caspase-3 activation in differentiated PC12 cells. Copyright © 2016 Elsevier B.V. All rights reserved.

Neuromodulatory propensity of Bacopa monniera against scopolamine-induced cytotoxicity in PC12 cells via down-regulation of AChE and up-regulation of BDNF and muscarnic-1 receptor expression.

PubMed

Pandareesh, M D; Anand, T

2013-10-01

Scopolamine is a competitive antagonist of muscarinic acetylcholine receptors, and thus classified as an anti-muscarinic and anti-cholinergic drug. PC12 cell lines possess muscarinic receptors and mimic the neuronal cells. These cells were treated with different concentrations of scopolamine for 24 h and were protected from the cellular damage by pretreatment with Bacopa monniera extract (BME). In current study, we have explored the molecular mechanism of neuromodulatory and antioxidant propensity of (BME) to attenuate scopolamine-induced cytotoxicity using PC12 cells. Our results elucidate that pretreatment of PC12 cells with BME ameliorates the mitochondrial and plasma membrane damage induced by 3 μg/ml scopolamine to 54.83 and 30.30 % as evidenced by MTT and lactate dehydrogenase assays respectively. BME (100 μg/ml) ameliorated scopolamine effect by down-regulating acetylcholine esterase and up-regulating brain-derived neurotropic factor and muscarinic muscarinic-1 receptor expression. BME pretreated cells also showed significant protection against scopolamine-induced toxicity by restoring the levels of antioxidant enzymes and lipid peroxidation. This result indicates that the scopolamine-induced cytotoxicity and neuromodulatory changes were restored with the pretreatment of BME.

PCSK1 Variants and Human Obesity.

PubMed

Ramos-Molina, B; Martin, M G; Lindberg, I

2016-01-01

PCSK1, encoding prohormone convertase 1/3 (PC1/3), was one of the first genes linked to monogenic early-onset obesity. PC1/3 is a protease involved in the biosynthetic processing of a variety of neuropeptides and prohormones in endocrine tissues. PC1/3 activity is essential for the activating cleavage of many peptide hormone precursors implicated in the regulation of food ingestion, glucose homeostasis, and energy homeostasis, for example, proopiomelanocortin, proinsulin, proglucagon, and proghrelin. A large number of genome-wide association studies in a variety of different populations have now firmly established a link between three PCSK1 polymorphisms frequent in the population and increased risk of obesity. Human subjects with PC1/3 deficiency, a rare autosomal-recessive disorder caused by the presence of loss-of-function mutations in both alleles, are obese and display a complex set of endocrinopathies. Increasing numbers of genetic diagnoses of infants with persistent diarrhea has recently led to the finding of many novel PCSK1 mutations. PCSK1-deficient infants experience severe intestinal malabsorption during the first years of life, requiring controlled nutrition; these children then become hyperphagic, with associated obesity. The biochemical characterization of novel loss-of-function PCSK1 mutations has resulted in the discovery of new pathological mechanisms affecting the cell biology of the endocrine cell beyond simple loss of enzyme activity, for example, dominant-negative effects of certain mutants on wild-type PC1/3 protein, and activation of the cellular unfolded protein response by endoplasmic reticulum-retained mutants. A better understanding of these molecular and cellular pathologies may illuminate possible treatments for the complex endocrinopathy of PCSK1 deficiency, including obesity. Copyright © 2016 Elsevier Inc. All rights reserved.

Novel theranostic zinc phthalocyanine-phospholipid complex self-assembled nanoparticles for imaging-guided targeted photodynamic treatment with controllable ROS production and shape-assisted enhanced cellular uptake.

PubMed

Ma, Jinyuan; Li, Yang; Liu, Guihua; Li, Ai; Chen, Yilin; Zhou, Xinyi; Chen, Dengyue; Hou, Zhenqing; Zhu, Xuan

2018-02-01

The novel drug delivery system based on self-assembly of zinc phthalocyanine-soybean phosphatidylcholine (ZnPc-SPC) complex was developed by a co-solvent method followed by a nanoprecipitaion technique. DSPE-PEG-methotrexate (DSPE-PEG-MTX) was introduced on the surface of ZnPc-SPC self-assembled nanoparticles (ZS) to endow them with folate receptor-targeting property. NMR, XRD, FTIR, and UV-vis-NIR analysis demonstrated the weak molecular interaction between ZnPc and SPC. The ZS functionalized with DSPE-PEG-MTX (ZSPM) was successfully constructed with an average particle size of ∼170nm, a narrow size distribution, and could remain physiologically stable for at least 7days. In vitro cellular uptake and cytotoxicity studies demonstrated that ZSPM exhibited stronger cellular uptake efficacy and photodynamic cytotoxicity against HeLa and MCF-7 cells than ZS functionalized with DSPE-mPEG (ZSP) and free ZnPc. More importantly, ZSPM showed the enhanced accumulation effect at the tumor region compared with ZSP by the active-plus-passive targeting via enhanced permeability and retention (EPR) effect and folate receptor-mediated endocytosis. Furthermore, in vivo antitumor effect and histological analysis demonstrated the superior tumor growth inhibition effect of ZSPM. In addition, the needle-shape ZSP (ZSPN) exhibited better in vitro cellular uptake and in vivo tumor accumulation compared with ZSP due to the shape-assisted effect. Moreover, the interesting off-on switch effect of reactive oxygen species (ROS) production of ZnPc-SPC complex-based nanoparticles was discovered to achieve photodynamic treatment in a controllable way. These findings suggested that the ZnPc-SPC complex-based self-assembled nanoparticles could serve as a promising and effective formulation to achieve tumor-targeting fluorescence imaging and enhanced photodynamic treatment. Copyright © 2017. Published by Elsevier B.V.

Osthole Attenuates Doxorubicin-Induced Apoptosis in PC12 Cells through Inhibition of Mitochondrial Dysfunction and ROS Production

PubMed Central

Shokoohinia, Yalda; Hosseinzadeh, Leila; Moieni-Arya, Maryam; Mostafaie, Ali; Mohammadi-Motlagh, Hamid-Reza

2014-01-01

Doxorubicin (DOX) is a potent, broad-spectrum chemotherapeutic drug used for treatment of several types of cancers. Despite its effectiveness, it has a wide range of toxic side effects, many of which most likely result from its inherent prooxidant activity. It has been reported that DOX has toxic effects on normal tissues, including brain tissue. In the current study, we investigated the protective effect of osthole isolated from Prangos ferulacea (L.) Lindl. on oxidative stress and apoptosis induced by DOX in PC12 as a neuronal model cell line. PC12 cells were pretreated with osthole 2 h after treatment with different concentrations of DOX. 24 h later, the cell viability, mitochondrial membrane potential (MMP), the activity of caspase-3, the expression ratio of Bax/Bcl-2, and the generation of intracellular ROS were detected. We found that pretreatment with osthole on PC12 cells significantly reduced the loss of cell viability, the activity of caspase-3, the increase in Bax/Bcl-2 ratio, and the generation of intracellular ROS induced by DOX. Moreover, pretreatment with osthole led to an increase in MMP in PC12 cells. In conclusion, our results indicated that pretreatment with nontoxic concentrations of osthole protected PC12 cells from DOX-mediated apoptosis by inhibition of ROS production. PMID:25013759

(2S)-2-(3-(1-Carboxy-5-(4-211At-Astatobenzamido)Pentyl)Ureido)-Pentanedioic Acid for PSMA-Targeted α-Particle Radiopharmaceutical Therapy

PubMed Central

Kiess, Ana P.; Minn, Il; Vaidyanathan, Ganesan; Hobbs, Robert F.; Josefsson, Anders; Shen, Colette; Brummet, Mary; Chen, Ying; Choi, Jaeyeon; Koumarianou, Eftychia; Baidoo, Kwamena; Brechbiel, Martin W.; Mease, Ronnie C.; Sgouros, George; Zalutsky, Michael R.

2016-01-01

Alpha-particle emitters have a high linear energy transfer and short range, offering the potential for treating micrometastases while sparing normal tissues. We developed a urea-based, 211At-labeled small molecule targeting prostate-specific membrane antigen (PSMA) for the treatment of micrometastases due to prostate cancer (PC). Methods: PSMA-targeted (2S)-2-(3-(1-carboxy-5-(4-211At-astatobenzamido)pentyl)ureido)-pentanedioic acid (211At-6) was synthesized. Cellular uptake and clonogenic survival were tested in PSMA-positive (PSMA+) PC3 PIP and PSMA-negative (PSMA−) PC3 flu human PC cells after 211At-6 treatment. The antitumor efficacy of 211At-6 was evaluated in mice bearing PSMA+ PC3 PIP and PSMA– PC3 flu flank xenografts at a 740-kBq dose and in mice bearing PSMA+, luciferase-expressing PC3-ML micrometastases. Biodistribution was determined in mice bearing PSMA+ PC3 PIP and PSMA– PC3 flu flank xenografts. Suborgan distribution was evaluated using α-camera images, and microscale dosimetry was modeled. Long-term toxicity was assessed in mice for 12 mo. Results: 211At-6 treatment resulted in PSMA-specific cellular uptake and decreased clonogenic survival in PSMA+ PC3 PIP cells and caused significant tumor growth delay in PSMA+ PC3 PIP flank tumors. Significantly improved survival was achieved in the newly developed PSMA+ micrometastatic PC model. Biodistribution showed uptake of 211At-6 in PSMA+ PC3 PIP tumors and in kidneys. Microscale kidney dosimetry based on α-camera images and a nephron model revealed hot spots in the proximal renal tubules. Long-term toxicity studies confirmed that the dose-limiting toxicity was late radiation nephropathy. Conclusion: PSMA-targeted 211At-6 α-particle radiotherapy yielded significantly improved survival in mice bearing PC micrometastases after systemic administration. 211At-6 also showed uptake in renal proximal tubules resulting in late nephrotoxicity, highlighting the importance of long-term toxicity studies and microscale dosimetry. PMID:27230930

Phthalocyanines as photosensitizing agents for tumors--mechanism of action

NASA Astrophysics Data System (ADS)

Ben-Hur, Ehud

1994-03-01

Aluminum phthalocyanine (AlPc) is a second-generation photosensitizer under study for photodynamic therapy (PDT) of cancer. Its mechanism of action is not known. Fluoride appears to be a powerful probe for the mechanistic study of AlPc derivatives. F- forms a complex with the Al ligand, resulting in drastically reduced AlPc-induced phototoxicity. This is due to a modified binding of AlPc with certain target proteins, resulting in inhibition of electron transfer reactions (type I) but not singlet oxygen reactions (type II). In Chinese hamster ovary (CHO) cell membranes, Na+/K+-ATPase activity is selectively protected by F- from photosensitized inhibition by AlPc, suggesting that this enzyme may be a critical target for AlPc-PDT. Another cellular response, not interfered with by F-, is a transient increase of cytoplasmic free Ca2+ after AlPc-PDT. This increase was shown to trigger the induction of a recovery process.

Cholecystokinin-8 inhibits methamphetamine-induced neurotoxicity via an anti-oxidative stress pathway.

PubMed

Wen, Di; An, Meiling; Gou, Hongyan; Liu, Xia; Liu, Li; Ma, Chunling; Cong, Bin

2016-12-01

As a powerful addictive psychostimulant drug, coupled with its neurotoxicity, methamphetamine (METH) abuse may lead to long-lasting abnormalities in brain structure and function. We found that pretreatment of cholecystokinin-8 (CCK-8) inhibited METH-induced brain cellular dopaminergic (DA) damage in the striatum and substantia nigra, and related behavioural deficits and hyperthermia. However, the mechanism of CCK-8 action on METH-induced toxicity is not clear. The aim of this study was to explore whether the possible protective effect of CCK-8 on METH-induced neurotoxicity involved anti-oxidative stress mechanisms. The subtypes of CCK receptors mediating the regulatory action of CCK-8 were also investigated. The present results revealed that CCK-8 dose-dependently inhibited METH-induced cytotoxic effect by activating the CCK2 receptor subtype in PC12 cells and CCK2 receptor stable transfected-HEK293 cells. Pre-treatment of CCK-8 before METH stimulation significantly attenuated the generation of reactive oxygen species and NADPH oxidase activation in PC12 cells. In conclusion, our study demonstrated a protective effect of CCK-8 on METH-induced neurotoxicity in vitro and suggested that a possible mechanism of this action was dependent on the activation of the CCK2 receptor to reduce the neurotoxicity and oxidative stress induced by METH stimulation. Copyright © 2016 Elsevier B.V. All rights reserved.

Development of a Sono-Assembled, Bifunctional Soy Peptide Nanoparticle for Cellular Delivery of Hydrophobic Active Cargoes.

PubMed

Zhang, Yuanhong; Zhao, Mouming; Ning, Zhengxiang; Yu, Shujuan; Tang, Ning; Zhou, Feibai

2018-04-25

Soy proteins are prone to aggregate upon proteolysis, hindering their sustainable development in food processing. Here, a continuous work on the large insoluble peptide aggregates was carried out, aiming to develop a new type of soy peptide-based nanoparticle (SPN) for active cargo delivery. Sono-assembled SPN in spherical appearance and core-shell structure maintained by noncovalent interactions was successfully fabricated, exhibiting small particle size (103.95 nm) in a homogeneous distribution state (PDI = 0.18). Curcumin as a model cargo was efficiently encapsulated into SPN upon sonication, showing high water dispersity (129.6 mg/L, 10 4 higher than its water solubility) and storage stability. Additionally, the pepsin-resistant SPN contributed to the controlled release of curcumin at the intestinal phase and thus significantly improved the bioaccessibility. Encapsulated curcumin was effective in protecting glutamate-induced toxicity in PC12 cells, where the matrix SPN can simultaneously reduce lipid peroxidation and elevate antioxidant enzymes levels, innovatively demonstrating its bifunctionality during cellular delivery.

Comparison of PC12 and Cerebellar Granule Cell Cultures for Evaluating Neurite Outgrowth Using High Content Screening

EPA Science Inventory

Development of high-throughput assays for chemical screening and hazard identification is a pressing priority worldwide. One approach uses in vitro, cell-based assays which recapitulate biological events observed in vivo. Neurite outgrowth is one such critical cellular process un...

BAG3 is involved in neuronal differentiation and migration.

PubMed

Santoro, Antonietta; Nicolin, Vanessa; Florenzano, Fulvio; Rosati, Alessandra; Capunzo, Mario; Nori, Stefania L

2017-05-01

Bcl2-associated athanogene 3 (BAG3) protein belongs to the family of co-chaperones interacting with several heat shock proteins. It plays a key role in protein quality control and mediates the clearance of misfolded proteins. Little is known about the expression and cellular localization of BAG3 during nervous system development and differentiation. Therefore, we analyze the subcellular distribution and expression of BAG3 in nerve-growth-factor-induced neurite outgrowth in PC12 cells and in developing and adult cortex of mouse brain. In differentiated PC12 cells, BAG3 was localized mainly in the neuritic domain rather than the cell body, whereas in control cells, it appeared to be confined to the cytoplasm near the nuclear membrane. Interestingly, the change of BAG3 localization during neuronal differentiation was associated only with a slight increase in total BAG3 expression. These data were coroborated by transmission electron microscopy showing that BAG3 was confined mainly within large dense-core vesicles of the axon in differentiated PC12 cells. In mouse developing cortex, BAG3 appeared to be intensely expressed in cellular processes of migrating cells, whereas in adult brain, a diffuse expression of low to medium intensity was detected in neuronal cell bodies. These findings suggest that BAG3 expression is required for neuronal differentiation and migration and that its role is linked to a change in its distribution pattern rather than to an increase in its protein expression levels.

Assessment of tris (1, 3-dichloro-2-propyl) phosphate toxicology in PC12 cells by using digital gene expression profiling.

PubMed

Li, Li; Jiang, Shuai; Li, Kang; Lin, Bencheng; Wang, Ziyu; Zhang, Zhiqing; Fang, Yanjun

2017-09-01

Tris (1,3-dichloro-2-propyl) phosphate (TDCIPP), one of the most universally used organophosphate flame retardants (OPFRs), is an environmental pollutant. However, limited information is available regarding its toxicity and environmental health risk. In the present study, PC12 cells provided a useful model for the evaluation of the toxic effects of TDCIPP. Exposure to 7.5, 15, 30, or 60 μM TDCIPP for 72 h inhibited cell viability, and enhanced cellular apoptosis and oxidative stress. To further explore the underlying mechanisms, digital gene expression (DGE) technology was used to identify early transcriptional changes following TDCIPP exposure. Expression of the transcripts of 161 genes was significantly altered upon treatment with TDCIPP. Functional and pathway analysis of the transcriptional profile demonstrated that genes showing significant TDCIPP-associated changes in expression were involved in the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway, extracellular matrix-receptor interactions, protein digestion and absorption, and microRNAs in cancer. Using quantitative real-time PCR, we validated the differential expression of selected genes. These results showed that the expression profiles of cells exposed to 60 μM TDCIPP were consistent with the DGE data. Furthermore, western blotting showed that treatment with TDCIPP reduced the Bcl-2/Bax ratio and attenuated PI3K/Akt/Myc signaling. Taken together, these data suggest that TDCIPP exposure can reduce cell viability and induce apoptosis in PC12 cells by inhibiting activation of the PI3K/Akt/Myc signaling pathway. These observations provide valuable preliminary information regarding the mechanisms of TDCIPP-induced toxicity in PC12 cells and indicate that further study of the toxicity of other environmental OPFRs is warranted. Copyright © 2017 Elsevier Ltd. All rights reserved.

Ninjin'yoeito and ginseng extract prevent oxaliplatin-induced neurodegeneration in PC12 cells.

PubMed

Suzuki, Toshiaki; Yamamoto, Ayano; Ohsawa, Masahiro; Motoo, Yoshiharu; Mizukami, Hajime; Makino, Toshiaki

2015-10-01

Ninjin'yoeito (NYT) is a formula of Japanese traditional kampo medicine composed of 12 crude drugs, and is designed to improve the decline in constitution after recovery from disease, fatigue, anemia, anorexia, perspiration during sleep, cold limbs, slight fever, chills, persistent cough, malaise, mental disequilibrium, insomnia, and constipation. Oxaliplatin (L-OHP) is a platinum-based anticancer drug used to treat colorectal, pancreatic, and stomach cancers. However, it often causes acute and chronic peripheral neuropathies including cold allodynia and mechanical hyperalgesia. In this study, we investigated the preventive effects of NYT on neuronal degeneration caused by L-OHP using PC12 cells, which are derived from the rat adrenal medulla and differentiate into nerve-like cells after exposure to nerve growth factor. L-OHP treatment decreased the elongation of neurite-like projection outgrowths in differentiated PC12 cells. When PC12 cells were treated with NYT hot water extract, neurodegeneration caused by L-OHP was significantly prevented in a concentration-dependent manner. Among the 12 crude drugs composing NYT, the extract of Ginseng (the root of Panax ginseng) exhibited the strongest preventive effects on neurodegeneration in differentiated PC12 cells. By activity-guided fractionation, we found that the fraction containing ginsenosides displayed preventive activity and, among several ginsenosides, ginsenoside F2 exhibited significant preventive effects on L-OHP-induced decreases in neurite-like outgrowths in differentiated PC12 cells. These results suggest that NYT and ginseng are promising agents for preventing L-OHP-induced neuropathies and present ginsenoside F2 as one of the active ingredients in ginseng.

Protective effects of fractions from Artemisia biennis hydro-ethanolic extract against doxorubicin-induced oxidative stress and apoptosis in PC12 cells

PubMed Central

Mojarrab, Mahdi; Mehrabi, Mehran; Ahmadi, Farahnaz; Hosseinzadeh, Leila

2016-01-01

Objective(s): This study was designed to indicate whether different fractions from Artemisia biennis hydroethanolic extract could provide cytoprotection against oxidative stress and apoptosis induced by doxorubicin (DOX) in rat pheochromocytoma cell line (PC12). Material and Methods: Cell viability was determined by MTT assay. Also, activation of caspase-3 and superoxide dismutase were evaluated by spectrophotometry. Detection of reactive oxygen species (ROS) and measurement of mitochondrial membrane potential (MMP) were performed by flowcytometry. Results: Treatment of PC12 cells with DOX reduced viability dose dependently. For evaluation of the effect of fractions (A-G) on DOX-induced cytotoxicity, PC12 cells were pretreated for 24 hr with the A. biennis fractions and then cells were treated with DOX. The fractions C and D increased PC12 cells viability significantly compared to DOX treated cells. Moreover, pretreatment with fractions C and D for 24 hr attenuated DOX-mediated apoptosis and the anti-apoptotic action of A. biennis fractions was partially dependent on inhibition of caspase 3 activity and also increasing the mitochondrial membrane potential (MMP). Selected A. biennis fractions also suppressed the generation of ROS and increased superoxide dismutase (SOD) activity. Conclusion: Taken together our observation indicated that subtoxic concentration of aforementioned fractions of A. biennis hydroetanolic extract has protective effect against apoptosis induced by DOX in PC12 cell. The results highlighted that fractions C and D may exert cytoprotective effects through their antioxidant actions. PMID:27403257

Protective effects of fractions from Artemisia biennis hydro-ethanolic extract against doxorubicin-induced oxidative stress and apoptosis in PC12 cells.

PubMed

Mojarrab, Mahdi; Mehrabi, Mehran; Ahmadi, Farahnaz; Hosseinzadeh, Leila

2016-05-01

This study was designed to indicate whether different fractions from Artemisia biennis hydroethanolic extract could provide cytoprotection against oxidative stress and apoptosis induced by doxorubicin (DOX) in rat pheochromocytoma cell line (PC12). Cell viability was determined by MTT assay. Also, activation of caspase-3 and superoxide dismutase were evaluated by spectrophotometry. Detection of reactive oxygen species (ROS) and measurement of mitochondrial membrane potential (MMP) were performed by flowcytometry. Treatment of PC12 cells with DOX reduced viability dose dependently. For evaluation of the effect of fractions (A-G) on DOX-induced cytotoxicity, PC12 cells were pretreated for 24 hr with the A. biennis fractions and then cells were treated with DOX. The fractions C and D increased PC12 cells viability significantly compared to DOX treated cells. Moreover, pretreatment with fractions C and D for 24 hr attenuated DOX-mediated apoptosis and the anti-apoptotic action of A. biennis fractions was partially dependent on inhibition of caspase 3 activity and also increasing the mitochondrial membrane potential (MMP). Selected A. biennis fractions also suppressed the generation of ROS and increased superoxide dismutase (SOD) activity. Taken together our observation indicated that subtoxic concentration of aforementioned fractions of A. biennis hydroetanolic extract has protective effect against apoptosis induced by DOX in PC12 cell. The results highlighted that fractions C and D may exert cytoprotective effects through their antioxidant actions.

Development of microarray device for functional evaluation of PC12D cell axonal extension ability

NASA Astrophysics Data System (ADS)

Nakamachi, Eiji; Yanagimoto, Junpei; Murakami, Shinya; Morita, Yusuke

2014-04-01

In this study, we developed a microarray bio-MEMS device that could trap PC12D (rat pheochromocytoma cells) cells to examine the intercellular interaction effect on the cell activation and the axonal extension ability. This is needed to assign particular patterns of PC12D cells to establish a cell functional evaluation technique. This experimental observation-based technique can be used for design of the cell sheet and scaffold for peripheral and central nerve regeneration. We have fabricated a micropillar-array bio-MEMS device, whose diameter was approximately 10 μm, by using thick photoresist SU-8 on the glass slide substrate. A maximum trapped PC12D cell ratio, 48.5%, was achieved. Through experimental observation of patterned PC12D "bi-cells" activation, we obtained the following results. Most of the PC12D "bi-cells" which had distances between 40 and 100 μm were connected after 24 h with a high probability. On the other hand, "bi-cells" which had distances between 110 and 200 μm were not connected. In addition, we measured axonal extension velocities in cases where the intercellular distance was between 40 and 100 μm. A maximum axonal extension velocity, 86.4 μm/h, was obtained at the intercellular distance of 40 μm.

Fisetin Protects PC12 Cells from Tunicamycin-Mediated Cell Death via Reactive Oxygen Species Scavenging and Modulation of Nrf2-Driven Gene Expression, SIRT1 and MAPK Signaling in PC12 Cells.

PubMed

Yen, Jui-Hung; Wu, Pei-Shan; Chen, Shu-Fen; Wu, Ming-Jiuan

2017-04-17

Fisetin (3,7,3',4'-tetrahydroxyflavone) is a dietary flavonol and exhibits antioxidant, anti-inflammatory, and neuroprotective activities. However, high concentration of fisetin is reported to produce reactive oxygen species (ROS), induce endoplasmic reticulum (ER) stress and cause cytotoxicity in cancer cells. The aim of this study is to investigate the cytoprotective effects of low concentration of fisetin against tunicamycin (Tm)-mediated cytotoxicity in neuronal-like catecholaminergic PC12 cells. Cell viability was assayed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and apoptotic and autophagic markers were analyzed by Western blot. Gene expression of unfolded protein response (UPR) and Phase II enzymes was further investigated using RT-Q-PCR or Western blotting. Intracellular ROS level was measured using 2',7'-dichlorodihydrofluorescein diacetate (H₂DCFDA) by a fluorometer. The effects of fisetin on mitogen activated protein kinases (MAPKs) and SIRT1 (Sirtuin 1) signaling pathways were examined using Western blotting and specific inhibitors. Fisetin (<20 µM) restored cell viability and repressed apoptosis, autophagy and ROS production in Tm-treated cells. Fisetin attenuated Tm-mediated expression of ER stress genes, such as glucose-regulated proteins 78 (GRP78), C/EBP homologous protein (CHOP also known as GADD153) and Tribbles homolog 3 (TRB3), but induced the expression of nuclear E2 related factor (Nrf)2-targeted heme oxygenase (HO)-1, glutamate cysteine ligase (GCL) and cystine/glutamate transporter (xCT/SLC7A11), in both the presence and absence of Tm. Moreover, fisetin enhanced phosphorylation of ERK (extracellular signal-regulated kinase), JNK (c-JUN NH₂-terminal protein kinase), and p38 MAPK. Addition of JNK and p38 MAPK inhibitor significantly antagonized its cytoprotective activity and modulatory effects on UPR. Fisetin also restored Tm-inhibited SIRT1 expression and addition of sirtinol (SIRT1 activation inhibitor) significantly blocked fisetin-mediated cytoprotection. In conclusion, this result shows that fisetin activates Nrf2, MAPK and SIRT1, which may elicit adaptive cellular stress response pathways so as to protect cells from Tm-induced cytotoxicity.

Design and synthesis of formononetin-dithiocarbamate hybrids that inhibit growth and migration of PC-3 cells via MAPK/Wnt signaling pathways.

PubMed

Fu, Dong-Jun; Zhang, Li; Song, Jian; Mao, Ruo-Wang; Zhao, Ruo-Han; Liu, Ying-Chao; Hou, Yu-Hui; Li, Jia-Huan; Yang, Jia-Jia; Jin, Cheng-Yun; Li, Ping; Zi, Xiao-Lin; Liu, Hong-Min; Zhang, Sai-Yang; Zhang, Yan-Bing

2017-02-15

A series of novel formononetin-dithiocarbamate derivatives were designed, synthesized and evaluated for antiproliferative activity against three selected cancer cell line (MGC-803, EC-109, PC-3). The first structure-activity relationship (SAR) for this formononetin-dithiocarbamate scaffold is explored in this report with evaluation of 14 variants of the structural class. Among these analogues, tert-butyl 4-(((3-((3-(4-methoxyphenyl)-4-oxo-4H-chromen-7-yl)oxy)propyl)thio)carbonothioyl)piperazine-1-carboxylate (8i) showed the best inhibitory activity against PC-3 cells (IC 50  = 1.97 μM). Cellular mechanism studies elucidated 8i arrests cell cycle at G1 phase and regulates the expression of G1 checkpoint-related proteins in concentration-dependent manners. Furthermore, 8i could inhibit cell growth via MAPK signaling pathway and inhibit migration via Wnt pathway in PC-3 cells. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

NEURODEVELOPMENTAL TOXICITY AND METABOLISM OF THE FLAME RETARDANT, TRIS (1,3-DICHLORO-2-PROPYL) PHOSPHATE

EPA Science Inventory

In the PC12 cell studies, it was found that exposure to an equimolar concentration of TDCPP resulted in deficits equivalent to or greater than that of CPF on measures of cellular replication and cell number, without affecting cell viability. TDCPP also altered the pattern o...

3-Bromopyruvate induces rapid human prostate cancer cell death by affecting cell energy metabolism, GSH pool and the glyoxalase system.

PubMed

Valenti, Daniela; Vacca, Rosa A; de Bari, Lidia

2015-12-01

3-bromopyruvate (3-BP) is an anti-tumour drug effective on hepatocellular carcinoma and other tumour cell types, which affects both glycolytic and mitochondrial targets, depleting cellular ATP pool. Here we tested 3-BP on human prostate cancer cells showing, differently from other tumour types, efficient ATP production and functional mitochondrial metabolism. We found that 3-BP rapidly induced cultured androgen-insensitive (PC-3) and androgen-responsive (LNCaP) prostate cancer cell death at low concentrations (IC(50) values of 50 and 70 μM, respectively) with a multimodal mechanism of action. In particular, 3-BP-treated PC-3 cells showed a selective, strong reduction of glyceraldeide 3-phosphate dehydrogenase activity, due to the direct interaction of the drug with the enzyme. Moreover, 3-BP strongly impaired both glutamate/malate- and succinate-dependent mitochondrial respiration, membrane potential generation and ATP synthesis, concomitant with the inhibition of respiratory chain complex I, II and ATP synthase activities. The drastic reduction of cellular ATP levels and depletion of GSH pool, associated with significant increase in cell oxidative stress, were found after 3-BP treatment of PC-3 cells. Interestingly, the activity of both glyoxalase I and II, devoted to the elimination of the cytotoxic methylglyoxal, was strongly inhibited by 3-BP. Both N-acetylcysteine and aminoguanidine, GSH precursor and methylglyoxal scavenger, respectively, prevented 3-BP-induced PC-3 cell death, showing that impaired cell antioxidant and detoxifying capacities are crucial events leading to cell death. The provided information on the multi-target cytotoxic action of 3-BP, finally leading to PC-3 cell necrosis, might be useful for future development of 3-BP as a therapeutic option for prostate cancer treatment.

Schisandrin B protects PC12 cells by decreasing the expression of amyloid precursor protein and vacuolar protein sorting 35★

PubMed Central

Yan, Mingmin; Mao, Shanping; Dong, Huimin; Liu, Baohui; Zhang, Qian; Pan, Gaofeng; Fu, Zhiping

2012-01-01

PC12 cell injury was induced using 20 μM amyloid β-protein 25–35 to establish a model of Alzheimer's disease. The cells were then treated with 5, 10, and 25 μM Schisandrin B. Methylthiazolyldiphenyl-tetrazolium bromide assays and Hoechst 33342 staining results showed that with increasing Schisandrin B concentration, the survival rate of PC12 cells injured by amyloid β-protein 25–35 gradually increased and the rate of apoptosis gradually decreased. Reverse transcription-PCR, immunocytochemical staining and western blot results showed that with increasing Schisandrin B concentration, the mRNA and protein expression of vacuolar protein sorting 35 and amyloid precursor protein were gradually decreased. Vacuolar protein sorting 35 and amyloid precursor protein showed a consistent trend for change. These findings suggest that 5, 10, and 25 μM Schisandrin B antagonizes the cellular injury induced by amyloid β-protein 25–35 in a dose-dependent manner. This may be caused by decreasing the expression of vacuolar protein sorting 35 and amyloid precursor protein. PMID:25745458

Neuronal responses to vector-averaged gravity: a search for gravisensing and adaptation mechanisms--a preliminary report.

PubMed

Gruener, R

1998-01-01

This paper serves as a milepost in our work using the clinostat as a tool for mimicking certain aspects of altered gravity conditions (vector-nulled gravity) in order to gain insights into the adaptation of cells (and hence organisms) to the microgravity environment of space. I review here recent data, limited to cellular adaptation to altered gravity environments, from others in the field, and including some of our work using the clinostat and from spaceflight experiments. Finally, I report here preliminary results of experiments, carried out initially at Nagoya University's RIEM with follow-up experiments at the University of Arizona, to test the applicability of PC12 cells as neuronal models in which to assess adaptation to altered gravity conditions. PC12 (phaeochromocytoma) cells were used to examine two central hypotheses. The first is that the ubiquity of the cytoskeletally tethered nucleus of cells serves as a general gravisensing device which may be incidental to its other, more central genomic control-role. The second hypothesis is that the clinostat is a useful, earthbound platform on which to carry out space-biology relevant experiments in preparation for testing in space flights. PC12 cells were triggered to differentiate, into neuron-like cells, by the addition of Nerve Growth Factor (NGF) to the culture medium within 4-6 hours after cell plating and just before mounting cultures on the clinostat and control devices. Cultures, in 60 mm or 35 mm polylysine-coated dishes, were subjected to clinorotation, centrifugal force, motional controls and shear-turbulence control conditions for varying periods. Experiments were carried out at 37 degrees C. Cell morphology (including neurite characteristics) and gene activation were examined. Cytoskeletal integrity was assessed from the staining of tubulin and actin filaments. Confocal microscopy in combination with fluorescence monitoring was undertaken. At this point of the investigation, only preliminary data can be presented. This is due to various technical problems and the need to carry out rigorous statistical tests. Still, the preliminary data are of interest because they form the foundation for interpretation against the background of cellular gravisensing and adaptation to gravitational perturbations.

The selective and inducible activation of endogenous PI 3-kinase in PC12 cells results in efficient NGF-mediated survival but defective neurite outgrowth.

PubMed

Ashcroft, M; Stephens, R M; Hallberg, B; Downward, J; Kaplan, D R

1999-08-12

The Trk/Nerve Growth Factor receptor mediates the rapid activation of a number of intracellular signaling proteins, including phosphatidylinositol 3-kinase (PI 3-kinase). Here, we describe a novel, NGF-inducible system that we used to specifically address the signaling potential of endogenous PI 3-kinase in NGF-mediated neuronal survival and differentiation processes. This system utilizes a Trk receptor mutant (Trk(def)) lacking sequences Y490, Y785 and KFG important for the activation of the major Trk targets; SHC, PLC-gammal, Ras, PI 3-kinase and SNT. Trk(def) was kinase active but defective for NGF-induced responses when stably expressed in PC12nnr5 cells (which lack detectable levels of TrkA and are non-responsive to NGF). The PI 3-kinase consensus binding site, YxxM (YVPM), was introduced into the insert region within the kinase domain of Trk(def). NGF-stimulated tyrosine phosphorylation of the Trk(def)+PI 3-kinase addback receptor, resulted in the direct association and selective activation of PI 3-kinase in vitro and the production of PI(3,4)P2 and PI(3,4,5)P3 in vivo (comparable to wild-type). PC12nnr5 cells stably expressing Trk(def) + PI 3-kinase, initiated neurite outgrowth but failed to stably extend and maintain these neurites in response to NGF as compared to PC12 parental cells, or PC12nnr5 cells overexpressing wild-type Trk. However, Trk(def) + PI 3-kinase was fully competent in mediating NGF-induced survival processes. We propose that while endogenous PI 3-kinase can contribute in part to neurite initiation processes, its selective activation and subsequent signaling to downstream effectors such as Akt, functions mainly to promote cell survival in the PC12 system.

Therapeutic Molecules and Endogenous Ligands Regulate the Interaction between Brain Cellular Prion Protein (PrPC) and Metabotropic Glutamate Receptor 5 (mGluR5)*

PubMed Central

Haas, Laura T.; Kostylev, Mikhail A.; Strittmatter, Stephen M.

2014-01-01

Soluble Amyloid-β oligomers (Aβo) can trigger Alzheimer disease (AD) pathophysiology by binding to cell surface cellular prion protein (PrPC). PrPC interacts physically with metabotropic glutamate receptor 5 (mGluR5), and this interaction controls the transmission of neurotoxic signals to intracellular substrates. Because the interruption of the signal transduction from PrPC to mGluR5 has therapeutic potential for AD, we developed assays to explore the effect of endogenous ligands, agonists/antagonists, and antibodies on the interaction between PrPC and mGluR5 in cell lines and mouse brain. We show that the PrPC segment of amino acids 91–153 mediates the interaction with mGluR5. Agonists of mGluR5 increase the mGluR5-PrPC interaction, whereas mGluR5 antagonists suppress protein association. Synthetic Aβo promotes the protein interaction in mouse brain and transfected HEK-293 cell membrane preparations. The interaction of PrPC and mGluR5 is enhanced dramatically in the brains of familial AD transgenic model mice. In brain homogenates with Aβo, the interaction of PrPC and mGluR5 is reversed by mGluR5-directed antagonists or antibodies directed against the PrPC segment of amino acids 91–153. Silent allosteric modulators of mGluR5 do not alter Glu or basal mGluR5 activity, but they disrupt the Aβo-induced interaction of mGluR5 with PrPC. The assays described here have the potential to identify and develop new compounds that inhibit the interaction of PrPC and mGluR5, which plays a pivotal role in the pathogenesis of Alzheimer disease by transmitting the signal from extracellular Aβo into the cytosol. PMID:25148681

A pH-sensitive micelle composed of heparin, phospholipids, and histidine as the carrier of photosensitizers: Application to enhance photodynamic therapy of cancer.

PubMed

Debele, Tilahun Ayane; Mekuria, Shewaye Lakew; Tsai, Hsieh-Chih

2017-05-01

In this study, we describe the synthesis of a stable, pH-sensitive micelle composed of heparin, 1, 2-distearoyl-sn-glycerol-3-phosphoethanolamine, and l-histidine (HDH) through 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)/N-hydroxysuccinimide (NHS) chemistry. 1 H-Nuclear magnetic resonance (NMR) and Fourier transform infrared spectroscopy (FTIR) analyses confirmed the formation of HDH copolymers and dynamic light scattering (DLS) measurements indicated a particle size of 111.57±12.36nm and zeta potential of -59.8±5.2mV for the nanoparticles. The drug-loading and encapsulation efficiency of the micelles were 14.52±1.2% and 65.47±1.87%, respectively. Drug release studies showed approximately 91% zinc phthalocyanine (ZnPc) release from micelles in acidic conditions (pH 5.0) in comparison with 63% in physiological conditions (pH 7.4) after 96h of incubation. Singlet oxygen ( 1 O 2 ) detection revealed that the micelles prevented ZnPc aggregation and enhanced 1 O 2 generation. Cellular uptake of ZnPc-loaded micelles (ZnPc-HDH) was observed using confocal microscopy. Phototoxicity experiments in HeLa cells showed that ZnPc-loaded micelles had higher toxicity than that shown by the same concentration of free ZnPc. Hence, pH-sensitive HDH micelles are a promising carrier for hydrophobic ZnPc and improving PDT efficacy. Copyright © 2017 Elsevier B.V. All rights reserved.

Phosphatidylinositol transfer protein beta displays minimal sphingomyelin transfer activity and is not required for biosynthesis and trafficking of sphingomyelin.

PubMed

Ségui, Bruno; Allen-Baume, Victoria; Cockcroft, Shamshad

2002-08-15

Mammalian phosphatidylinositol transfer proteins (PITPs) alpha and beta, which share 77% identity, have been shown to exhibit distinct lipid-transfer activities. In addition to transferring phosphatidylinositol (PI) and phosphatidylcholine (PC), PITPbeta has been shown to transfer sphingomyelin (SM), and this has led to the suggestion that PITPbeta is important for the regulation of SM metabolism. In the present study, we have analysed the ability of human PITPbeta to transfer and regulate the metabolism of cellular SM. We report that, in vitro, the two PITP isoforms were comparable in mediating PI, PC or SM transfer. Using permeabilized HL-60 cells as the donor compartment, both PITP isoforms efficiently transferred PI and PC, and were slightly active towards SM, with the activity of PITPbeta being slightly greater. To identify which cellular lipids were selected by PITPs, PITPalpha and PITPbeta were exposed to permeabilized HL-60 cells, and subsequently repurified and analysed for their bound lipids. Both PITPs were able to select only PI and PC, but not SM. SM synthesis takes place at the Golgi, and PITPbeta was shown to localize in that compartment. To examine the role of PITPbeta in SM biosynthesis, Golgi membranes were used. Purified Golgi membranes had lost their endogenous PITPbeta, but were able to recruit PITPbeta when added exogenously. However, PITPbeta did not enhance the activities of either SM synthase or glucosylceramide synthase. Further analysis in COS-7 cells overexpressing PITPbeta showed no effects on (a) SM and glucosylceramide biosynthesis, (b) diacylglycerol or ceramide levels, (c) SM transport from the Golgi to the plasma membrane, or (d) resynthesis of SM after exogenous sphingomyelinase treatment. Altogether, these observations do not support the suggestion that PITPbeta participates in the transfer of SM, the regulation of SM biosynthesis or its intracellular trafficking.

Cellular prion protein promotes glucose uptake through the Fyn-HIF-2α-Glut1 pathway to support colorectal cancer cell survival.

PubMed

Li, Qing-Quan; Sun, Yan-Ping; Ruan, Can-Ping; Xu, Xin-Yun; Ge, Jun-Hui; He, Jin; Xu, Zu-De; Wang, Qiang; Gao, Wen-Chao

2011-02-01

Cellular prion protein (PrPc) is a glycosylphosphatidylinositol-anchored membrane protein that has various physical functions, including protection against apoptotic and oxidative stress, cellular uptake of copper ions, transmembrane signaling, and adhesion to the extracellular matrix. In this study, we show that PrPc is highly expressed in colorectal adenocarcinomas. Transcriptome profiling of PrPc-depleted DLD-1 cells revealed downregulation of glucose transporter 1 (Glut1). PrPc is shown to be involved in regulating Glut1 expression through the Fyn-HIF-2α pathway. As Glut1 is the natural transporter of glucose and is required for the high glycolytic rate seen in colorectal tumors, silencing of PrPc reduced the proliferation and survival rate of colorectal cancer cells in vitro. In vivo, knockdown of PrPc by hydrodynamic injection with a cocktail of PrPc-shRNA-encoding plasmids also inhibited tumorigenicity in a xenograft model in nude mice. In summary, our data characterize a novel molecular mechanism that links PrPc expression to the regulation of glycolysis. Targeting PrPc will therefore be a promising strategy to overcome the growth and survival advantage in colorectal tumors. © 2010 Japanese Cancer Association.

Design, synthesis, and biological evaluation of novel EF24 and EF31 analogs as potential IκB kinase β inhibitors for the treatment of pancreatic cancer.

PubMed

Xie, Xuemeng; Tu, Jinfu; You, Heyi; Hu, Bingren

2017-01-01

Given the important role that inhibitory kappa B (IκB) kinase β (IKKβ) plays in pancreatic cancer (PC) development and progression, inhibitors targeting IKKβ are believed to be increasingly popular as novel anti-PC therapies. Two synthetic molecules, named EF24 and EF31 , exhibited favorable potential in terms of inhibition of both IKKβ activity and PC cell proliferation. Aiming to enhance their cellular efficacy and to analyze their structure-activity relationship, four series of EF24 and EF31 analogs were designed and synthesized. Through kinase activity and vitality screening of cancer cells, D6 displayed excellent inhibition of both IKKβ activity and PC cell proliferation. Additionally, multiple biological evaluations showed that D6 was directly bound to IKKβ and significantly suppressed the activation of the IKKβ/nuclear factor κB pathway induced by tumor necrosis factor-α, as well as effectively inducing cancer cell apoptosis. Moreover, molecular docking and molecular dynamics simulation analysis indicated that the dominant force between D6 and IKKβ comprised hydrophobic interactions. In conclusion, D6 may be a promising therapeutic agent for PC treatment and it also provides a structural lead for the design of novel IKKβ inhibitors.

Design, synthesis, and biological evaluation of novel EF24 and EF31 analogs as potential IκB kinase β inhibitors for the treatment of pancreatic cancer

PubMed Central

Xie, Xuemeng; Tu, Jinfu; You, Heyi; Hu, Bingren

2017-01-01

Given the important role that inhibitory kappa B (IκB) kinase β (IKKβ) plays in pancreatic cancer (PC) development and progression, inhibitors targeting IKKβ are believed to be increasingly popular as novel anti-PC therapies. Two synthetic molecules, named EF24 and EF31, exhibited favorable potential in terms of inhibition of both IKKβ activity and PC cell proliferation. Aiming to enhance their cellular efficacy and to analyze their structure–activity relationship, four series of EF24 and EF31 analogs were designed and synthesized. Through kinase activity and vitality screening of cancer cells, D6 displayed excellent inhibition of both IKKβ activity and PC cell proliferation. Additionally, multiple biological evaluations showed that D6 was directly bound to IKKβ and significantly suppressed the activation of the IKKβ/nuclear factor κB pathway induced by tumor necrosis factor-α, as well as effectively inducing cancer cell apoptosis. Moreover, molecular docking and molecular dynamics simulation analysis indicated that the dominant force between D6 and IKKβ comprised hydrophobic interactions. In conclusion, D6 may be a promising therapeutic agent for PC treatment and it also provides a structural lead for the design of novel IKKβ inhibitors. PMID:28553074

Role of the Cellular Prion Protein in Oligodendrocyte Precursor Cell Proliferation and Differentiation in the Developing and Adult Mouse CNS

PubMed Central

Bribián, Ana; Gavín, Rosalina; Reina, Manuel; García-Verdugo, José Manuel; Torres, Juan María; de Castro, Fernando; del Río, José Antonio

2012-01-01

There are numerous studies describing the signaling mechanisms that mediate oligodendrocyte precursor cell (OPC) proliferation and differentiation, although the contribution of the cellular prion protein (PrPc) to this process remains unclear. PrPc is a glycosyl-phosphatidylinositol (GPI)-anchored glycoprotein involved in diverse cellular processes during the development and maturation of the mammalian central nervous system (CNS). Here we describe how PrPc influences oligodendrocyte proliferation in the developing and adult CNS. OPCs that lack PrPc proliferate more vigorously at the expense of a delay in differentiation, which correlates with changes in the expression of oligodendrocyte lineage markers. In addition, numerous NG2-positive cells were observed in cortical regions of adult PrPc knockout mice, although no significant changes in myelination can be seen, probably due to the death of surplus cells. PMID:22529900

Intracellular distribution and mechanisms of actions of photosensitizer Zinc(II)-phthalocyanine solubilized in Cremophor EL against human hepatocellular carcinoma HepG2 cells.

PubMed

Shao, Jingwei; Dai, Yongchao; Zhao, Wenna; Xie, Jingjing; Xue, Jinping; Ye, Jianhui; Jia, Lee

2013-03-01

Zinc(II)-phthalocyanine (ZnPc) is a metal photosensitizer. In the present study, we formulated the poorly-soluble ZnPc in Cremophor EL solution to enhance its solubility and determined its intracellular distribution and mechanisms of action on human hepatocellular carcinoma HepG2 cells. ZnPc uptake by the cells reached a plateau by 8h. ZnPc primarily located in mitochondria, lysosome and endoplasmic reticulum. The concentration-growth inhibition curves of ZnPc on the cell lines were pharmacodynamically enhanced by 10-50 folds by irradiation. Once irradiated, ZnPc produced significant amount of reactive oxygen species (ROS), activated caspase-3 and caspase-9, arrested cell cycle mainly at G2/M stage, and decreased membrane potential (ΔΨm) of HepG2 cells. In conclusion, the present study first elucidated cellular and molecular mechanisms of ZnPc on HepG2 cells. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Synergistic effects of hydrogen peroxide and ethanol on cell viability loss in PC12 cells by increase in mitochondrial permeability transition.

PubMed

Lee, Chung Soo; Kim, Yun Jeong; Ko, Hyun Hee; Han, Eun Sook

2005-07-15

The promoting effect of ethanol against the cytotoxicity of hydrogen peroxide (H2O2) in differentiated PC12 cells was assessed by measuring the effect on the mitochondrial membrane permeability. Treatment of PC12 cells with H2O2 resulted in the nuclear damage, decrease in the mitochondrial transmembrane potential, cytosolic accumulation of cytochrome c, activation of caspase-3, increase in the formation of reactive oxygen species (ROS) and depletion of GSH. In PC12 cells and dopaminergic neuroblastoma SH-SY5Y cells, the promoting effect of ethanol on the H2O2-induced cell death was increased with exposure time. Ethanol promoted the nuclear damage, change in the mitochondrial membrane permeability, ROS formation and decrease in GSH contents due to H2O2 in PC12 cells. Catalase, carboxy-PTIO, Mn-TBAP, N-acetylcysteine, cyclosporin A and trifluoperazine inhibited the H2O2 and ethanol-induced mitochondrial dysfunction and cell injury. The results show that the ethanol treatment promotes the cytotoxicity of H2O2 against PC12 cells. Ethanol may enhance the H2O2-induced viability loss in PC12 cells by promoting the mitochondrial membrane permeability change, release of cytochrome c and subsequent activation of caspase-3, which is associated with the increased formation of ROS and depletion of GSH. The findings suggest that ethanol as a promoting agent for the formation of mitochondrial permeability transition may enhance the neuronal cell injury caused by oxidants.

Three new cyathane diterpenes with neurotrophic activity from the liquid cultures of Hericium erinaceus.

PubMed

Zhang, Yuting; Liu, Li; Bao, Li; Yang, Yanlong; Ma, Ke; Liu, Hongwei

2018-05-21

Three new cyathane diterpenes erinacines T-V (1-3), and two known cyathane diterpenes erinacine A (4) and erinacine P (5) were isolated from the liquid cultures of Hericium erinaceus. The structures of 1-3 were determined by extensive spectroscopic analysis. All isolated compounds were evaluated for the cytotoxicity, and neurite-promoting activities using PC12 cell line. Compounds 1-3, and 5 exhibited pronounced neurite outgrowth-promoting effects on PC12 cells in the range of 2.5-10 μM. Compound 4 showed weak cytotoxicity against PC12 cells with IC 50 of 73.7 μM.

A quantitative image cytometry technique for time series or population analyses of signaling networks.

PubMed

Ozaki, Yu-ichi; Uda, Shinsuke; Saito, Takeshi H; Chung, Jaehoon; Kubota, Hiroyuki; Kuroda, Shinya

2010-04-01

Modeling of cellular functions on the basis of experimental observation is increasingly common in the field of cellular signaling. However, such modeling requires a large amount of quantitative data of signaling events with high spatio-temporal resolution. A novel technique which allows us to obtain such data is needed for systems biology of cellular signaling. We developed a fully automatable assay technique, termed quantitative image cytometry (QIC), which integrates a quantitative immunostaining technique and a high precision image-processing algorithm for cell identification. With the aid of an automated sample preparation system, this device can quantify protein expression, phosphorylation and localization with subcellular resolution at one-minute intervals. The signaling activities quantified by the assay system showed good correlation with, as well as comparable reproducibility to, western blot analysis. Taking advantage of the high spatio-temporal resolution, we investigated the signaling dynamics of the ERK pathway in PC12 cells. The QIC technique appears as a highly quantitative and versatile technique, which can be a convenient replacement for the most conventional techniques including western blot, flow cytometry and live cell imaging. Thus, the QIC technique can be a powerful tool for investigating the systems biology of cellular signaling.

Novel substrate (algal protein) for cultivation of Rhodospirillum rubrum.

PubMed

Vatsala, T M; Rekha, R; Srividhya, R

2011-10-01

Rhodospirillum rubrum was grown under light anaerobic conditions with phycocyanin (C-pc) extracted from Spirulina platensis as the sole source of carbon and nitrogen. When grown under these conditions cellular components like lipids, carbohydrates, protein, carotenoids, bacteriochlorophyll were similar to the one grown with malic acid and ammonium chloride. Growth of R. rubrum increased with increase in concentration of C-pc (200 to 1000 mg/l). R. rubrum also utilized C-pc under dark anaerobic condition. With both malic acid and C-pc as carbon sources C-pc was consumed only after exhaustion of malic acid under light anaerobic condition. No aberration of cell morphology was seen under scanning electron microscope (SEM). R. rubrum utilized both phycocyanobilin and phycoprotein individually as well as in combination. When grown with 1000 mg/l of phycoprotein 450 mg/l of biomass was obtained, and with combination of phycocyanobilin (75 mg/l) and phycoprotein (925 mg/l) 610 mg/l of biomass was obtained. Phycocyanobilin alone did not inhibit the growth of R. rubrum. Utilization of C-pc with protease like activity was observed in plate assay. Protease like activity was also observed as zones around the colonies in plates containing sterilized casein, gelatin and filter sterilized bovine serum albumin. No amino acids were detected in the supernatant when analyzed with ninhydrin. Extracellular protease like activity was highest when C-pc was used as substrate (2.8 U/ml). Intracellular protease like activity was not detected in cell free extracts.

Phosphatidylcholine hydrolysis and c-myc expression are in collaborating mitogenic pathways activated by colony-stimulating factor 1.

PubMed

Xu, X X; Tessner, T G; Rock, C O; Jackowski, S

1993-03-01

Stimulation of diglyceride production via phospholipase C (PLC) hydrolysis of phosphatidylcholine was an early event in the mitogenic action of colony-stimulating factor 1 (CSF-1) in the murine macrophage cell line BAC1.2F5 and was followed by a second phase of diglyceride production that persisted throughout the G1 phase of the cell cycle. Addition of phosphatidylcholine-specific PLC (PC-PLC) from Bacillus cereus to the medium of quiescent cells raised the intracellular diglyceride concentration and stimulated [3H]thymidine incorporation, although PC-PLC did not support continuous proliferation. PC-PLC treatment did not induce tyrosine phosphorylation or turnover of the CSF-1 receptor. The major protein kinase C (PKC) isotype in BAC1.2F5 cells was PKC-delta. Diglyceride production from PC-PLC did not target PKC-delta, since unlike phorbol esters, PC-PLC treatment neither decreased the electrophoretic mobility of PKC-delta nor increased the amount of GTP bound to Ras, and PC-PLC was mitogenically active in BAC1.2F5 cells in which PKC-delta was downregulated by prolonged treatment with phorbol ester. PC-PLC mimicked CSF-1 action by elevating c-fos and junB mRNAs to 40% of the level induced by CSF-1; however, PC-PLC induced c-myc mRNA to only 5% of the level in CSF-1-stimulated cells. PC-PLC addition to CSF-1-dependent BAC1.2F5 clones that constitutively express c-myc increased [3H]thymidine incorporation to 86% of the level evoked by CSF-1 and supported slow growth in the absence of CSF-1. Therefore, PC-PLC is a component of a signal transduction pathway leading to transcription of c-fos and junB that collaborates with c-myc and is independent of PKC-delta and Ras activation.

Acorus tatarinowii Schott extract protects PC12 cells from amyloid-beta induced neurotoxicity.

PubMed

An, Hong-Mei; Li, Guo-Wen; Lin, Chen; Gu, Chao; Jin, Miao; Sun, Wen-Xian; Qiu, Ming-Feng; Hu, Bing

2014-05-01

Amyloid-beta induced neurotoxicity has been identified as a major cause of Alzheimer's disease. Acorus tatarinowii Schott is one of the most frequently used Chinese herbs for Alzheimer's disease treatment. However, the effects of Acorus tatarinowii Schott on amyloid-beta mediated nerve cell damage remains unknown. In the present study, neuronal differentiated PC12 cells were used as a model to evaluate the effects of A. tatarinowii Schott extract (ATSE) against Abeta25-35 induced neurotoxicity. The results showed pretreatment with ATSE significantly protected PC12 cells from Abeta25-35 induced cell death, lactate dehydrogenase release, DNA damage, mitochondrial dysfunction and cytochrome c release from mitochondria. In addition, pretreatment with ATSE also significantly inhibited Abeta25-35 induced caspase-3 activation and reactive oxygen species generation in PC12 cells. These observations suggested that ATSE protects PC12 cells from amyloid-beta induced neurotoxicity.

Tl(I) and Tl(III) activate both mitochondrial and extrinsic pathways of apoptosis in rat pheochromocytoma (PC12) cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hanzel, Cecilia Eliana; Verstraeten, Sandra Viviana

2009-04-01

Thallium (Tl) is a highly toxic metal though yet its mechanisms are poorly understood. Previously, we demonstrated that rat pheochromocytoma (PC12) cells exposure to thallous (Tl(I)) or thallic (Tl(III)) cations leads to mitochondrial damage and reduced cell viability. In the present work we comparatively characterized the possible pathways involved in Tl(I)- and Tl(III)- (10-100 {mu}M) mediated decrease in PC12 cells viability. We observed that these cations do not cause cell necrosis but significantly increased the number of cells with apoptotic features. Both cations lead to Bax oligomerization and caused apoptosis inducing factor (AIF), endonuclease G (Endo G), and cytochrome cmore » release from mitochondria, but they did not activate caspase dependent DNAse (CAD). Tl(I)- and Tl(III)-dependent caspases 9 and 3 activation followed similar kinetics, with maximal effects at 18 h of incubation. In addition, Tl(I) promoted phosphatidylserine (PS) exposure. Tl(III) induced 2- and 18-fold increase in Fas content and caspase 8 activity, respectively. Together, experimental results show that Tl(I) and Tl(III) induce PC12 cells apoptosis, although differential pathways are involved. While Tl(I)-mediated cell apoptosis was mainly associated with mitochondrial damage, Tl(III) showed a mixed effect triggering both the intrinsic and extrinsic pathways of apoptosis. These findings contribute to a better understanding of the mechanisms underlying Tl-induced loss of cell viability in PC12 cells.« less

DA-9801 promotes neurite outgrowth via ERK1/2-CREB pathway in PC12 cells.

PubMed

Won, Jong Hoon; Ahn, Kyong Hoon; Back, Moon Jung; Ha, Hae Chan; Jang, Ji Min; Kim, Ha Hyung; Choi, Sang-Zin; Son, Miwon; Kim, Dae Kyong

2015-01-01

In the present study, we examined the mechanisms underlying the effect of DA-9801 on neurite outgrowth. We found that DA-9801 elicits its effects via the mitogen-activated protein kinase (MEK) extracellular signal-regulated kinase (ERK)1/2-cAMP response element-binding protein (CREB) pathway. DA-9801, an extract from a mixture of Dioscorea japonica and Dioscorea nipponica, was reported to promote neurite outgrowth in PC12 cells. The effects of DA-9801 on cell viability and expression of neuronal markers were evaluated in PC12 cells. To investigate DA-9801 action, specific inhibitors targeting the ERK signaling cascade were used. No cytotoxicity was observed in PC12 cells at DA-9801 concentrations of less than 30 µg/mL. In the presence of nerve growth factor (NGF, 2 ng/mL), DA-9801 promoted neurite outgrowth and increased the relative mRNA levels of neurofilament-L (NF-L), a marker of neuronal differentiation. The Raf-1 inhibitor GW5074 and MEK inhibitor PD98059 significantly attenuated DA-9801-induced neurite outgrowth. Additionally, the MEK1 and MEK2 inhibitor SL327 significantly attenuated the increase in the percentage of neurite-bearing PC12 cells induced by DA-9801 treatment. Conversely, the selective p38 mitogen-activated protein kinase inhibitor SB203580 did not attenuate the DA-9801 treatment-induced increase in the percentage of neurite-bearing PC12 cells. DA-9801 enhanced the phosphorylation of ERK1/2 and CREB in PC12 cells incubated with and without NGF. Pretreatment with PD98059 blocked the DA-9801-induced phosphorylation of ERK1/2 and CREB. In conclusion, DA-9801 induces neurite outgrowth by affecting the ERK1/2-CREB signaling pathway. Insights into the mechanism underlying this effect of DA-9801 may suggest novel potential strategies for the treatment of peripheral neuropathy.

A WRKY transcription factor, PcWRKY33, from Polygonum cuspidatum reduces salt tolerance in transgenic Arabidopsis thaliana.

PubMed

Bao, Wenqi; Wang, Xiaowei; Chen, Mo; Chai, Tuanyao; Wang, Hong

2018-07-01

PcWRKY33 is a transcription factor which can reduce salt tolerance by decreasing the expression of stress-related genes and increasing the cellular levels of reactive oxygen species (ROS). WRKY transcription factors play important roles in the regulation of biotic and abiotic stresses. Here, we report a group I WRKY gene from Polygonum cuspidatum, PcWRKY33, that encodes a nucleoprotein, which specifically binds to the W-box in the promoter of target genes to regulate their expression. The results from qPCR and promoter analysis show that expression of PcWRKY33 can be induced by various abiotic stresses, including NaCl and plant hormones. Overexpression of PcWRKY33 in Arabidopsis thaliana reduced tolerance to salt stress. More specifically, several physiological parameters (such as root length, seed germination rate, seedling survival rate, and chlorophyll concentration) of the transgenic lines were significantly lower than those of the wild type under salt stress. In addition, following exposure to salt stress, transgenic plants showed decreased expression of stress-related genes, a weakened ability to maintain Na + /K + homeostasis, decreased activities of reactive oxygen species- (ROS-) scavenging enzymes, and increased accumulation of ROS. Taken together, these results suggest that PcWRKY33 negatively regulates the salt tolerance in at least two ways: by down-regulating the induction of stress-related genes and by increasing the level of cellular ROS. In sum, our results indicate that PcWRKY33 is a group I WRKY transcription factor involved in abiotic stress regulation.

Phase II enzyme induction by a carotenoid, lutein, in a PC12D neuronal cell line

DOE Office of Scientific and Technical Information (OSTI.GOV)

Miyake, Seiji; Department of Ophthalmology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582; Wakasa Seikatsu Co., Ltd., 134 Chudoujiminami-cho, Shimogyo-ku, Kyoto 600-8813

Highlights: • Lutein reduced ROS levels in a PC12D neuronal cell line. • Lutein induced mRNAs of phase II antioxidative enzymes in PC12D neuronal cells. • Lutein increased protein levels of HO-1, SOD2, and NQO-1 in PC12D neuronal cells. • Lutein had no effect on intranuclear Nrf2 levels in PC12D neuronal cells. • Lutein did not activate potential upstream Nrf2 nuclear translocation pathways. - Abstract: The mechanism by which lutein, a carotenoid, acts as an antioxidant in retinal cells is still not fully understood. Here, lutein treatment of a neuronal cell line (PC12D) immediately resulted in reduced intracellular ROS levels,more » implying that it has a direct role in ROS scavenging. Significantly, lutein treatment also induced phase II antioxidative enzyme expression, probably via a nuclear factor-like 2 (Nrf2) independent pathway. This latter mechanism could explain why lutein acts diversely to protect against oxidative/cytotoxic stress, and why it is physiologically involved in the human neural tissue, such as the retina.« less

Activation of Nrf2 target enzymes conferring protection against oxidative stress in PC12 cells by ginger principal constituent 6-shogaol.

PubMed

Peng, Shoujiao; Yao, Juan; Liu, Yaping; Duan, Dongzhu; Zhang, Xiaolong; Fang, Jianguo

2015-08-01

Natural compounds containing phenoxyl groups and/or Michael acceptor units appear to possess antioxidant and cytoprotective properties. The ginger principal constituent 6-shogaol (6-S) represents one of such compounds. In this study, we reported that 6-S efficiently scavenges various free radicals in vitro, and displays remarkable cytoprotection against oxidative stress-induced cell damage in the neuron-like rat pheochromocytoma cell line, PC12 cells. Pretreatment of PC12 cells with 6-S significantly upregulates a series of phase II antioxidant molecules, such as glutathione, heme oxygenase 1, NAD(P)H: quinone oxidoreductase 1, thioredoxin reductase 1, and thioredoxin 1. A mechanistic study revealed that 6-S enhanced the translocation of Nrf2 from the cytosol to the nucleus and knockdown of Nrf2 abolished such protection, indicating that this cytoprotection is mediated by the activation of the transcription factor Nrf2. Another ginger constituent 6-gingerol (6-G), having a similar structure of 6-S but lacking the alpha,beta-unsaturated ketone structure (Michael acceptor moiety), failed to shelter PC12 cells from oxidative stress. Our results demonstrate that 6-S is a novel small molecule activator of Nrf2 in PC12 cells, and suggest that 6-S might be a potential candidate for the prevention of oxidative stress-mediated neurodegenerative disorders.

Small GTPase Tc10 and its homologue RhoT induce N-WASP-mediated long process formation and neurite outgrowth.

PubMed

Abe, Tomoyuki; Kato, Masayoshi; Miki, Hiroaki; Takenawa, Tadaomi; Endo, Takeshi

2003-01-01

Rho family small GTPases regulate multiple cellular functions through reorganization of the actin cytoskeleton. Among them, Cdc42 and Tc10 induce filopodia or peripheral processes in cultured cells. We have identified a member of the family, designated as RhoT, which is closely related to Tc10. Tc10 was highly expressed in muscular tissues and brain and remarkably induced during differentiation of C2 skeletal muscle cells and neuronal differentiation of PC12 and N1E-115 cells. On the other hand, RhoT was predominantly expressed in heart and uterus and induced during neuronal differentiation of N1E-115 cells. Tc10 exogenously expressed in fibroblasts generated actin-filament-containing peripheral processes longer than the Cdc42-formed filopodia, whereas RhoT produced much longer and thicker processes containing actin filaments. Furthermore, both Tc10 and RhoT induced neurite outgrowth in PC12 and N1E-115 cells, but Cdc42 did not do this by itself. Tc10 and RhoT as well as Cdc42 bound to the N-terminal CRIB-motif-containing portion of N-WASP and activated N-WASP to induce Arp2/3-complex-mediated actin polymerization. The formation of peripheral processes and neurites by Tc10 and RhoT was prevented by the coexpression of dominant-negative mutants of N-WASP. Thus, N-WASP is essential for the process formation and neurite outgrowth induced by Tc10 and RhoT. Neuronal differentiation of PC12 and N1E-115 cells induced by dibutyryl cyclic AMP and by serum starvation, respectively, was prevented by dominant-negative Cdc42, Tc10 and RhoT. Taken together, all these Rho family proteins are required for neuronal differentiation, but they exert their functions differentially in process formation and neurite extension. Consequently, N-WASP activated by these small GTPases mediates neuronal differentiation in addition to its recently identified role in glucose uptake.

Postprandial antioxidant effect of the Mediterranean diet supplemented with coenzyme Q10 in elderly men and women.

PubMed

Yubero-Serrano, Elena M; Delgado-Casado, Nieves; Delgado-Lista, Javier; Perez-Martinez, Pablo; Tasset-Cuevas, Inmaculada; Santos-Gonzalez, Monica; Caballero, Javier; Garcia-Rios, Antonio; Marin, Carmen; Gutierrez-Mariscal, Francisco M; Fuentes, Francisco; Villalba, Jose M; Tunez, Isaac; Perez-Jimenez, Francisco; Lopez-Miranda, Jose

2011-12-01

Postprandial oxidative stress is characterized by an increased susceptibility of the organism towards oxidative damage after consumption of a meal rich in lipids and/or carbohydrates. We have investigated whether the quality of dietary fat alters postprandial cellular oxidative stress and whether the supplementation with coenzyme Q(10) (CoQ) lowers postprandial oxidative stress in an elderly population. In this randomized crossover study, 20 participants were assigned to receive three isocaloric diets for periods of 4 week each: (1) Mediterranean diet supplemented with CoQ (Med+CoQ diet), (2) Mediterranean diet (Med diet), and (3) saturated fatty acid-rich diet (SFA diet). After a 12-h fast, the volunteers consumed a breakfast with a fat composition similar to that consumed in each of the diets. CoQ, lipid peroxides (LPO), oxidized low-density lipoprotein (oxLDL), protein carbonyl (PC), total nitrite, nitrotyrosine plasma levels, catalase, superoxide dismutase (SOD), and glutathione peroxidase (GPx) activities and ischemic reactive hyperaemia (IRH) were determined. Med diet produced a lower postprandial GPx activity and a lower decrease in total nitrite level compared to the SFA diet. Med and Med+CoQ diets induced a higher postprandial increase in IRH and a lower postprandial LPO, oxLDL, and nitrotyrosine plasma levels than the SFA diet. Moreover, the Med+CoQ diet produced a lower postprandial decrease in total nitrite and a greater decrease in PC levels compared to the other two diets and lower SOD, CAT, and GPx activities than the SFA diet.In conclusion, Med diet reduces postprandial oxidative stress by reducing processes of cellular oxidation and increases the action of the antioxidant system in elderly persons and the administration of CoQ further improves this redox balance.

Autism-associated R451C mutation in neuroligin3 leads to activation of the unfolded protein response in a PC12 Tet-On inducible system

PubMed Central

Ulbrich, Lisa; Favaloro, Flores Lietta; Trobiani, Laura; Marchetti, Valentina; Patel, Vruti; Pascucci, Tiziana; Comoletti, Davide; Marciniak, Stefan J.; De Jaco, Antonella

2015-01-01

Several forms of monogenic heritable autism spectrum disorders are associated with mutations in the neuroligin genes. The autism-linked substitution R451C in neuroligin3 induces local misfolding of its extracellular domain, causing partial retention in the ER (endoplasmic reticulum) of expressing cells. We have generated a PC12 Tet-On cell model system with inducible expression of wild-type or R451C neuroligin3 to investigate whether there is activation of the UPR (unfolded protein response) as a result of misfolded protein retention. As a positive control for protein misfolding, we also expressed the mutant G221R neuroligin3, which is known to be completely retained within the ER. Our data show that overexpression of either R451C or G221R mutant proteins leads to the activation of all three signalling branches of the UPR downstream of the stress sensors ATF6 (activating transcription factor 6), IRE1 (inositol-requiring enzyme 1) and PERK [PKR (dsRNA-dependent protein kinase)-like endoplasmic reticulum kinase]. Each branch displayed different activation profiles that partially correlated with the degree of misfolding caused by each mutation. We also show that up-regulation of BiP (immunoglobulin heavy-chain-binding protein) and CHOP [C/EBP (CCAAT/enhancer-binding protein)-homologous protein] was induced by both mutant proteins but not by wild-type neuroligin3, both in proliferative cells and cells differentiated to a neuron-like phenotype. Collectively, our data show that mutant R451C neuroligin3 activates the UPR in a novel cell model system, suggesting that this cellular response may have a role in monogenic forms of autism characterized by misfolding mutations. PMID:26621873

Neuroprotective Effect of CeO2@PAA-LXW7 Against H2O2-Induced Cytotoxicity in NGF-Differentiated PC12 Cells.

PubMed

Jia, Jingjing; Zhang, Ting; Chi, Jieshan; Liu, Xiaoma; Sun, Jingjing; Xie, Qizhi; Peng, Sijia; Li, Changyan; Yi, Li

2018-06-07

CeO 2 nanoparticles (nanoceria) have been used in many studies as a powerful free radical scavenger, and LXW7, a small-molecule peptide, can specifically target the integrin αvβ3, whose neuroprotective effects have also been demonstrated. The objective of this study is to observe the neuroprotective effect and potential mechanism of CeO 2 @PAA-LXW7, a new compound that couples CeO 2 @PAA (nanoceria modified with the functional group of polyacrylic acid) with LXW7 via a series of chemical reactions, in H 2 O 2 -induced NGF-differentiated PC12 cells. We examined the effects of LXW7, CeO 2 @PAA, and CeO 2 @PAA-LXW7 on the viability of primary hippocampal neurons and found that there was no significant difference under control conditions, but increased cellular viability was observed in the case of H 2 O 2 -induced injury. We used H 2 O 2 -induced NGF-differentiated PC12 cells as the classical injury model to investigate the neuroprotective effect of CeO 2 @PAA-LXW7. In this study, LXW7, CeO 2 @PAA, and CeO 2 @PAA-LXW7 inhibit H 2 O 2 -induced oxidative stress by reducing the production of reactive oxygen species (ROS) and regulating Bax/Bcl-2, cleaved caspase-3 and mitochondrial cytochrome C (cyto C) in the apoptotic signaling pathways. We found that the levels of phosphorylation of focal adhesion kinase (FAK) and of signal transducer and activator of transcription 3 (STAT3) increased significantly in H 2 O 2 -induced NGF-differentiated PC12 cells, whereas LXW7, CeO 2 @PAA, and CeO 2 @PAA-LXW7 suppressed the increase to different degrees. Among the abovementioned changes, the inhibitory effect of CeO 2 @PAA-LXW7 on H 2 O 2 -induced changes, including the increases in the levels of p-FAK and p-STAT3, is more obvious than that of LXW7 or CeO 2 @PAA alone. In summary, these results suggest that integrin signaling participates in the regulation of apoptosis via the regulation of ROS and of the apoptosis pathway in H 2 O 2 -induced NGF-differentiated PC12 cells. LXW7, CeO 2 @PAA, and CeO 2 @PAA-LXW7 can play neuroprotective roles by counteracting the oxidative stress and apoptosis induced by H 2 O 2 in NGF-differentiated PC12 cells. CeO 2 @PAA-LXW7 exerting a more powerful synergistic effect via the conjunction of LXW7 and CeO 2 @PAA.

Rab3A Inhibition of Ca2+ -Dependent Dopamine Release From PC12 Cells Involves Interaction With Synaptotagmin I.

PubMed

Dai, Zhipan; Tang, Xia; Chen, Jia; Tang, Xiaochao; Wang, Xianchun

2017-11-01

Rab3 and synaptotagmin have been suggested to play important roles in the regulation of neurotransmitter release and, however, the molecular mechanism has not been completely clear. Here, we studied the effects of Rab3A and synaptotagmin I (Syt I) on dopamine release using PC12 cells as a model system. Rab3A was demonstrated to have effects on both Ca 2+ -independent and Ca 2+ -dependent dopamine releases from the PC12 cells. Application of Rab3A (up to 2500 nM) gradually decreased the amount of Ca 2+ -dependently released dopamine, indicating that Rab3A is a negative modulator that was further supported by the increase in dopamine release caused by Rab3A knockdown. Syt I knockdown weakened the Ca 2+ -dependent dopamine release, suggesting that Syt I plays a positive regulatory role in the cellular process. Treatment of the Syt I-knocked down PC12 cells with Rab3A further decreased Ca 2+ -dependent dopamine release and, however, the decrease magnitude was significantly reduced compared with that before Syt I knockdown, thus for the first time demonstrating that the inhibitory effect of Rab3A on Ca 2+ -dependent dopamine release involves the interaction with Syt I. This work has shed new light on the molecular mechanism for Rab3 and synaptotamin regulation of neurotransmitter release. J. Cell. Biochem. 118: 3696-3705, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Polycomb purification by in vivo biotinylation tagging reveals cohesin and Trithorax group proteins as interaction partners

PubMed Central

Strübbe, Gero; Popp, Christian; Schmidt, Alexander; Pauli, Andrea; Ringrose, Leonie; Beisel, Christian; Paro, Renato

2011-01-01

The maintenance of specific gene expression patterns during cellular proliferation is crucial for the identity of every cell type and the development of tissues in multicellular organisms. Such a cellular memory function is conveyed by the complex interplay of the Polycomb and Trithorax groups of proteins (PcG/TrxG). These proteins exert their function at the level of chromatin by establishing and maintaining repressed (PcG) and active (TrxG) chromatin domains. Past studies indicated that a core PcG protein complex is potentially associated with cell type or even cell stage-specific sets of accessory proteins. In order to better understand the dynamic aspects underlying PcG composition and function we have established an inducible version of the biotinylation tagging approach to purify Polycomb and associated factors from Drosophila embryos. This system enabled fast and efficient isolation of Polycomb containing complexes under near physiological conditions, thereby preserving substoichiometric interactions. Novel interacting proteins were identified by highly sensitive mass spectrometric analysis. We found many TrxG related proteins, suggesting a previously unrecognized extent of molecular interaction of the two counteracting chromatin regulatory protein groups. Furthermore, our analysis revealed an association of PcG protein complexes with the cohesin complex and showed that Polycomb-dependent silencing of a transgenic reporter depends on cohesin function. PMID:21415365

Crystal Structures of Human and Staphylococcus aureus Pyruvate Carboxylase and Molecular Insights into the Carboxyltransfer Reaction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xiang,S.; Tong, L.

2008-01-01

Pyruvate carboxylase (PC) catalyzes the biotin-dependent production of oxaloacetate and has important roles in gluconeogenesis, lipogenesis, insulin secretion and other cellular processes. PC contains the biotin carboxylase (BC), carboxyltransferase (CT) and biotin-carboxyl carrier protein (BCCP) domains. We report here the crystal structures at 2.8-Angstroms resolution of full-length PC from Staphylococcus aureus and the C-terminal region (missing only the BC domain) of human PC. A conserved tetrameric association is observed for both enzymes, and our structural and mutagenesis studies reveal a previously uncharacterized domain, the PC tetramerization (PT) domain, which is important for oligomerization. A BCCP domain is located in themore » active site of the CT domain, providing the first molecular insights into how biotin participates in the carboxyltransfer reaction. There are dramatic differences in domain positions in the monomer and the organization of the tetramer between these enzymes and the PC from Rhizobium etli.« less

Cedrin identified from Cedrus deodara (Roxb.) G. Don protects PC12 cells against neurotoxicity induced by Aβ1-42.

PubMed

Zhao, Zhiwei; Dong, Zhanfei; Ming, Jie; Liu, Yan

2018-06-01

Alzheimer's disease is a severe neurodegenerative disease affecting elder worldwide and closely related to the neurotoxicity induced by amyloid β. To find efficient therapeutics, we have investigated the protective effects of cedrin from Cedrus deodara (Roxb.) G. Don on PC12 cells against the neurotoxicity induced by amyloid β 1-42 . The results have shown the viability of PC12 cells injured by amyloid β 1-42 can be improved by cedrin. Cedrin can reduce reacrive oxygen species overproduction, increase the activity of superoxide dismutase and decrease malondialdehyde content. Meanwhile, the loss of mitochondrial membrane potential and mitochondrial permeability transition pore opening in PC12 cells, and elevated Caspase-3 activity, downregulated Bcl-2 and upregulated Bax are meliorated. These results demonstrate the protective effect of cedrin is related to the inhibition of oxidative stress, improvement of mitochondrial dysfunction and suppression of apoptosis. This investigation gives evidences for the application of cedrin in practice and further investigation in vivo.

Artemisinin protects PC12 cells against β-amyloid-induced apoptosis through activation of the ERK1/2 signaling pathway.

PubMed

Zeng, Zhiwen; Xu, Jinying; Zheng, Wenhua

2017-08-01

Accumulating evidence displays that an abnormal deposition of amyloid beta-peptide (Aβ) is the primary cause of the pathogenesis of Alzheimer's disease (AD). And therefore the elimination of Aβ is regarded as an important strategy for AD treatment. The discovery of drug candidates using culture neuronal cells against Aβ peptide toxicity is believed to be an effective approach to develop drug for the treatment of AD patients. We have previously showed that artemisinin, a FDA-approved anti-malaria drug, has neuroprotective effects recently. In the present study, we aimed to investigate the effects and potential mechanism of artemisinin in protecting neuronal PC12 cells from toxicity of β amyloid peptide. Our studies revealed that artemisinin, in clinical relevant concentration, protected and rescued PC12 cells from Aβ25-35-induced cell death. Further study showed that artemisinin significantly ameliorated cell death due to Aβ25-35 insult by restoring abnormal changes in nuclear morphology, lactate dehydrogenase, intracellular ROS, mitochondrial membrane potential and activity of apoptotic caspase. Western blotting analysis demonstrated that artemisinin activated extracellular regulated kinase ERK1/2 but not Akt survival signaling. Consistent with the role of ERK1/2, preincubation of cells with ERK1/2 pathway inhibitor PD98059 blocked the effect of artemisinin while PI3K inhibitor LY294002 has no effect. Moreover, Aβ1-42 also caused cells death of PC12 cells while artemisinin suppressed Aβ1-42 cytotoxicity in PC12 cells. Taken together, these results, at the first time, suggest that artemisinin is a potential protectant against β amyloid insult through activation of the ERK1/2 pathway. Our finding provides a potential application of artemisinin in prevention and treatment of AD. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Cyclo(His-Pro) promotes cytoprotection by activating Nrf2-mediated up-regulation of antioxidant defence

PubMed Central

Minelli, Alba; Conte, Carmela; Grottelli, Silvia; Bellezza, Maria; Cacciatore, Ivana; Bolaños, Juan P

2009-01-01

Hystidyl-proline [cyclo(His-Pro)] is an endogenous cyclic dipeptide produced by the cleavage of thyrotropin releasing hormone. Previous studies have shown that cyclo(His-Pro) protects against oxidative stress, although the underlying mechanism has remained elusive. Here, we addressed this issue and found that cyclo(His-Pro) triggered nuclear accumulation of NF-E2-related factor-2 (Nrf2), a transcription factor that up-regulates antioxidant-/electrophile-responsive element (ARE-EpRE)-related genes, in PC12 cells. Cyclo(His-Pro) attenuated reactive oxygen species production, and prevented glutathione depletion caused by glutamate, rotenone, paraquat and β-amyloid treatment. Moreover, real-time PCR analyses revealed that cyclo(His-Pro) induced the expression of a number of ARE-related genes and protected cells against hydrogen peroxide-mediated apoptotic death. Furthermore, these effects were abolished by RNA interference-mediated Nrf2 knockdown. Finally, pharmacological inhibition of p-38 MAPK partially prevented both cyclo(His-Pro)-mediated Nrf2 activation and cellular protection. These results suggest that the signalling mechanism responsible for the cytoprotective actions of cyclo(His-Pro) would involve p-38 MAPK activation leading to Nrf2-mediated up-regulation of antioxidant cellular defence. PMID:18373731

PrPC has nucleic acid chaperoning properties similar to the nucleocapsid protein of HIV-1.

PubMed

Derrington, Edmund; Gabus, Caroline; Leblanc, Pascal; Chnaidermann, Jonas; Grave, Linda; Dormont, Dominique; Swietnicki, Wieslaw; Morillas, Manuel; Marck, Daniel; Nandi, Pradip; Darlix, Jean-Luc

2002-01-01

The function of the cellular prion protein (PrPC) remains obscure. Studies suggest that PrPC functions in several processes including signal transduction and Cu2+ metabolism. PrPC has also been established to bind nucleic acids. Therefore we investigated the properties of PrPC as a putative nucleic acid chaperone. Surprisingly, PrPC possesses all the nucleic acid chaperoning properties previously specific to retroviral nucleocapsid proteins. PrPC appears to be a molecular mimic of NCP7, the nucleocapsid protein of HIV-1. Thus PrPC, like NCP7, chaperones the annealing of tRNA(Lys) to the HIV-1 primer binding site, the initial step of retrovirus replication. PrPC also chaperones the two DNA strand transfers required for production of a complete proviral DNA with LTRs. Concerning the functions of NCP7 during budding, PrPC also mimices NCP7 by dimerizing the HIV-1 genomic RNA. These data are unprecedented because, although many cellular proteins have been identified as nucleic acid chaperones, none have the properties of retroviral nucleocapsid proteins.

KCl stimulation increases norepinephrine transporter function in PC12 cells.

PubMed

Mandela, Prashant; Ordway, Gregory A

2006-09-01

The norepinephrine transporter (NET) plays a pivotal role in terminating noradrenergic signaling and conserving norepinephrine (NE) through the process of re-uptake. Recent evidence suggests a close association between NE release and regulation of NET function. The present study evaluated the relationship between release and uptake, and the cellular mechanisms that govern these processes. KCl stimulation of PC12 cells robustly increased [3H]NE uptake via the NET and simultaneously increased [3H]NE release. KCl-stimulated increases in uptake and release were dependent on Ca2+. Treatment of cells with phorbol-12-myristate-13-acetate (PMA) or okadaic acid decreased [3H]NE uptake but did not block KCl-stimulated increases in [3H]NE uptake. In contrast, PMA increased [3H]NE release and augmented KCl-stimulated release, while okadaic acid had no effects on release. Inhibition of Ca2+-activated signaling cascades with KN93 (a Ca2+ calmodulin-dependent kinase inhibitor), or ML7 and ML9 (myosin light chain kinase inhibitors), reduced [3H]NE uptake and blocked KCl-stimulated increases in uptake. In contrast, KN93, ML7 and ML9 had no effect on KCl-stimulated [3H]NE release. KCl-stimulated increases in [3H]NE uptake were independent of transporter trafficking to the plasma membrane. While increases in both NE release and uptake mediated by KCl stimulation require Ca2+, different intracellular mechanisms mediate these two events.

A superoxide anion-scavenger, 1,3-selenazolidin-4-one suppresses serum deprivation-induced apoptosis in PC12 cells by activating MAP kinase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nishina, Atsuyoshi, E-mail: nishina@yone.ac.jp; Kimura, Hirokazu; Kozawa, Kunihisa

Synthetic organic selenium compounds, such as ebselen, may show glutathione peroxidase-like antioxidant activity and have a neurotrophic effect. We synthesized 1,3-selenazolidin-4-ones, new types of synthetic organic selenium compounds (five-member ring compounds), to study their possible applications as antioxidants or neurotrophic-like molecules. Their superoxide radical scavenging effects were assessed using the quantitative, highly sensitive method of real-time kinetic chemiluminescence. At 166 {mu}M, the O{sub 2}{sup -} scavenging activity of 1,3-selenazolidin-4-ones ranged from 0 to 66.2%. 2-[3-(4-Methoxyphenyl)-4-oxo-1,3-selenazolidin-2-ylidene]malononitrile (compound b) showed the strongest superoxide anion-scavenging activity among the 6 kinds of 2-methylene-1,3-selenazolidin-4-ones examined. Compound b had a 50% inhibitory concentration (IC{sub 50}) atmore » 92.4 {mu}M and acted as an effective and potentially useful O{sub 2}{sup -} scavenger in vitro. The effect of compound b on rat pheochromocytome cell line PC12 cells was compared with that of ebselen or nerve growth factor (NGF) by use of the MTT [3-(4, 5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] assay. When ebselen was added at 100 {mu}M or more, toxicity toward PC12 cells was evident. On the contrary, compound b suppressed serum deprivation-induced apoptosis in PC12 cells more effectively at a concentration of 100 {mu}M. The activity of compound b to phosphorylate mitogen-activated protein kinase/extracellular signal-regulated protein kinase (ERK) 1/2 (MAP kinase) in PC12 cells was higher than that of ebselen, and the former at 100 {mu}M induced the phosphorylation of MAP kinase to a degree similar to that induced by NGF. From these results, we conclude that this superoxide anion-scavenger, compound b, suppressed serum deprivation-induced apoptosis by promoting the phosphorylation of MAP kinase. -- Highlights: Black-Right-Pointing-Pointer We newly synthesized 1,3-selenazolidin-4-ones to study their possible applications. Black-Right-Pointing-Pointer Among new compounds, compound b showed the strongest SOSA. Black-Right-Pointing-Pointer Compound b suppressed serum deprivation-induced apoptosis in PC12 cells. Black-Right-Pointing-Pointer Compound b suppressed apoptosis by promoting the activation of MAP kinase.« less

Stimulation of surfactant phospholipid biosynthesis in the lungs of rats treated with silica.

PubMed Central

Miller, B E; Hook, G E

1988-01-01

The effects of intratracheally instilled silica (10 mg/rat) on the biosynthesis of surfactant phospholipids was investigated in the lungs of rats. The sizes of the intracellular and extracellular pools of surfactant phospholipids were measured 7, 14 and 28 days after silica exposure. The ability of lung slices to incorporate [14C]choline and [3H]palmitate into surfactant phosphatidylcholine (PC) and disaturated phosphatidylcholine (DSPC) was also investigated. Both intra- and extra-cellular pools of surfactant phospholipids were increased by silica treatment. The intracellular pool increased linearly over the 28-day time period, ultimately reaching a size 62-fold greater than controls. The extracellular pool also increased, but showed a pattern different from that of the intracellular pool. The extracellular pool increased non-linearly up to 14 days, and then declined. At its maximum, the extracellular pool was increased 16-fold over the control. The ability of lung slices to incorporate phospholipid precursors into surfactant-associated PC and DSPC was elevated at all time periods. The rate of incorporation of [14C]choline into surfactant PC and DSPC was maximal at 14 days and was nearly 3-fold greater than the rate in controls. The rate of incorporation of [3H]palmitate was also maximal at 14 days, approx. 5-fold above controls for PC and 3-fold for DSPC. At this same time point, the microsomal activity of cholinephosphate cytidylyltransferase was increased 4.5-fold above controls, but cytosolic activity was not significantly affected by silica treatment. These data indicate that biosynthesis of surfactant PC is elevated after treatment of lungs with silica and that this increased biosynthesis probably underlies the expansion of the intra- and extra-cellular pools of surfactant phospholipids. PMID:2845927

A method to measure cellular adhesion utilizing a polymer micro-cantilever

NASA Astrophysics Data System (ADS)

Gaitas, Angelo; Malhotra, Ricky; Pienta, Kenneth

2013-09-01

In the present study we engineered a micro-machined polyimide cantilever with an embedded sensing element to investigate cellular adhesion, in terms of its relative ability to stick to a cross-linker, 3,3'-dithiobis[sulfosuccinimidylpropionate], coated on the cantilever surface. To achieve this objective, we investigated adhesive properties of three human prostate cancer cell lines, namely, a bone metastasis derived human prostate cancer cell line (PC3), a brain metastasis derived human prostate cancer cell line (DU145), and a subclone of PC3 (PC3-EMT14). We found that PC3-EMT14, which displays a mesenchymal phenotype, has the least adhesion compared to PC3 and DU145, which exhibit an epithelial phenotype.

Indocarbazostatin and indocarbazostatin B, novel inhibitors of NGF-induced neuronal differentiation in PC12 cells. I. Screening, taxonomy, fermentation and biological activities.

PubMed

Matsuura, Nobuyasu; Tamehiro, Norimasa; Andoh, Tsutomu; Kawashima, Akira; Ubukata, Makoto

2002-04-01

During the course of our screening for modulators of signal transduction of mammalian cells, we discovered two novel indolocarbazole antibiotics, indocarbazostatin and indocabazostatin B, from a culture broth of a Streptomyces sp. as inhibitors of NGF-induced neuronal differentiation in rat pheochromocytoma PC12 cells. Indocarbazostatin and indocarbazostatin B inhibited NGF-induced neurite outgrowth from PC12 cells at 6 nM and 24 nM, respectively, whereas K-252a inhibited at 200 nM under our assay conditions.

Glycyl-alanyl-histidine protects PC12 cells against hydrogen peroxide toxicity.

PubMed

Shimura, Hideki; Tanaka, Ryota; Shimada, Yoshiaki; Yamashiro, Kazuo; Hattori, Nobutaka; Urabe, Takao

2017-11-22

Peptides with cytoprotective functions, including antioxidants and anti-infectives, could be useful therapeutics. Carnosine, β-alanine-histidine, is a dipeptide with anti-oxidant properties. Tripeptides of Ala-His-Lys, Pro-His-His, or Tyr-His-Tyr are also of interest in this respect. We synthesized several histidine-containing peptides including glycine or alanine, and tested their cytoprotective effects on hydrogen peroxide toxicity for PC12 cells. Of all these peptides (Gly-His-His, Ala-His-His, Ala-His-Ala, Ala-Ala-His, Ala-Gly-His, Gly-Ala-His (GAH), Ala-His-Gly, His-Ala-Gly, His-His-His, Gly-His-Ala, and Gly-Gly-His), GAH was found to have the strongest cytoprotective activity. GAH decreased lactate dehydrogenase (LDH) leakage, apoptosis, morphological changes, and nuclear membrane permeability changes against hydrogen peroxide toxicity in PC12 cells. The cytoprotective activity of GAH was superior to that of carnosine against hydrogen peroxide toxicity in PC12 cells. GAH also protected PC12 cells against damage caused by actinomycin D and staurosporine. Additionally, it was found that GAH also protected SH-SY5Y and Jurkat cells from damage caused by hydrogen peroxide, as assessed by LDH leakage. Thus, a novel tripeptide, GAH, has been identified as having broad cytoprotective effects against hydrogen peroxide-induced cell damage.

Luteolin and Apigenin Attenuate 4-Hydroxy-2-Nonenal-Mediated Cell Death through Modulation of UPR, Nrf2-ARE and MAPK Pathways in PC12 Cells

PubMed Central

Wu, Pei-Shan; Yen, Jui-Hung; Kou, Mei-Chun; Wu, Ming-Jiuan

2015-01-01

Luteolin and apigenin are dietary flavones and exhibit a broad spectrum of biological activities including antioxidant, anti-inflammatory, anti-cancer and neuroprotective effects. The lipid peroxidation product 4-hydroxy-2-nonenal (4-HNE) has been implicated as a causative agent in the development of neurodegenerative disorders. This study investigates the cytoprotective effects of luteolin and apigenin against 4-HNE-mediated cytotoxicity in neuronal-like catecholaminergic PC12 cells. Both flavones restored cell viability and repressed caspase-3 and PARP-1 activation in 4-HNE-treated cells. Luteolin also mitigated 4-HNE-mediated LC3 conversion and reactive oxygen species (ROS) production. Luteolin and apigenin up-regulated 4-HNE-mediated unfolded protein response (UPR), leading to an increase in endoplasmic reticulum chaperone GRP78 and decrease in the expression of UPR-targeted pro-apoptotic genes. They also induced the expression of Nrf2-targeted HO-1 and xCT in the absence of 4-HNE, but counteracted their expression in the presence of 4-HNE. Moreover, we found that JNK and p38 MAPK inhibitors significantly antagonized the increase in cell viability induced by luteolin and apigenin. Consistently, enhanced phosphorylation of JNK and p38 MAPK was observed in luteolin- and apigenin-treated cells. In conclusion, this result shows that luteolin and apigenin activate MAPK and Nrf2 signaling, which elicit adaptive cellular stress response pathways, restore 4-HNE-induced ER homeostasis and inhibit cytotoxicity. Luteolin exerts a stronger cytoprotective effect than apigenin possibly due to its higher MAPK, Nrf2 and UPR activation, and ROS scavenging activity. PMID:26087007

77 FR 42225 - Airworthiness Directives; PILATUS AIRCRAFT LTD. Airplanes

Federal Register 2010, 2011, 2012, 2013, 2014

2012-07-18

... (AD) for all PILATUS AIRCRAFT LTD. Models PC 12, PC 12/45, PC 12/47, and PC 12/47E airplanes that... instructions and airworthiness limitations applicable to the Structure and Components of the PC-12 are... Pilatus Aircraft LTD. Models PC 12, PC 12/45, PC 12/47, and PC-12/47E airplanes, all manufacturer serial...

Microarray analysis of circular RNA expression profiles associated with gemcitabine resistance in pancreatic cancer cells.

PubMed

Xu, Chao; Yu, Yue; Ding, Fei

2018-07-01

Pancreatic cancer (PC) is one of the most malignant tumors of the digestive system due to its rapid progression, metastasis and resistance to chemotherapy. Gemcitabine (GEM) chemotherapy is the first‑choice treatment for advanced PC. However, the effect of GEM‑based chemotherapy on PC is limited due to the development of chemoresistance, and the molecular mechanisms underlying this resistance have yet to be investigated. Circular RNAs (circRNAs), which can function as microRNA sponges, have been found to be involved in the development of several types of cancer. However, research on circRNAs in PC drug resistance is limited. In the present study, the GEM‑resistant PC cell line, SWl990/GZ, was successfully established by treating parental SWl990 cells in vitro with increasing concentrations of GEM in culture medium intermittently for 10 months. By analyzing the expression profiles of circRNAs in microarray between SWl990/GZ and parental SW1990 cells, we identified 26 upregulated and 55 downregulated circRNAs (fold change ≥2 and P<0.05) among 12,866 detected circRNAs in SWl990/GZ compared with SW1990 cells. Furthermore, the changes in the expression of six representative circRNAs was validated by reverse transcription‑quantitative PCR. In addition, Kyoto Encyclopedia of Genes and Genomes pathway analysis and Gene Ontology analysis were performed. These analyses revealed that the dysregulated circRNAs regulated several cancer‑related pathways, such as the mitogen‑activated protein kinase (MAPK) and mammalian target of rapamycin (mTOR) signaling pathways, and may be involved in the biological process of the regulation of chemoresistance, including nucleic acid metabolic process and cellular response to stress. The present study undertook a comprehensive expression analysis and revealed the functional profiles of differentially expressed circRNAs associated with GEM‑resistance in PC, thereby indicating the possible participation of these dysregulated circRNAs in the development of chemoresistance and providing novel potential therapeutic targets for PC.

Protective Effect of Quercetin against Oxidative Stress-Induced Cytotoxicity in Rat Pheochromocytoma (PC-12) Cells.

PubMed

Bao, Dengke; Wang, Jingkai; Pang, Xiaobin; Liu, Hongliang

2017-07-06

Oxidative stress has been implicated in the pathogenesis of many kinds of neurodegenerative disorders, particularly Parkinson's disease. Quercetin is a bioflavonoid found ubiquitously in fruits and vegetables, and has antioxidative activity. However, the underlying mechanism of the antioxidative effect of quercetin in neurodegenerative diseases has not been well explored. Here, we investigated the antioxidative effect and underlying molecular mechanisms of quercetin on PC-12 cells. We found that PC-12 cells pretreated with quercetin exhibited an increased cell viability and reduced lactate dehydrogenase (LDH) release when exposed to hydrogen peroxide (H₂O₂). The significantly-alleviated intracellular reactive oxygen species (ROS), malondialdehyde (MDA), and lipoperoxidation of the cell membrane of PC-12 cells induced by H₂O₂ were observed in the quercetin pretreated group. Furthermore, quercetin pretreatment markedly reduced the apoptosis of PC-12 cells and hippocampal neurons. The inductions of antioxidant enzyme catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) in PC-12 cells exposed to H₂O₂ were significantly reduced by preatment with quercetin. In addition, quercetin pretreatment significantly increased Bcl-2 expression, and reduced Bax, cleaved caspase-3 and p53 expressions. In conclusion, this study demonstrated that quercetin exhibited a protective effect against oxidative stress-induced apoptosis in PC-12 cells. Our findings suggested that quercetin may be developed as a novel therapeutic agent for neurodegenerative diseases induced by oxidative stress.

Fisetin Protects PC12 Cells from Tunicamycin-Mediated Cell Death via Reactive Oxygen Species Scavenging and Modulation of Nrf2-Driven Gene Expression, SIRT1 and MAPK Signaling in PC12 Cells

PubMed Central

Yen, Jui-Hung; Wu, Pei-Shan; Chen, Shu-Fen; Wu, Ming-Jiuan

2017-01-01

Background: Fisetin (3,7,3′,4′-tetrahydroxyflavone) is a dietary flavonol and exhibits antioxidant, anti-inflammatory, and neuroprotective activities. However, high concentration of fisetin is reported to produce reactive oxygen species (ROS), induce endoplasmic reticulum (ER) stress and cause cytotoxicity in cancer cells. The aim of this study is to investigate the cytoprotective effects of low concentration of fisetin against tunicamycin (Tm)-mediated cytotoxicity in neuronal-like catecholaminergic PC12 cells. Methods: Cell viability was assayed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and apoptotic and autophagic markers were analyzed by Western blot. Gene expression of unfolded protein response (UPR) and Phase II enzymes was further investigated using RT-Q-PCR or Western blotting. Intracellular ROS level was measured using 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) by a fluorometer. The effects of fisetin on mitogen activated protein kinases (MAPKs) and SIRT1 (Sirtuin 1) signaling pathways were examined using Western blotting and specific inhibitors. Results: Fisetin (<20 µM) restored cell viability and repressed apoptosis, autophagy and ROS production in Tm-treated cells. Fisetin attenuated Tm-mediated expression of ER stress genes, such as glucose-regulated proteins 78 (GRP78), C/EBP homologous protein (CHOP also known as GADD153) and Tribbles homolog 3 (TRB3), but induced the expression of nuclear E2 related factor (Nrf)2-targeted heme oxygenase (HO)-1, glutamate cysteine ligase (GCL) and cystine/glutamate transporter (xCT/SLC7A11), in both the presence and absence of Tm. Moreover, fisetin enhanced phosphorylation of ERK (extracellular signal-regulated kinase), JNK (c-JUN NH2-terminal protein kinase), and p38 MAPK. Addition of JNK and p38 MAPK inhibitor significantly antagonized its cytoprotective activity and modulatory effects on UPR. Fisetin also restored Tm-inhibited SIRT1 expression and addition of sirtinol (SIRT1 activation inhibitor) significantly blocked fisetin-mediated cytoprotection. In conclusion, this result shows that fisetin activates Nrf2, MAPK and SIRT1, which may elicit adaptive cellular stress response pathways so as to protect cells from Tm-induced cytotoxicity. PMID:28420170

PC5-A-mediated processing of pro-neurotensin in early compartments of the regulated secretory pathway of PC5-transfected PC12 cells.

PubMed

Barbero, P; Rovère, C; De Bie, I; Seidah, N; Beaudet, A; Kitabgi, P

1998-09-25

Among the members of the proprotein convertase (PC) family, PC1 and PC2 have well established roles as prohormone convertases. Another good candidate for this role is PC5-A that has been shown to be present in the regulated secretory pathway of certain neuroendocrine tissues, but evidence that it can process prohormones is lacking. To determine whether PC5-A could function as a prohormone convertase and to compare its cleavage specificity with that of PC1 and PC2, we stably transfected the rat pheochromocytoma PC12 cell line with PC5-A and analyzed the biosynthesis and subcellular localization of the enzyme, as well as its ability to process pro-neurotensin/neuromedin N (pro-NT/NN) into active peptides. Our data showed that in transfected PC12 cells, PC5-A was converted from its 126-kDa precursor form into a 117-kDa mature form and, to a lesser extent, into a C-terminally truncated 65-kDa form of the 117-kDa product. Metabolic and immunochemical studies showed that PC5-A was sorted to early compartments of the regulated secretory pathway where it colocalized with immunoreactive NT. Furthermore, pro-NT/NN was processed in these compartments according to a pattern that differed from that previously described in PC1- and PC2-transfected PC12 cells. This pattern resembled that previously reported for pro-NT/NN processing in the adrenal medulla, a tissue known to express high levels of PC5-A. Altogether, these data demonstrate for the first time the ability of PC5-A to function as a prohormone convertase in the regulated secretory pathway and suggest a role for this enzyme in the physiological processing of pro-NT/NN.

Effects of asarinin on dopamine biosynthesis and 6-hydroxydopamine-induced cytotoxicity in PC12 cells.

PubMed

Park, Hyun Jin; Lee, Kyung Sook; Zhao, Ting Ting; Lee, Kyung Eun; Lee, Myung Koo

2017-05-01

This study investigated the effects of asarinin on dopamine biosynthesis and 6-hydroxydopamine (6-OHDA)-induced cytotoxicity in rat adrenal pheochromocytoma (PC12) cells. Treatment with asarinin (25-50 μM) increased intracellular dopamine levels and enhanced L-DOPA-induced increases in dopamine levels. Asarinin (25 μM) induced cyclic AMP-dependent protein kinase A (PKA) signaling, leading to increased cyclic AMP-response element binding protein (CREB) and tyrosine hydroxylase (TH) phosphorylation, which in turn stimulated dopamine production. Asarinin (25 μM) also activated transient phosphorylation of extracellular signal-regulated kinase (ERK1/2) and Bad phosphorylation at Ser 112, both of which have been shown to promote cell survival. In contrast, asarinin (25 μM) inhibited sustained ERK1/2, Bax, c-Jun N-terminal kinase (JNK1/2) and p38 mitogen-activated protein kinase (p38MAPK) phosphorylation and caspase-3 activity, which were induced by 6-OHDA (100 μM). These results suggest that asarinin induces dopamine biosynthesis via activation of the PKA-CREB-TH system and protects against 6-OHDA-induced cytotoxicity by inhibiting the sustained activation of the ERK-p38MAPK-JNK1/2-caspase-3 system in PC12 cells.

Purification of kavalactones from Alpinia zerumbet and their protective actions against hydrogen peroxide-induced cytotoxicity in PC12 cells.

PubMed

Rao, Yerra Koteswara; Shih, Hui-Nung; Lee, Yi-Ching; Cheng, Wen-Tai; Hung, Hui-Chin; Wang, Huang-Chi; Chen, Ching Jung; Tzeng, Yew-Min; Lee, Meng-Jen

2014-12-01

This study found that fruit shells of shell ginger (Alpinia zerumbet) are a rich source of the kavalactones dihydro-5,6-dehydrokavain (DDK) and 5,6-dehydrokavain (DK). The fruit shell extraction with hexane resulted in good purity and higher yields of DDK and DK than did chloroform, ethanol, 10% ethanol, methanol or water. Additionally, this study examined the neuroprotective effects of DDK and DK against H2O2-induced cytotoxicity in PC12 cells and the possible molecular mechanisms involved. 16 h after stimulation with 400 μM H2O2, the viability (MTT reduction) of PC12 cells decreased while membrane damage (LDH release) was noticeably increased. However, pretreatment for 6 h with DDK and DK (1 μM, 5 μM, 10 μM and 50 μM) rescued PC12 cells from H2O2-induced cytotoxicity, as evidenced by decreased LDH release and increased cell viability. DDK and DK inhibit the MAPK family member p38, activate AKT, and reduce caspase-3 activity. DDK also reduced the oxidative status in H2O2-treated PC12 cells. Together, our data indicate that the A. zerumbet constituents, DDK and DK, exert a protective effect against oxidative stress-induced PC12 cell death and that the regulation of p-Akt and the p38 MAPK, and of oxidative states may be involved. Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Cellular uptake and retention measurements of alkylphosphocholines in the SK-BR-3 breast cancer and Molt-4 leukemia cell line using capillary gas chromatography.

PubMed

Brochez, V; Van Heuverswyn, D; Diniz, J A; De Potter, C R; Van den Eeckhout, E G

1999-05-01

The determination of cellular content of octadecylphosphocholine (D-19391) and hexadecylphosphocholine (HePC, D-18506), two anticancer agents of the alkylphosphocholine group, using capillary gas chromatography is described. The compounds' cytotoxicity was first determined by the MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium] assay, being indicative for the concentration used in the uptake and retention measurements. D-19391 was added to the SK-BR-3 breast cancer cell line and HePC to the Molt-4 leukemia cell line in concentrations of, respectively, 18.6 and 15.0 microM, during a 36-h incubation period at 37 degrees C, 5% CO2. HePC uptake in the leukemia cells was followed by a 24-h reversibility test in drug-free medium. Subsequently, sample clean-up was performed on a weak cation-exchange column. For the quantitative analysis, HePC was used as internal standard for the D-19391 measurements and vice versa. Derivatization of the samples with trimethylsilylbromide was followed by capillary gas chromatographic analysis. From these data we conclude that our uptake results are quite similar with those of a previous study of HePC cellular uptake in the more resistant Caco-2T colon cancer cell line. Without having investigated the mechanism that underlies the cellular uptake results obtained, our study points to no direct correlation between the compounds' cellular uptake and their cytotoxic effects.

Enantioselective induction of cytotoxicity by o,p'-DDD in PC12 cells: implications of chirality in risk assessment of POPs metabolites.

PubMed

Wang, Cui; Li, Zhuoyu; Zhang, Quan; Zhao, Meirong; Liu, Weiping

2013-04-16

The increased release of chiral persistent organic pollutants (POPs) into the environment has resulted in more attention to the role of enantioselectivity in the fate and ecotoxicological effects of these compounds. Although the enantioselectivity of chiral POPs has been considered in previous studies, little effort has been expended to discern the enantiospecific effects of chiral POPs metabolites, which may impede comprehensive risk assessments of these chemicals. In the present study, o,p'-DDD, the chiral metabolite of o,p'-DDT, was used as a model chiral metabolite. First, a preferential chiral separation at 100% ethanol was employed to obtain a pure enantiomer. The enantioselective cytotoxicity of o,p'-DDD in rat cells (PC12) was evaluated by detecting activation of the cellular apoptosis and oxidative stress systems and microarray analysis. We have documented for the first time that R-(+)-o,p'-DDD increases apoptosis by selectively disturbing the oxidative system (enzymes and molecules) and regulating the transcription of Aven, Bid, Cideb and Tp53. By comparing the data from the present study to data derived from the parent compound, we concluded that the R-enantiomer is the more detrimental stereostructure for both o,p'-DDT and o,p'-DDD. This observed stereostructural effect is in line with the structure-activity relationship formulated at other structural levels. Biological activities of the chiral metabolites are likely to occur in the same absolute configuration between chiral POPs and their metabolites provided that they have the similar stereostructures.

High vascular delivery of EGF, but low receptor binding rate is observed in AsPC-1 tumors as compared to normal pancreas.

PubMed

Samkoe, Kimberley S; Sexton, Kristian; Tichauer, Kenneth M; Hextrum, Shannon K; Pardesi, Omar; Davis, Scott C; O'Hara, Julia A; Hoopes, P Jack; Hasan, Tayyaba; Pogue, Brian W

2012-08-01

Cellular receptor targeted imaging agents present the potential to target extracellular molecular expression in cancerous lesions; however, the image contrast in vivo does not reflect the magnitude of overexpression expected from in vitro data. Here, the in vivo delivery and binding kinetics of epidermal growth factor receptor (EGFR) was determined for normal pancreas and AsPC-1 orthotopic pancreatic tumors known to overexpress EGFR. EGFR in orthotopic xenograft AsPC-1 tumors was targeted with epidermal growth factor (EGF) conjugated with IRDye800CW. The transfer rate constants (k(e), K₁₂, k₂₁, k₂₃, and k₃₂) associated with a three-compartment model describing the vascular delivery, leakage rate and binding of targeted agents were determined experimentally. The plasma excretion rate, k (e), was determined from extracted blood plasma samples. K₁₂, k₂₁, and k₃₂ were determined from ex vivo tissue washing studies at time points ≥ 24 h. The measured in vivo uptake of IRDye800CW-EGF and a non-targeted tracer dye, IRDye700DX-carboxylate, injected simultaneously was used to determined k₂₃. The vascular exchange of IRDye800CW-EGF in the orthotopic tumor (K₁₂ and k₂₁) was higher than in the AsPC-1 tumor as compared to normal pancreas, suggesting that more targeted agent can be taken up in tumor tissue. However, the cellular associated (binding) rate constant (k₂₃) was slightly lower for AsPC-1 pancreatic tumor (4.1 × 10(-4) s(-1)) than the normal pancreas (5.5 × 10(-4) s(-1)), implying that less binding is occurring. Higher vascular delivery but low cellular association in the AsPC-1 tumor compared to the normal pancreas may be indicative of low receptor density due to low cellular content. This attribute of the AsPC-1 tumor may indicate one contributing cause of the difficulty in treating pancreatic tumors with cellular targeted agents.

Proliferation of prostate cancer cells and activity of neutral endopeptidase is regulated by bombesin and IL-1beta with IL-1beta acting as a modulator of cellular differentiation.

PubMed

Albrecht, Martin; Doroszewicz, Jolanta; Gillen, Sonja; Gomes, Iara; Wilhelm, Beate; Stief, Thomas; Aumüller, Gerhard

2004-01-01

Neutral endopeptidase (NEP) is a cell-surface bound enzyme that cleaves and inactivates neuropeptides such as bombesin and substance P and is involved in the transition from hormonally regulated androgen-dependent prostate cancer (PC) to androgen-independent PC. Neuropeptides are implicated in growth regulation of different cell types and function as transmitters between the neuroendocrine and the immune system. NEP-expression, enzymatic activity of the membrane bound protein, cell proliferation, procalcitonin (PCT) production, and secretion as well as changes in cell morphology of prostatic cells were evaluated after treatment with the immunomodulatory cytokine interleukin-1beta (IL-1beta), neuropeptides (bombesin, substance P), and neuropeptide-conditioned media derived from a human neuroendocrine cell line. Incubation of LNCaP tumor cells with IL-1beta resulted in a diminished proliferative activity, induction of neurite-like outgrowth which was accompanied by the formation of tubular-type mitochondria typical for neuronal/neuroendocrine cells, and an increased production and secretion of PCT. Conversely, proliferation of prostatic stromal cells was enhanced by the cytokine coming along with an increased number of Golgi-apparatuses and ER-cisternae. Bombesin had an antimitotic effect on LNCaP, but not on stromal cells. Substance P did not influence the growth of any of the cell types investigated, whereas neuropeptide-conditioned media exerted a slightly mitogenic effect on both cell types. The activity of LNCaP cell-surface bound NEP was enhanced by bombesin, but was diminished by substance P and neuropeptide-conditioned media. Proliferation and activity of neuropeptide degrading NEP is regulated differently by immunomodulatory substances in PC cells and cells derived from the prostatic stroma with IL-1beta being a potent modulator of cellular differentiation and a potential target for anticancer drug design in PC cells. Copyright 2003 Wiley-Liss, Inc.

Structure-function relationships in reconstituted HDL: Focus on antioxidative activity and cholesterol efflux capacity.

PubMed

Cukier, Alexandre M O; Therond, Patrice; Didichenko, Svetlana A; Guillas, Isabelle; Chapman, M John; Wright, Samuel D; Kontush, Anatol

2017-09-01

High-density lipoprotein (HDL) contains multiple components that endow it with biological activities. Apolipoprotein A-I (apoA-I) and surface phospholipids contribute to these activities; however, structure-function relationships in HDL particles remain incompletely characterised. Reconstituted HDLs (rHDLs) were prepared from apoA-I and soy phosphatidylcholine (PC) at molar ratios of 1:50, 1:100 and 1:150. Oxidative status of apoA-I was varied using controlled oxidation of Met112 residue. HDL-mediated inactivation of PC hydroperoxides (PCOOH) derived from mildly pre-oxidized low-density lipoprotein (LDL) was evaluated by HPLC with chemiluminescent detection in HDL+LDL mixtures and re-isolated LDL. Cellular cholesterol efflux was characterised in RAW264.7 macrophages. rHDL inactivated LDL-derived PCOOH in a dose- and time-dependent manner. The capacity of rHDL to both inactivate PCOOH and efflux cholesterol via ATP-binding cassette transporter A1 (ABCA1) increased with increasing apoA-I/PC ratio proportionally to the apoA-I content in rHDL. Controlled oxidation of apoA-I Met112 gradually decreased PCOOH-inactivating capacity of rHDL but increased ABCA1-mediated cellular cholesterol efflux. Increasing apoA-I content in rHDL enhanced its antioxidative activity towards oxidized LDL and cholesterol efflux capacity via ABCA1, whereas oxidation of apoA-I Met112 decreased the antioxidative activity but increased the cholesterol efflux. These findings provide important considerations in the design of future HDL therapeutics. Non-standard abbreviations and acronyms: AAPH, 2,2'-azobis(-amidinopropane) dihydrochloride; ABCA1, ATP-binding cassette transporter A1; apoA-I, apolipoprotein A-I; BHT, butylated hydroxytoluene; CV, cardiovascular; EDTA, ethylenediaminetetraacetic acid; HDL-C, high-density lipoprotein cholesterol; LOOH, lipid hydroperoxides; Met(O), methionine sulfoxide; Met112, methionine 112 residue; Met86, methionine 86 residue; oxLDL, oxidized low-density lipoprotein; PBS, phosphate-buffered saline; PC, phosphatidylcholine; PL, phospholipid; PCOOH, phosphatidylcholine hydroperoxide; PLOOH, phospholipid hydroperoxide. Copyright © 2017 Elsevier B.V. All rights reserved.

Biochemical properties of the nerve growth factor-inducible large external (NILE) glycoprotein.

PubMed

Salton, S R; Shelanski, M L; Greene, L A

1983-12-01

In the presence of nerve growth factor (NGF), PC12 pheochromocytoma cells undergo neuronal differentiation with a concomitant 3- to 5-fold increase in the specific level of an Mr = 230,000 cell surface component named the NGF-inducible large external, or NILE, glycoprotein. Antisera raised against NILE glycoprotein (NILE GP) purified from PC12 cells have been found to recognize most, if not all, neurons derived from the peripheral and central nervous systems. In the current studies several of the biochemical properties of NILE GP were investigated. NILE GP was found to be phosphorylated in NGF-treated and -untreated PC12 cells and in cultured rat sympathetic neurons. The phosphate moiety of NILE GP is almost completely alkali labile, suggesting that phosphoserine groups predominate. Immunoprecipitation experiments revealed that incorporation of [32P]phosphate into NILE GP relative to total PC12 cell phosphoprotein was not significantly altered at 12 and 24 hr of NGF treatment but was enhanced 3-fold after 7 days and up to 5-fold after 2 to 3 weeks of NGF exposure. These changes in phosphorylated NILE GP paralleled, and therefore appeared to be mainly a consequence of, the NGF-induced increase in total cellular levels of NILE GP. By two-dimensional gel analysis, anti-NILE GP selectively immunoprecipitated two NGF-inducible spots (apparent Mr = 230,000; pI = 6.4 to 6.6) from PC12 cells labeled with either [3H] fucose, [35S]methionine, or [32P]phosphate. Anti-NILE GP immunoprecipitated a single band (apparent Mr = 205,000) from extracts of rat brain labeled with [3H] glucosamine. This confirms the previously established apparent molecular weight difference between central and peripheral NILE GP cross-reactive material. When PC12 cells, cerebellar cultures, and cultured cerebral cortex were treated with tunicamycin and labeled with [35S]methionine, nonglycosylated bands each with Mr = 160,000 were immunoprecipitated, implying that the differences in the mobilities on sodium dodecyl sulfate gels of cross-reactive NILE GP from different tissues is due to variation in glycosylation rather than to large differences in apoprotein structure. Prolonged treatment of PC12 cells with trypsin produced an immunoreactive fragment of NILE GP of apparent Mr = 28,000 that was phosphorylated but not glycosylated, and that remained in the membrane. NILE GP remained predominantly membrane associated under a variety of aqueous extraction conditions, suggesting that it is an integral membrane protein.

In Vitro Neuroprotective and Anti-Inflammatory Activities of Natural and Semi-Synthetic Spirosteroid Analogues.

PubMed

García-Pupo, Laura; Zaldo-Castro, Armando; Exarchou, Vassiliki; Tacoronte-Morales, Juan Enrique; Pieters, Luc; Vanden Berghe, Wim; Nuñez-Figueredo, Yanier; Delgado-Hernández, René

2016-07-29

Two spirosteroid analogues were synthesized and evaluated for their in vitro neuroprotective activities in PC12 cells, against glutamate-induced excitotoxicity and mitochondrial damage in glucose deprivation conditions, as well as their anti-inflammatory potential in LPS/IFNγ-stimulated microglia primary cultures. We also evaluated the in vitro anti-excitotoxic and anti-inflammatory activities of natural and endogenous steroids. Our results show that the plant-derived steroid solasodine decreased PC12 glutamate-induced excitotoxicity, but not the cell death induced by mitochondrial damage and glucose deprivation. Among the two synthetic spirosteroid analogues, only the (25R)-5α-spirostan-3,6-one (S15) protected PC12 against ischemia-related in vitro models and inhibited NO production, as well as the release of IL-1β by stimulated primary microglia. These findings provide further insights into the role of specific modifications of the A and B rings of sapogenins for their neuroprotective potential.

Mammalian Target of Rapamycin Repression by 3,3'-Diindolylmethane Inhibits Invasion and Angiogenesis in Platelet-Derived Growth Factor-D–Overexpressing PC3 Cells

PubMed Central

Kong, Dejuan; Banerjee, Sanjeev; Huang, Wei; Li, Yiwei; Wang, Zhiwei; Kim, Hyeong-Reh Choi; Sarkar, Fazlul H.

2013-01-01

Platelet-derived growth factor-D (PDGF-D) is a newly recognized growth factor known to regulate many cellular processes, including cell proliferation, transformation, invasion, and angiogenesis. Recent studies have shown that PDGF-D and its cognate receptor PDGFR-β are expressed in prostate tumor tissues, suggesting that PDGF-D might play an important role in the development and progression of prostate cancer. However, the biological role of PDGF-D in tumorigenesis remains elusive. In this study, we found that PDGF-D–overexpressing PC3 cells (PC3 cells stably transfected with PDGF-D cDNA and referred to as PC3 PDGF-D) exhibited a rapid growth rate and enhanced cell invasion that was associated with the activation of mammalian target of rapamycin (mTOR) and reduced Akt activity. Rapamycin repressed mTOR activity and concomitantly resulted in the activation of Akt, which could attenuate the therapeutic effects of mTOR inhibitors. In contrast, B-DIM (BR-DIM from Bioresponse, Inc.; a chemopreventive agent) significantly inhibited both mTOR and Akt in PC3 PDGF-D cells, which were correlated with decreased cell proliferation and invasion. Moreover, conditioned medium from PC3 PDGF-D cells significantly increased the tube formation of human umbilical vein endothelial cells, which was inhibited by B-DIM treatment concomitant with reduced full-length and active form of PDGF-D. Our results suggest that B-DIM could serve as a novel and efficient chemopreventive and/or therapeutic agent by inactivation of both mTOR and Akt activity in PDGF-D–overexpressing prostate cancer. PMID:18339874

Non-monotonic changes in clonogenic cell survival induced by disulphonated aluminum phthalocyanine photodynamic treatment in a human glioma cell line

PubMed Central

2010-01-01

Background Photodynamic therapy (PDT) involves excitation of sensitizer molecules by visible light in the presence of molecular oxygen, thereby generating reactive oxygen species (ROS) through electron/energy transfer processes. The ROS, thus produced can cause damage to both the structure and the function of the cellular constituents resulting in cell death. Our preliminary investigations of dose-response relationships in a human glioma cell line (BMG-1) showed that disulphonated aluminum phthalocyanine (AlPcS2) photodynamically induced loss of cell survival in a concentration dependent manner up to 1 μM, further increases in AlPcS2concentration (>1 μM) were, however, observed to decrease the photodynamic toxicity. Considering the fact that for most photosensitizers only monotonic dose-response (survival) relationships have been reported, this result was unexpected. The present studies were, therefore, undertaken to further investigate the concentration dependent photodynamic effects of AlPcS2. Methods Concentration-dependent cellular uptake, sub-cellular localization, proliferation and photodynamic effects of AlPcS2 were investigated in BMG-1 cells by absorbance and fluorescence measurements, image analysis, cell counting and colony forming assays, flow cytometry and micronuclei formation respectively. Results The cellular uptake as a function of extra-cellular AlPcS2 concentrations was observed to be biphasic. AlPcS2 was distributed throughout the cytoplasm with intense fluorescence in the perinuclear regions at a concentration of 1 μM, while a weak diffuse fluorescence was observed at higher concentrations. A concentration-dependent decrease in cell proliferation with accumulation of cells in G2+M phase was observed after PDT. The response of clonogenic survival after AlPcS2-PDT was non-monotonic with respect to AlPcS2 concentration. Conclusions Based on the results we conclude that concentration-dependent changes in physico-chemical properties of sensitizer such as aggregation may influence intracellular transport and localization of photosensitizer. Consequent modifications in the photodynamic induction of lesions and their repair leading to different modes of cell death may contribute to the observed non-linear effects. PMID:20433757

Contra-directional Coupling of Nur77 and Nurr1 in Neurodegeneration: A Novel Mechanism for Memantine-Induced Anti-inflammation and Anti-mitochondrial Impairment.

PubMed

Wei, Xiaobo; Gao, Huimin; Zou, Jing; Liu, Xu; Chen, Dan; Liao, Jinchi; Xu, Yunqi; Ma, Long; Tang, Beisha; Zhang, Zhuohua; Cai, Xiang; Jin, Kunling; Xia, Ying; Wang, Qing

2016-11-01

Recent evidence suggests that nerve growth factor IB (Nur77) and nuclear receptor related1 (Nurr1) are differentially involved in dopaminergic neurodegeneration. Since memantine has shown clinically relevant efficacy in Parkinson's disease (PD) and displayed a potent protective effect on dopaminergic neurons in experimental PD models, we asked if it exerts its neuroprotection by regulating Nur77 and Nurr1 signaling. We adopted a well-established in vitro PD model, 6-hydroxydopamine (OHDA)-lesioned PC12 cells, to test our hypothesis. Different concentrations of memantine were incubated with 6-OHDA-lesioned PC12 cells, and Nur77/Nurr1 and their related signaling molecules were examined by Western blot and immunocytochemistry. Nur77-deficient PC12 cells were used to verify the influences of Nur77 on neurodegeneration and memantine-mediated neuroprotection. We found that memantine reversed Nur77 upregulation and restored Nurr1 downregulation in 6-OHDA-lesioned PC12 cells. 6-OHDA incubation caused Nur77 translocation from the nucleus to cytosol and induced co-localization of Cyt c/HSP60/Nur77 in the cytosol. Memantine strongly reduced the sub-cellular translocations of Nur77/Cyt c/HSP60 under 6-OHDA-induced oxidative condition. Knockdown of Nur77 enhanced the viability of PC12 cells exposed to 6-OHDA, while memantine-induced neuroprotection was much less in the cells with Nur77 knockdown than in those without it. We conclude that Nur77 plays a crucial role in modulating mitochondrial impairment and contributes to neurodegeneration under the experimental PD condition. Memantine effectively suppresses such Nur77-mediated neurodegeneration and promotes survival signaling through post-translational modification of Nurr1. Nur77 and Nurr1 present a contra-directionally coupling interaction in memantine-mediated neuroprotection.

Protective effect of cinnamaldehyde against glutamate-induced oxidative stress and apoptosis in PC12 cells.

PubMed

Lv, Chao; Yuan, Xing; Zeng, Hua-Wu; Liu, Run-Hui; Zhang, Wei-Dong

2017-11-15

Cinnamaldehyde is a main ingredient of cinnamon oils from the stem bark of Cinnamomum cassia, which has been widely used in food and traditional herbal medicine in Asia. In the present study, the neuroprotective effects and the potential mechanisms of cinnamaldehyde against glutamate-induced oxidative stress in PC12 cells were investigated. Exposure to 4mM glutamate altered the GSH, MDA levels and SOD activity, caused the generation of reactive oxygen species, resulted in the induction of oxidative stress in PC12 cell, ultimately induced cell death. However, pretreatment with cinnamaldehyde at 5, 10 and 20μM significantly attenuated cell viability loss, reduced the generation of reactive oxygen species, stabilised mitochondrial membrane potential (MMP), decreased the release of cytochrome c and limited the activities of caspase-9 and -3. In addition, cinnamaldehyde also markedly increased Bcl-2 while inhibiting Bax expression，and decreased the LC3-II/LC3-I ratio. These results indicate that cinnamaldehyde exists a potential protective effect against glutamate-induced oxidative stress and apoptosis in PC12 cells. Copyright © 2017. Published by Elsevier B.V.

Minocycline Promotes Neurite Outgrowth of PC12 Cells Exposed to Oxygen-Glucose Deprivation and Reoxygenation Through Regulation of MLCP/MLC Signaling Pathways.

PubMed

Tao, Tao; Feng, Jin-Zhou; Xu, Guang-Hui; Fu, Jie; Li, Xiao-Gang; Qin, Xin-Yue

2017-04-01

Minocycline, a semi-synthetic second-generation derivative of tetracycline, has been reported to exert neuroprotective effects both in animal models and in clinic trials of neurological diseases. In the present study, we first investigated the protective effects of minocycline on oxygen-glucose deprivation and reoxygenation-induced impairment of neurite outgrowth and its potential mechanism in the neuronal cell line, PC12 cells. We found that minocycline significantly increased cell viability, promoted neurite outgrowth and enhanced the expression of growth-associated protein-43 (GAP-43) in PC12 cells exposed to oxygen-glucose deprivation/reoxygenation injury. In addition, immunoblots revealed that minocycline reversed the overexpression of phosphorylated myosin light chain (MLC) and the suppression of activated extracellular signal-regulated kinase 1/2 (ERK1/2) caused by oxygen-glucose deprivation/reoxygenation injury. Moreover, the minocycline-induced neurite outgrowth was significantly blocked by Calyculin A (1 nM), an inhibitor of myosin light chain phosphatase (MLCP), but not by an ERK1/2 inhibitor (U0126; 10 μM). These findings suggested that minocycline activated the MLCP/MLC signaling pathway in PC12 cells after oxygen-glucose deprivation/reoxygenation injury, which resulted in the promotion of neurite outgrowth.

Hybrid ZnPc@TiO2 nanostructures for targeted photodynamic therapy, bioimaging and doxorubicin delivery.

PubMed

Flak, Dorota; Yate, Luis; Nowaczyk, Grzegorz; Jurga, Stefan

2017-09-01

In this study ZnPc@TiO 2 hybrid nanostructures, both nanoparticles and nanotubes, as potential photosensitizers for the photodynamic therapy, fluorescent bioimaging agents, as well as anti-cancer drug nanocarriers, were prepared via zinc phthalocyanine (ZnPc) deposition on TiO 2 . In order to provide the selectivity of prepared hybrid nanostructures towards cancer cells they were modified with folic acid molecules (FA). The efficient attachment of both ZnPc and FA molecules was confirmed with dynamic light scattering (DLS), zeta potential measurements and X-ray photoelectron spectroscopy (XPS). It was presented that ZnPc and FA attachment has a strong effect on fluorescence emission properties of TiO 2 nanostructures, which can be further used for their simultaneous visualization upon cellular uptake. ZnPc@TiO 2 and FA/ZnPc@TiO 2 hybrid nanotubes were then employed as doxorubicin nanocarriers. It was demonstrated that doxorubicin can be easily loaded on these hybrid nanostructures via an electrostatic interaction and then released. In vitro cytotoxicity and photo-cytotoxic activity studies showed that prepared hybrid nanostructures were selectively targeting to cancer cells. Doxorubicin loaded hybrid nanostructures were significantly more cytotoxic than un-loaded ones and their cytotoxic effect was even more severe upon irradiation. The cellular uptake of prepared hybrid nanostructures and their localization in cells was monitored in vitro in 2D cell culture and tumor-like 3D multicellular culture environment with fluorescent confocal microscopy. These hybrid nanostructures preferentially penetrated into human cervical cancer cells (HeLa) than into normal fibroblasts (MSU-1.1) and were mainly localized within the cell cytoplasm. HeLa cells spheroids were also efficiently labelled by prepared hybrid nanostructures. Fluorescent imaging of Hela cells treated with doxorubicin loaded hybrid nanostructures showed that doxorubicin was effectively delivered into cells, released and evenly distributed in the cytoplasm. In conclusion, prepared hybrid nanostructures exhibit high potential as selective bioimaging agents next to their photodynamic activity and drug delivery ability. Copyright © 2017 Elsevier B.V. All rights reserved.

Phototoxic effect of aluminium-chlorine and aluminium-hydroxide phthalocyanines on Leishmania (l.) amazonensis.

PubMed

Nesi-Reis, V; Navasconi, T R; Lera-Nosone, D S S L; Oliveira, E L; Barbosa, P M; Caetano, W; Silveira, T G V; Aristides, S M A; Hioka, N; Lonardoni, M V C

2018-03-01

This study investigated the activity of photosensitive phthalocyanines on promastigotes and amastigotes of Leishmania (L.) amazonensis. Aluminum phthalocyanine chloride (AlPcCl), Aluminum phthalocyanine hydroxide (AlPcOH) and zinc phthalocyanine (PcZn) were tested in the presence (matte red LED, potency of 2.5-2.3 μW for 30 min) and absence of light against L. amazonensis promastigotes and the parasite viability was evaluated after 24, 48 and 72 h. The amastigote forms were treated with AlPcCl and AlPcOH, following the same lighting protocols described for the promastigote forms, being evaluated after 24 h. Cytotoxicity to human erythrocytes and peritoneal macrophages was also evaluated. The results showed that AlPcCl and AlPcOH in the presence of light have antileishmania activity, with leishmanistatic effects on promastigotes and amastigotes of L. amazonensis, without causing cytotoxicity to peritoneal macrophages and erythrocytes. The concentrations that inhibited 50% of the promastigote forms after 24 h of light exposure were 0.21 ± 0.08 μM for AlPcCl and 0.23 ± 0.06 μM for AlPcOH. In 48 h and 72 h after the treatment, the IC 50 of AlPcCl was 0.13 ± 0.02 and 0.12 ± 0.03 μM and for AlPcOH was 0.14 ± 0.01 μM and 0.11 ± 0.01 μM, respectively. PcZn showed no activity on promastigotes of L. amazonensis. This study showed a substantial photodynamic activity of the phthalocyanines AlPcCl and AlPcOH against intracellular amastigotes forms of L. amazonensis after irradiation, presenting IC 50 values of 0.62 ± 0.06 μM and 0.92 ± 0.12 μM, respectively. These results support the possibility of using photodynamic therapy for the treatment of cutaneous leishmaniasis. Copyright © 2017 Elsevier B.V. All rights reserved.

Chimeric Anti-Human Podoplanin Antibody NZ-12 of Lambda Light Chain Exerts Higher Antibody-Dependent Cellular Cytotoxicity and Complement-Dependent Cytotoxicity Compared with NZ-8 of Kappa Light Chain.

PubMed

Kaneko, Mika K; Abe, Shinji; Ogasawara, Satoshi; Fujii, Yuki; Yamada, Shinji; Murata, Takeshi; Uchida, Hiroaki; Tahara, Hideaki; Nishioka, Yasuhiko; Kato, Yukinari

2017-02-01

Podoplanin (PDPN), a type I transmembrane 36-kDa glycoprotein, is expressed not only in normal cells, such as renal epithelial cells (podocytes), lymphatic endothelial cells, and pulmonary type I alveolar cells, but also in cancer cells, including brain tumors and lung squamous cell carcinomas. Podoplanin activates platelet aggregation by binding to C-type lectin-like receptor-2 (CLEC-2) on platelets, and the podoplanin/CLEC-2 interaction facilitates blood/lymphatic vessel separation. We previously produced neutralizing anti-human podoplanin monoclonal antibody (mAb), clone NZ-1 (rat IgG 2a , lambda), which neutralizes the podoplanin/CLEC-2 interaction and inhibits platelet aggregation and cancer metastasis. Human-rat chimeric antibody, NZ-8, was previously developed using variable regions of NZ-1 and human constant regions of heavy chain (IgG 1 ) and light chain (kappa chain). Although NZ-8 showed high antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against human podoplanin-expressing cancer cells, the binding affinity of NZ-8 was lower than that of NZ-1. Herein, we produced a novel human-rat chimeric antibody, NZ-12, the constant regions of which consist of IgG 1 heavy chain and lambda light chain. Using flow cytometry, we demonstrated that the binding affinity of NZ-12 was much higher than that of NZ-8. Furthermore, ADCC and CDC activities of NZ-12 were significantly increased against glioblastoma cell lines (LN319 and D397) and lung cancer cell line (PC-10). These results suggested that NZ-12 could become a promising therapeutic antibody against podoplanin-expressing brain tumors and lung cancers.

NBD-labelled phospholipid accelerates apolipoprotein C-II amyloid fibril formation but is not incorporated into mature fibrils

PubMed Central

Ryan, Timothy M.; Griffin, Michael D. W.; Bailey, Michael F.; Schuck, Peter; Howlett, Geoffrey J.

2014-01-01

Human apolipoprotein (apo) C-II is one of several lipid-binding proteins that self-assemble into fibrils and accumulate in disease-related amyloid deposits. A general characteristic of these amyloid deposits is the presence of lipids, known to modulate individual steps in amyloid fibril formation. ApoC-II fibril formation is activated by sub-micellar phospholipids but inhibited by micellar lipids. We examined the mechanism for the activation by sub-micellar lipids using the fluorescently-labelled, short-chain phospholipid, 1-dodecyl-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]-2-hydroxy-glycero-3-phosphocholine (NBD-lyso-12-PC). Addition of submicellar NBD-lyso-12-PC increased the rate of fibril formation by apoC-II approximately two-fold. Stopped flow kinetic analysis using fluorescence detection and low, non-fibril forming concentrations of apoC-II indicated NBD-Lyso-12-PC binds rapidly, in the millisecond timescale, followed by the slower formation of discrete apoC-II tetramers. Sedimentation velocity analysis showed NBD-Lyso-12-PC binds to both apoC-II monomers and tetramers at approximately 5 sites per monomer with an average dissociation constant of approximately 10 μM. Mature apoC-II fibrils formed in the presence of NBD-Lyso-12-PC were devoid of lipid indicating a purely catalytic role for sub-micellar lipids in the activation of apoC-II fibril formation. These studies demonstrate the catalytic potential of small amphiphilic molecules to control protein folding and fibril assembly pathways. PMID:21985034

Protective effects of red wine flavonols on 4-hydroxynonenal-induced apoptosis in PC12 cells.

PubMed

Jang, Young Jin; Kang, Nam Joo; Lee, Ki Won; Lee, Hyong Joo

2009-08-01

There is accumulating evidence that a moderate consumption of red wine has health benefits, such as the inhibition of neurodegenerative diseases. Although this is generally attributed to resveratrol, the protective mechanisms and the active substance(s) remain unclear. We examined whether and how red wine extract (RWE) and red wine flavonols quercetin and myricetin inhibited 4-hydroxynonenal (HNE)-induced apoptosis of rat pheochromocytoma PC12 cells. RWE attenuated HNE-induced PC12 cell death in a dose-dependent manner. HNE induced cleavage of poly(ADP-ribose) polymerase, which is involved in DNA repair in the nucleus, and this was inhibited by RWE treatment. Treatment with RWE also inhibited HNE-induced nuclear condensation in PC12 cells. Data of 2',7'-dichlorofluorescin diacetate showed that RWE protected against apoptosis of PC12 cells by attenuating intracellular reactive oxygen species. The cytoprotective effects on HNE-induced cell death were stronger for quercetin and myricetin than for resveratrol. HNE-induced nuclear condensation was attenuated by quercetin and myricetin. These results suggest that the neuroprotective potential of red wine is attributable to flavonols rather than to resveratrol.

Effects of rearrangement and allelic exclusion of JJAZ1/SUZ12 on cell proliferation and survival

PubMed Central

Li, Hui; Ma, XianYong; Wang, Jinglan; Koontz, Jason; Nucci, Marisa; Sklar, Jeffrey

2007-01-01

Polycomb group genes (PcGs) have been implicated in cancer based on altered levels of expression observed in certain tumors and the behavior of cultured cells containing inserted PcG transgenes. Endometrial stromal tumors provide evidence for a direct causal relationship because they contain several chromosomal translocations and resultant gene fusions involving PcGs, the most common of which joins portions of theJAZF1 gene to the PcGJJAZ1/SUZ12. We show here that both benign and malignant forms of this tumor have theJAZF1–JJAZ1 fusion but only the malignant form also exhibits exclusion of the unrearrangedJJAZ1 allele. To evaluate the effects of both theJJAZ1/SUZ12 fusion and allelic exclusion on functions related to cell growth, we studied HEK293 cells that were modified with respect toJJAZ1 expression. We found that theJAZF1–JJAZ1 fusion restored levels of the polycomb protein EZH2 and histone 3 lysine 27 trimethylation, which were reduced by knockdown of endogenous JJAZ1. At the same time, the presence ofJAZF1–JJAZ1 markedly inhibited apoptosis and induced above normal proliferation rates, although the latter effect occurred only when normalJJAZ1 was suppressed. Our findings suggest a genetic pathway for progression of a benign precursor to a sarcoma involving increased cell survival associated with acquisition of a PcG rearrangement, followed by accelerated cellular proliferation upon allelic exclusion of the unrearranged copy of that gene. Furthermore, these results indicate the likely functional importance of allelic exclusion of genes disrupted by chromosomal translocations, as seen in a variety of other cancers. PMID:18077430

Deficiency in methionine, tryptophan, isoleucine, or choline induces apoptosis in cultured cells.

PubMed

Yen, Chi-Liang E; Mar, Mei-Heng; Craciunescu, Corneliu N; Edwards, Lloyd J; Zeisel, Steven H

2002-07-01

Cells in culture die by apoptosis when deprived of the essential nutrient choline. We now report that cells (both proliferating PC12 cells and postmitotic neurons isolated from fetal rat brains) undergo apoptosis when deprived of other individual essential nutrients (methionine, tryptophan or isoleucine). In PC12 cells, deficiencies of each nutrient independently led to ceramide accumulation and to caspase activation, both recognized signals of several apoptotic pathways. A similar profile of caspases was activated in PC12 cells deprived of choline, methionine, tryptophan or isoleucine. More than one caspase was involved and these caspases appeared to transmit parallel signals for apoptosis induction because only broad-spectrum caspase inhibitors, but not inhibitors for specific individual caspases inhibited apoptosis in choline- or methionine-deprived cells. The induction of these caspase-dependent apoptosis pathways likely did not involve the same upstream signals. Choline deficiency perturbed choline metabolism but did not affect protein synthesis, whereas amino acid deficiencies inhibited protein synthesis but did not perturb choline metabolism. In addition, a subclone of PC12 cells that was resistant to choline deficiency-induced apoptosis was not resistant to tryptophan deficiency-induced apoptosis. These observations suggest that deficiency of each studied nutrient activates different pathways for signaling apoptosis that ultimately converge on a common execution pathway.

Rhus verniciflua Stokes Extract and Its Flavonoids Protect PC-12 Cells against H2O2-Induced Cytotoxicity.

PubMed

Nam, Tae Gyu; Lee, Bong Han; Choi, Hyo-Kyoung; Mansur, Ahmad Rois; Lee, Sang Gil; Kim, Dae-Ok

2017-06-28

Rhus verniciflua Stokes (RVS), an herbal medicine found in East Asia, was extracted and further fractionated to investigate its antioxidant capacity and neuroprotective effects. The RVS ethyl acetate (EtOAc) fraction had the highest level of total phenolics and antioxidant capacity among all solvent fractions tested. Pretreatment of PC-12 cells with the EtOAc fraction effectively attenuated H 2 O 2 -induced oxidative damage. Furthermore, the EtOAc fraction significantly attenuated caspase-3 activity, resulting in inhibition of H 2 O 2 -induced apoptosis. We identified and quantified fustin, sulfuretin, and butein in the EtOAc fraction using accurate mass quadrupole time-of-flight mass spectrometry and reversed-phase high-performance liquid chromatography. The intracellular antioxidant capacity and superoxide dismutase (SOD) activity were significantly increased in PC-12 cells treated with the EtOAc fraction and with individual flavonoids. When cells were pretreated with the EtOAc fraction or individual flavonoids and then co-incubated with diethyldithiocarbamic acid (an inhibitor of SOD activity), cell viability against H 2 O 2 -induced oxidative stress was attenuated. These results suggest that the RVS EtOAc fraction and its flavonoid constituents protect PC-12 cells against H 2 O 2 -induced neurotoxicity through their antioxidant properties.

Antioxidant and neuroprotector effect of Lepidium meyenii (maca) methanol leaf extract against 6-hydroxy dopamine (6-OHDA)-induced toxicity in PC12 cells.

PubMed

Rodríguez-Huamán, Ángel; Casimiro-Gonzales, Sandra; Chávez-Pérez, Jorge Antonio; Gonzales-Arimborgo, Carla; Cisneros-Fernández, Richard; Aguilar-Mendoza, Luis Ángel; Gonzales, Gustavo F

2017-05-01

Reactive oxygen species (ROS) are normally produced during cell metabolism, there is strong evidence to suggest that ROS produced in excess impair the cell and may be etiologically related to various neurodegenerative diseases. This study was undertaken to examine the effects of Lepidium meyenii (MACA) methanol leaf extract on neurotoxicity in PC12 cell exposed to 6-hydroxydopamine (6-OHDA). Fresh samples of "maca" leaves were processed in order to obtain foliar extracts and to evaluate the neurobiological activity on PC12 cells, subjected to the cytotoxic effect of 6-OHDA through the determination of the capacity antioxidant, cell viability and cytotoxicity assays on PC12 cells. The results of the tests of antioxidant activity, showed maximum values of 2262.37 and 1305.36 expressed in Trolox equivalents (TEAC), for the methanolic and aqueous fractions respectively. Cell viability assays at a dose of 10 μg extract showed an increase of 31% and 60% at 6 and 12 h of pretreatment, respectively. Cytotoxicity assays at the same dose and exposure time showed a 31.4% and 47.8% reduction in lactate dehydrogenase (LDH) activity and an increase in superoxide dismutase (SOD) activity. The results allow us to affirm that the methanolic foliar extract of "maca" presents in vitro neurobiological activity of antioxidant protection, increase in cell viability and reduction of cytotoxicity against oxidative stress generated by 6-OHDA. In conclusion, the present study shows a protective role for Lepidium meyenii leaf extract on 6-OHDA-induced toxicity by an antioxidant effect.

Regulation of Polycystin-1 Function by Calmodulin Binding

PubMed Central

Doerr, Nicholas; Wang, Yidi; Kipp, Kevin R.; Liu, Guangyi; Benza, Jesse J.; Pletnev, Vladimir; Pavlov, Tengis S.; Staruschenko, Alexander; Mohieldin, Ashraf M.; Takahashi, Maki; Nauli, Surya M.; Weimbs, Thomas

2016-01-01

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a common genetic disease that leads to progressive renal cyst growth and loss of renal function, and is caused by mutations in the genes encoding polycystin-1 (PC1) and polycystin-2 (PC2), respectively. The PC1/PC2 complex localizes to primary cilia and can act as a flow-dependent calcium channel in addition to numerous other signaling functions. The exact functions of the polycystins, their regulation and the purpose of the PC1/PC2 channel are still poorly understood. PC1 is an integral membrane protein with a large extracytoplasmic N-terminal domain and a short, ~200 amino acid C-terminal cytoplasmic tail. Most proteins that interact with PC1 have been found to bind via the cytoplasmic tail. Here we report that the PC1 tail has homology to the regulatory domain of myosin heavy chain including a conserved calmodulin-binding motif. This motif binds to CaM in a calcium-dependent manner. Disruption of the CaM-binding motif in PC1 does not affect PC2 binding, cilia targeting, or signaling via heterotrimeric G-proteins or STAT3. However, disruption of CaM binding inhibits the PC1/PC2 calcium channel activity and the flow-dependent calcium response in kidney epithelial cells. Furthermore, expression of CaM-binding mutant PC1 disrupts cellular energy metabolism. These results suggest that critical functions of PC1 are regulated by its ability to sense cytosolic calcium levels via binding to CaM. PMID:27560828

Factors influencing the expression of T15 idiotopes during an antigen-driven response to phosphorylcholine (PC)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Strickland, F.; Cronkhite, R.; Cerny, J.

1986-03-01

The idiotypy of the serum antibody response to PC was examined in twelve inbred strains of mice including several Igh- and h-2 congenics. The sera were collected after primary (1/sup 0/), and secondary (2/sup 0/), and tertiary (3/sup 0/) immunizations with either S. pneumoniae R36a (Pn) or PC coupled to keyhole limpet hemocyanine (PC-KLH). Concentrations of PC-binding activity and of four distinct T15 idiotopes (Id) detectable with monoclonal anti-Id AB1-2, MaId5-4, B36-82 and B24-44 were measured in solid-phase radioimmunoassay. These Id are independent clonotypic markers of the anti-PC antibody repertoire. The serum levels of the Id had kinetics distinct frommore » each other and from the total PC-binding activity. For example, the concentration of AB1-2 decreased after 2/sup 0/ and 3/sup 0/ challenge with PC-KLH while the levels of B36-82 and B24-44 remained stable in several strains. Marked differences in Id serum levels were observed among various strains; however, their genetic mapping has been difficult in that an apparent effect of H-2 locus seemed to be modified by the genetic background of the mice. The only unambiguous linkage was the absence of AB1-2 and MaId5-4 in mice bearing Igh/sup j/. The authors data indicate that idiotopically-distinct T15/sup +/ clones are selected during an antigen-driven response in genetically different mice. Mechanisms regulating these patterns are being studied.« less

Assessment of Parylene C Thin Films for Heart Valve Tissue Engineering

PubMed Central

Marei, Isra; Chester, Adrian; Carubelli, Ivan; Prodromakis, Themistoklis; Trantidou, Tatiana

2015-01-01

Background: Scaffolds are a key component of tissue-engineered heart valves (TEHVs). Several approaches had been adopted in the design of scaffolds using both natural and synthetic resources. We have investigated the suitability of parylene C (PC), a vapor deposited polymeric material, for the use as a scaffold in TEHV. Aims: To evaluate the adsorption of extracellular matrix components onto plasma-activated PC and study the biocompatibility of PC by measuring cellular adhesion, viability, apoptosis, and phenotypic expression of valve endothelial and interstitial cells. Finally, the mechanical properties of PC were compared with those of native aortic valve cusp tissue. Methods: PC slides were plasma activated and then coated with gelatin, type I collagen, or fibronectin. Porcine pulmonary valve endothelial and interstitial cells were then grown on plasma oxidized PC with different types of coatings and their adhesion was observed after 20 h of incubation. Cell viability was tested using the MTS assay, and apoptosis was estimated using TUNEL staining. The mechanical properties of PC and valve tissue were measured using a Bose Mechanical Tester. Finally, cell-seeded PC films were exposed to pulsatile pressure and aortic shear stress, respectively, to test their durability in a dynamic environment. Results: Our findings show that collagen and fibronectin could bind to plasma oxidized PC. Both valve endothelial and interstitial cells adhered to protein-coated ECM. PC had a profile of mechanical stiffness and ultimate tensile strength that were comparable with or in excess of those seen in porcine aortic valve cusps. Cells were still attached to PC films after 3 days of exposure to up to 50 mmHg pulsatile pressure or aortic levels of shear stress. Conclusion: PC is a promising candidate for use as a scaffold in tissue engineering heart valves. Additional studies are required to determine both the durability and long-term performance of cell-seeded PC when in a similar hemodynamic environment to that of the aortic valve. PMID:26101808

CXCR6-CXCL16 axis promotes prostate cancer by mediating cytoskeleton rearrangement via Ezrin activation and αvβ3 integrin clustering.

PubMed

Singh, Rajesh; Kapur, Neeraj; Mir, Hina; Singh, Nalinaksha; Lillard, James W; Singh, Shailesh

2016-02-09

Cytoskeletal rearrangement is required for migration and invasion, which are the key steps of cancer metastasis. Ezrin and integrin co-ordinate these processes by regulating cellular adhesion and cytoskeletal polymerization-depolymerization. It is also well established that chemokine-chemokine receptor axis plays a crucial role in regulating cancer cell migration and invasion. In this study, we show involvement of CXC chemokine receptor 6 (CXCR6) and its only natural ligand CXCL16 in pathobiology of prostate cancer (PCa). CXCR6 is highly expressed in PCa tissues and cell lines (LNCaP and PC3), relative to normal tissue and cells. CXCR6 expression in PCa tissues correlated with higher Gleason score. Similarly, aggressive PCa cells (PC3) show high CXCR6 compared to less aggressive LNCaP. Besides, PC3 cells show higher MMPs expression compared to LNCaP cells following CXCL16 stimulation. Intriguingly, CXCR6-CXCL16 interaction in PCa cells promotes Ezrin activation, αvβ3 integrin clustering and capping at the leading edge in FAK/PI3K/PKC dependent manner, thereby modifying cellular adhesion as well as motility. Together these results demonstrate that CXCL16 stimulation changes cytoskeletal dynamics resulting in enhanced migration, invasion and adhesion to endothelial cells, ultimately enabling PCa cells to achieve their metastatic goal.

CXCR6-CXCL16 axis promotes prostate cancer by mediating cytoskeleton rearrangement via Ezrin activation and αvβ3 integrin clustering

PubMed Central

Singh, Rajesh; Kapur, Neeraj; Mir, Hina; Singh, Nalinaksha; Lillard, James W.; Singh, Shailesh

2016-01-01

Cytoskeletal rearrangement is required for migration and invasion, which are the key steps of cancer metastasis. Ezrin and integrin co-ordinate these processes by regulating cellular adhesion and cytoskeletal polymerization-depolymerization. It is also well established that chemokine-chemokine receptor axis plays a crucial role in regulating cancer cell migration and invasion. In this study, we show involvement of CXC chemokine receptor 6 (CXCR6) and its only natural ligand CXCL16 in pathobiology of prostate cancer (PCa). CXCR6 is highly expressed in PCa tissues and cell lines (LNCaP and PC3), relative to normal tissue and cells. CXCR6 expression in PCa tissues correlated with higher Gleason score. Similarly, aggressive PCa cells (PC3) show high CXCR6 compared to less aggressive LNCaP. Besides, PC3 cells show higher MMPs expression compared to LNCaP cells following CXCL16 stimulation. Intriguingly, CXCR6-CXCL16 interaction in PCa cells promotes Ezrin activation, αvβ3 integrin clustering and capping at the leading edge in FAK/PI3K/PKC dependent manner, thereby modifying cellular adhesion as well as motility. Together these results demonstrate that CXCL16 stimulation changes cytoskeletal dynamics resulting in enhanced migration, invasion and adhesion to endothelial cells, ultimately enabling PCa cells to achieve their metastatic goal. PMID:26799186

Prion disease tempo determined by host-dependent substrate reduction

PubMed Central

Mays, Charles E.; Kim, Chae; Haldiman, Tracy; van der Merwe, Jacques; Lau, Agnes; Yang, Jing; Grams, Jennifer; Di Bari, Michele A.; Nonno, Romolo; Telling, Glenn C.; Kong, Qingzhong; Langeveld, Jan; McKenzie, Debbie; Westaway, David; Safar, Jiri G.

2014-01-01

The symptoms of prion infection can take years or decades to manifest following the initial exposure. Molecular markers of prion disease include accumulation of the misfolded prion protein (PrPSc), which is derived from its cellular precursor (PrPC), as well as downregulation of the PrP-like Shadoo (Sho) glycoprotein. Given the overlapping cellular environments for PrPC and Sho, we inferred that PrPC levels might also be altered as part of a host response during prion infection. Using rodent models, we found that, in addition to changes in PrPC glycosylation and proteolytic processing, net reductions in PrPC occur in a wide range of prion diseases, including sheep scrapie, human Creutzfeldt-Jakob disease, and cervid chronic wasting disease. The reduction in PrPC results in decreased prion replication, as measured by the protein misfolding cyclic amplification technique for generating PrPSc in vitro. While PrPC downregulation is not discernible in animals with unusually short incubation periods and high PrPC expression, slowly evolving prion infections exhibit downregulation of the PrPC substrate required for new PrPSc synthesis and as a receptor for pathogenic signaling. Our data reveal PrPC downregulation as a previously unappreciated element of disease pathogenesis that defines the extensive, presymptomatic period for many prion strains. PMID:24430187

Apple Procyanidins Suppress Amyloid β-Protein Aggregation

PubMed Central

Toda, Toshihiko; Sunagawa, Tadahiro; Kanda, Tomomasa; Tagashira, Motoyuki; Shirasawa, Takuji; Shimizu, Takahiko

2011-01-01

Procyanidins (PCs) are major components of the apple polyphenols (APs). We previously reported that treatment with PC extended the mean lifespan of Caenorhabditis elegans (Sunagawa et al., 2011). In order to estimate the neuroprotective effects of PC, we investigated the antiaggregative activity of PC on amyloid β-protein (Aβ) aggregation, which is a pathological hallmark of Alzheimer's disease. We herein report that PC significantly suppressed Aβ42 aggregation and dissociated Aβ42 aggregates in a dose-dependent manner, indicating that PC is a potent suppressor of Aβ aggregation. Furthermore, PC significantly inhibited Aβ42 neurotoxicity and stimulated proliferation in PC-12 cells. These results suggested that the PC and AP acted as neuroprotective factors against toxic Aβ aggregates. PMID:21826271

Enhancer of polycomb coordinates multiple signaling pathways to promote both cyst and germline stem cell differentiation in the Drosophila adult testis

PubMed Central

Feng, Lijuan; Shi, Zhen; Chen, Xin

2017-01-01

Stem cells reside in a particular microenvironment known as a niche. The interaction between extrinsic cues originating from the niche and intrinsic factors in stem cells determines their identity and activity. Maintenance of stem cell identity and stem cell self-renewal are known to be controlled by chromatin factors. Herein, we use the Drosophila adult testis which has two adult stem cell lineages, the germline stem cell (GSC) lineage and the cyst stem cell (CySC) lineage, to study how chromatin factors regulate stem cell differentiation. We find that the chromatin factor Enhancer of Polycomb [E(Pc)] acts in the CySC lineage to negatively control transcription of genes associated with multiple signaling pathways, including JAK-STAT and EGF, to promote cellular differentiation in the CySC lineage. E(Pc) also has a non-cell-autonomous role in regulating GSC lineage differentiation. When E(Pc) is specifically inactivated in the CySC lineage, defects occur in both germ cell differentiation and maintenance of germline identity. Furthermore, compromising Tip60 histone acetyltransferase activity in the CySC lineage recapitulates loss-of-function phenotypes of E(Pc), suggesting that Tip60 and E(Pc) act together, consistent with published biochemical data. In summary, our results demonstrate that E(Pc) plays a central role in coordinating differentiation between the two adult stem cell lineages in Drosophila testes. PMID:28196077

Ablation of Prion Protein in Wild Type Human Amyloid Precursor Protein (APP) Transgenic Mice Does Not Alter The Proteolysis of APP, Levels of Amyloid-β or Pathologic Phenotype

PubMed Central

Baybutt, Herbert; Diack, Abigail B.; Kellett, Katherine A. B.; Piccardo, Pedro; Manson, Jean C.

2016-01-01

The cellular prion protein (PrPC) has been proposed to play an important role in the pathogenesis of Alzheimer’s disease. In cellular models PrPC inhibited the action of the β-secretase BACE1 on wild type amyloid precursor protein resulting in a reduction in amyloid-β (Aβ) peptides. Here we have assessed the effect of genetic ablation of PrPC in transgenic mice expressing human wild type amyloid precursor protein (line I5). Deletion of PrPC had no effect on the α- and β-secretase proteolysis of the amyloid precursor protein (APP) nor on the amount of Aβ38, Aβ40 or Aβ42 in the brains of the mice. In addition, ablation of PrPC did not alter Aβ deposition or histopathology phenotype in this transgenic model. Thus using this transgenic model we could not provide evidence to support the hypothesis that PrPC regulates Aβ production. PMID:27447728

Modulation of the antioxidant/pro-oxidant balance, cytotoxicity and antiviral actions of grape seed extracts.

PubMed

Ignea, Codruţa; Dorobanţu, Cristina Mihaela; Mintoff, Christopher Paul; Branza-Nichita, Norica; Ladomery, Michael R; Kefalas, Panagiotis; Chedea, Veronica Sanda

2013-12-15

Grape seed extracts (GSEs) were investigated in yeast cells harbouring defects in their antioxidant system (regarding the cellular growth and growth recovery from H2O2 insult). GSEs antioxidant activity was detected in wild-type and mutant strains Δcta1, Δgsh1 and Δoye2glr1, while pro-oxidant activity in Δsod1 cells was seen. Assessment of proliferation of prostate cancer PC3 and HBV-replicating HepG2 2.2.15 cells treated with GSEs has shown higher cytotoxicity of red grape seed extract (RW) than white grape seed extract (WW) subjective to dose and period of administration. No antiviral effect was detected by measuring the secreted virion particles in HepG2 2.2.15 cells treated with GSEs. The GSEs play a dual antioxidant/pro-oxidant role in vivo according with the cellular antioxidant system deficiencies and exhibit cytotoxic properties in PC3 and HepG2 2.2.15 cell lines, but no antiviral action against HBV. Copyright © 2013 Elsevier Ltd. All rights reserved.

[Regulation of [12Asp]K-ras4B on transcriptional activity of estrogen receptor in endometrial carcinoma HEC-1A cell lines].

PubMed

Gui, Li-ming; Wei, Li-hui; Xu, Ming-xu; Wang, Jian-liu; Zhong, Ying-cheng; Li, Xiao-ping; Tu, Zheng; Sun, Peng-ming; Ma, Da-long

2004-01-01

To investigate the effect of mutant-type [(12)Asp]K-ras4B gene on the expression of estrogen receptor (ER) alpha and beta and their transcriptional activity as a transcription factor in endometrial carcinoma HEC-1A cell line. (1) Effect of [(12)Asp]K-ras4B on the expression of ER alpha and beta were determined using Western blot assay. (2) Eukaryotic expression plasmid pGL3-luciferase-ERE containing luciferase report gene and estrogen receptor element (ERE) was constructed, and co-transfected into NIH3T3 and HEC-1A cell lines with pEGFP-N1 to examine the effect of [(12)Asp]K-ras4B on ER transcription that is regulated by estradiol. In addition, they were transfected into pSV5-HER0 (containing full length wide type ERalpha cDNA) and pCMV-rafS621A (inhibiting raf kinase) plasmids to test the effect of [(12)Asp]K-ras4B/raf signal pathway on transcriptional activity of ER proteins. (1) Protein level of ERs expressed in pcDI transfected control cells was low while it was increased for 3.6-fold (97 +/- 25, 349 +/- 67, P < 0.01) and 1.9-fold (128 +/- 37, 349 +/- 30, P < 0.05) in ERalpha and ERbeta, respectively, in pcDI-[(12)Asp]K-ras4B NIH3T3 cells after transfection. (2) In pcDI-[(12)Asp]K-ras4B NIH3T3 cells, the ratios for ERalpha and and ERbeta levels before transfection of rafS621A plasmids to that after the transfection, were 2.4:1 (724 +/- 45, 310 +/- 46, P < 0.05) and 1.8:1 (493 +/- 20, 284 +/- 20, P < 0.01), respectively; In HEC-1A cells, these ratios were 2.1:1 (566 +/- 22, 279 +/- 30, P < 0.01) and 2.4:1 (405 +/- 33, 165 +/- 15, P < 0.01), respectively. (3) In low serum (2%) culture condition, estradiol (E(2)) stimulated luciferase activity with an increase of 13-fold (130 +/- 42, 1681 +/- 242, P < 0.01) in pcDI-[(12)Asp] K-ras4B NIH3T3 cells, 19-fold (141 +/- 39, 2644 +/- 331, P < 0.001) in HEC-1A cells, respectively, when compared with those in the absence of E(2). (4) In pSV5-HER0 transfected pcDI-[(12)Asp] K-ras4B NIH3T3 cells and HEC-1A cells, compared to the untransfected cells, the ER transcriptional activity in the transfected cells increased markedly. The luciferase activity was increased for 8-fold (1048 +/- 91, 8099 +/- 452, P < 0.01) and 6-fold (2148 +/- 259, 12,705 +/- 2670, P < 0.001), respectively. rafS621A mutant had suppressive effects on luciferase activities in HEC-1A cells and pcDI-[(12)Asp]K-ras4B NIH3T3 cells. The ratio of luciferase activities in pcDI-[(12)Asp]K-ras4B NIH3T3 and HEC-1A cells, before and after transfection was 7.8:1 (1184 +/- 168, 152 +/- 27, P < 0.05) and 6.4:1 (1949 +/- 212, 304 +/- 60, P < 0.01), respectively. (1) [(12)Asp]K-ras4B can enhance the expression of ERalpha and beta proteins. This may be correlated with [(12)Asp]K-ras4B/raf signaling pathway. (2) The effect of mutant-type [(12)Asp]K-ras4B gene on ERs transcriptional activity in HEC-1A cells appears to need E(2).

CXCR6 is expressed in human prostate cancer in vivo and is involved in the in vitro invasion of PC3 and LNCap cells.

PubMed

Hu, Weidong; Zhen, Xinming; Xiong, Bin; Wang, Bicheng; Zhang, Weibing; Zhou, Wenhui

2008-07-01

In spite of the clinical importance of prostate cancer (PCa) bone metastasis, the precise mechanisms for the directed migration of malignant cells remain unclear. In the present study, the expression of CXCR6 in human PCa and benign prostatic hyperplasia samples, and the expression of CXCL16 in human osseous tissues were determined by immunohistochemistry. It was found that the level of CXCR6 protein expression was elevated in human malignant prostate tumors, and CXCL16 was expressed positively by human osteocytes in vivo. The in vitro experiments further confirmed that the PCa cell lines PC3 and LNCap expressed CXCR6 at both the mRNA and protein levels, and exogenous CXCL16 has the potential to stimulate the invasion of PC3 and LNCap. To further elucidate the role of the CXCL16-CXCR6 axis in PCa progression, we compared the expression of CXCR6 and CXCR4 in human PCa tissues and the effects of CXCL16 and CXCL12 on the in vitro invasion of PC3 and LNCap cells. It was shown that CXCR6 and CXCR4 proteins were coexpressed and elevated in human PCa samples, and CXCL16 and CXCL12 promoted the invasion of PC3 and LNCap via their respective receptors. Furthermore, in contrast to CXCL12, which enhanced the activity of matrix metalloproteinase (MMP) 9 and MMP2 in PC3 and LNCap, CXCL16 ligation resulted in stronger MMP9 and MMP2 activity in LNCap but not in PC3. Our results suggest that besides CXCL12/CXCR4, CXCL16/CXCR6 might be another important factor involved in PCa bone metastasis.

Introducing a simple model system for binding studies of known and novel inhibitors of AMPK: a therapeutic target for prostate cancer.

PubMed

Kumar, Rakesh; Maurya, Ranjana; Saran, Shweta

2018-02-23

Prostate cancer (PC) is one of the leading cancers in men, raising a serious health issue worldwide. Due to lack of suitable biomarker, their inhibitors and the platform for testing those inhibitors result in poor prognosis of PC. AMP-activated protein kinase (AMPK) is a highly conserved protein kinase found in eukaryotes that is involved in growth and development, and also acts as a therapeutic target for PC. The aim of the present study is to identify novel potent inhibitors of AMPK and propose a simple cellular model system for understanding its biology. Structural modelling and MD simulations were performed to construct and refine the 3D models of Dictyostelium and human AMPK. Binding mechanisms of different drug compounds were studied by performing molecular docking, molecular dynamics and MM-PBSA methods. Two novel drugs were isolated having higher binding affinity over the known drugs and hydrophobic forces that played a key role during protein-ligand interactions. The study also explored the simple cellular model system for drug screening and understanding the biology of a therapeutic target by performing in vitro experiments.

Characterization of superparamagnetic iron oxide nanoparticle-induced apoptosis in PC12 cells and mouse hippocampus and striatum.

PubMed

Liu, Yutong; Li, Juan; Xu, Kaige; Gu, Jingjing; Huang, Lu; Zhang, Lei; Liu, N; Kong, Jiming; Xing, Malcolm; Zhang, Lin; Zhang, Lu

2018-08-01

Superparamagnetic iron oxide nanoparticles (SPIONs) have been widely used as theranostic drug-carrier and MRI contrast agent. Their potential effects are still in blank while SPIONs are used for brain. The present study aims to investigate SPIONs' neurotoxicity in vitro and in vivo using stereotaxic technique. By co-incubating SPIONs with dopaminergic neuronal PC12 cells, we found that SPIONs had a dose-dependent cytotoxic in PC12 cells at 60-200 ug/mL but not at 10-50 ug/mL, it reduced cell viability, decreased the capacity of PC12 cells to extend neurites in response to nerve growth factor (NGF), induced a reduction of the tyrosine hydroxylase protein, while increasing PC12 cell apoptosis. Accordingly, the no-observed-adverse-effect level (NOAEL) of current SPIONs was 50 ug/mL in vitro, which would be useful for human health risk assessment. While directly injecting the SPIONs into the dorsal striatum or hippocampus, 7 and 14 days after surgery, nanoparticles decreased the TH + fiber density in both the dorsal striatum and the hippocampus. A behavioral evaluation demonstrated that SPIONs attenuated the animals' motor coordination and spatial memory, as evaluated by the rotarod test and the Morris water maze. We further examined mitogen-activated protein kinase (MAPK) activation and found that c-Jun N-terminal kinase (JNK) was activated after SPIONs treatment. It suggests that the SPIONs-induced neurotoxicity might be mediated through the JNK signaling pathway. SPIONs could possibly induce neurotoxic effects on the dorsal striatum and hippocampus. Copyright © 2018 Elsevier B.V. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Qiwen; Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou 225009; Bijie Pilot Area Research Institute of Bijie University, Bijie 551700

Highlights: •Cadmium can promote early upregulation of autophagy in PC-12 cells. •Autophagy precedes apoptosis in cadmium-treated PC-12 cells. •Cadmium-induced autophagy is cytoprotective in PC-12 cells. •Class III PI3K/beclin-1/Bcl-2 signaling pathway plays a positive role in cadmium-triggered autophagy. -- Abstract: Laboratory data have demonstrated that cadmium (Cd) may induce neuronal apoptosis. However, little is known about the role of autophagy in neurons. In this study, cell viability decreased in a dose- and time-dependent manner after treatment with Cd in PC-12 cells. As cells were exposed to Cd, the levels of LC3-II proteins became elevated, specific punctate distribution of endogenous LC3-II increased,more » and numerous autophagosomes appeared, which suggest that Cd induced a high level of autophagy. In the late stages of autophagy, an increase in the apoptosis ratio was observed. Likewise, pre-treatment with chloroquine (an autophagic inhibitor) and rapamycin (an autophagic inducer) resulted in an increased and decreased percentage of apoptosis in contrast to other Cd-treated groups, respectively. The results indicate that autophagy delayed apoptosis in Cd-treated PC-12 cells. Furthermore, co-treatment of cells with chloroquine reduced autophagy and cell activity. However, rapamycin had an opposite effect on autophagy and cell activity. Moreover, class III PI3 K/beclin-1/Bcl-2 signaling pathways served a function in Cd-induced autophagy. The findings suggest that Cd can induce cytoprotective autophagy by activating class III PI3 K/beclin-1/Bcl-2 signaling pathways. In sum, this study strongly suggests that autophagy may serve a positive function in the reduction of Cd-induced cytotoxicity.« less

Long non-coding RNA GAS5 aggravates hypoxia injury in PC-12 cells via down-regulating miR-124.

PubMed

Hu, Xiaoli; Liu, Juan; Zhao, Gang; Zheng, Jiaping; Qin, Xia

2018-05-08

One important feature of cerebral ischemia is hypoxia injury in nerve cells. Growth arrest-specific transcript 5 (GAS5) is widely reported as a tumor suppressor gene; however, the investigations about its role in cerebrovascular disease are relatively rare. This study was aimed to explore the impact of GAS5 on hypoxia response in nervous cells. PC-12 cells were incubated under anoxic condition to induce hypoxia injury. Regulatory effects of GAS5 on miR-124 and miR-124 on ICAM-1 expression were assessed by qRT-PCR and/or Western blot. Targeting effect of miR-124 on ICAM-1 3'-untranslated regions (UTR) was evaluated through dual luciferase activity assay. The potential regulatory mechanism on hypoxia injury in PC-12 cells was assessed by detecting key elements of NF-κB and Notch signaling pathways using Western blot. GAS5 ectopic expression accentuated hypoxia injury in PC-12 cells. miR-124 expression was negatively regulated by GAS5 expression. Cells with overexpressions of GAS5 and miR-124 alleviated hypoxia injury as in compassion with cells only with GAS5 overexpression. ICAM-1 expression was negatively regulated by miR-124 expression. ICAM-1 was a functional target of miR-124. ICAM-1 overexpression aggravated hypoxia injury, but inversely, ICAM-1 silence diminished hypoxia damage. Besides, ICAM-1 expression was negatively related with activation of NF-κB and Notch pathways. GAS5-miR-124-ICAM-1 axis could regulate hypoxia injury in PC-12 cells. GAS5 might aggravate hypoxia injury via down-regulating miR-124, then up-regulating ICAM-1, and further enhancing activations of NF-κB and Notch pathways. © 2018 Wiley Periodicals, Inc.

Eriodictyol protects against H(2)O(2)-induced neuron-like PC12 cell death through activation of Nrf2/ARE signaling pathway.

PubMed

Lou, Haiyan; Jing, Xu; Ren, Dongmei; Wei, Xinbing; Zhang, Xiumei

2012-07-01

Eriodictyol, a flavonoid isolated from the Chinese herb Dracocephalum rupestre has long been established as an antioxidant. The present study was designed to explore the protective effects of eriodictyol against hydrogen peroxide (H(2)O(2))-induced neurotoxicity with cultured rat pheochromocytoma cells (PC12 cells) and the possible mechanisms involved. For this purpose, differentiated PC12 cells were cultured and exposed to 200 μM H(2)O(2) in the absence or presence of eriodictyol (20, 40 and 80 μM). In addition, the potential contribution of the Nrf2/ARE neuroprotective pathway in eriodictyol-mediated protection against H(2)O(2)-induced neurotoxicity was also investigated. The results showed that H(2)O(2)-induced cell death can be inhibited in the presence of eriodictyol as measured by assays for MTT and apoptosis. Further study revealed that eriodictyol induced the nuclear translocation of Nrf2, enhanced the expression of heme oxygenase (HO-1) and γ-glutamylcysteine synthetase (γ-GCS), and increased the levels of intracellular glutathione. Treatment of PC12 cells with Nrf2 small interference RNA abolished eriodictyol-induced HO-1 and γ-GCS expression and its protective effects. In conclusion, these results suggest that eriodictyol upregulates HO-1 and γ-GCS expression through the activation of Nrf2/ARE pathway and protects PC12 cells against H(2)O(2)-induced oxidative stress. Copyright © 2012 Elsevier Ltd. All rights reserved.

Short-term incubation of gabapentin or pregabalin does not affect chemically induced injury in neuronal cell models in vitro

PubMed Central

Baldewig, Malte; Goldbaum, Olaf; Richter-Landsberg, Christiane; Weyland, Andreas; Bantel, Carsten

2018-01-01

Purpose Gabapentinoids are currently the mainstay of pharmacological treatments for patients with neuropathic pain. Little is known about the effects of this therapy on the integrity of neuronal networks, especially in patients with an already-damaged nervous system. Since gabapentinoids can worsen cognitive functions and recent studies have shown alterations in the brains of patients with neuropathic pain, it may be possible that these drugs have neurotoxic effects. Methods Rat clonal PC12 pheochromocytoma (autonomic) and primary sensory dorsal-root ganglion (DRG) neurons from newborn Wistar rats were employed for this study. To mimic neuronal damage, cells were exposed to cytotoxins using either hydrogen peroxide (H2O2) or vincristine. Results No direct cytotoxic effects were observed after incubating PC12 cells for 24 hours with increasing concentrations of gabapentin or pregabalin using MTT cytotoxicity assays. Even a 7-day incubation did not cause cellular damage. Furthermore, in preinjured PC12 and DRG neurons, neither gabapentin nor pregabalin prevented or enhanced the cytotoxic effects of H2O2 or vincristine after incubation for 24 hours and 7 days, respectively. Cell morphology and integrity of the cytoskeleton assessed by employing immunostaining of cytoskeletal proteins (α-tubulin, neurofilament L) remained intact and were not altered by gabapentinoids. Conclusion Based on these results, gabapentinoids are unlikely to be neurotoxic in cultured autonomic (PC12) and sensory DRG cells, even when cells are preinjured. These results are of high clinical relevance, as it seems unlikely that the morphological changes recently observed in the brains of neuropathic pain patients are caused or worsened by gabapentinoids.

Composition and functional property of photosynthetic pigments under circadian rhythm in the cyanobacterium Spirulina platensis.

PubMed

Kumar, Deepak; Kannaujiya, Vinod K; Richa; Pathak, Jainendra; Sundaram, Shanthy; Sinha, Rajeshwar P

2018-05-01

Circadian rhythm is an important endogenous biological signal for sustainable growth and development of cyanobacteria in natural ecosystems. Circadian effects of photosynthetically active radiation (PAR), ultraviolet-A (UV-A) and ultraviolet-B (UV-B) radiations on pigment composition have been studied in the cyanobacterium Spirulina platensis under light (L)/dark (D) oscillation with a combination of 4/20, 8/16, 12/12, 16/8, 20/4 and 24/24 h time duration. Circadian exposure of PAR + UV-A (PA) and PAR + UV-A + UV-B (PAB) showed more than twofold decline in Chl a, total protein and phycocyanin (PC) in light phase and significant recovery was achieved in dark phase. The fluorescence emission wavelength of PC was shifted towards lower wavelengths in the light phase of PAB in comparison to P and PA whereas the same wavelength was retrieved in the dark phase. The production of free radicals was accelerated twofold in the light phase (24 h L) whereas the same was retrieved to the level of control during the dark phase. Oxidatively induced damage was alleviated by antioxidative enzymes such as catalase (CAT), peroxidase (POD), superoxide dismutase (SOD) and ascorbate peroxidase (APX) in the light phase (0-24-h L) whereas the dark phase showed significant inhibition of the same enzymes. Similar characteristic inhibition of free radicals and recovery of PC was observed inside cellular filament after circadian rhythm of 24/24 h (L/D). Circadian exposure of P, PA and PAB significantly altered the synthesis and recovery of pigments that could be crucial for optimization and sustainable production of photosynthetic products for human welfare.

Tyr phosphatase-mediated P-ERK inhibition suppresses senescence in EIA + v-raf transformed cells, which, paradoxically, are apoptosis-protected in a MEK-dependent manner.

PubMed

De Vitis, Stefania; Sonia Treglia, Antonella; Ulianich, Luca; Turco, Stefano; Terrazzano, Giuseppe; Lombardi, Angela; Miele, Claudia; Garbi, Corrado; Beguinot, Francesco; Di Jeso, Bruno

2011-02-01

Activation of the Ras-Raf-extracellular signal-regulated kinase (ERK) pathway causes not only proliferation and suppression of apoptosis but also the antioncogenic response of senescence. How these contrasting effects are reconciled to achieve cell transformation and cancer formation is poorly understood. In a system of two-step carcinogenesis (dedifferentiated PC EIA, transformed PC EIA-polyoma-middle T [PC EIA + Py] and PC EIA-v-raf [PC EIA + raf] cells], v-raf cooperated with EIA by virtue of a strong prosurvival effect, not elicited by Py-middle T, evident toward serum-deprivation-and H(2)O(2)-induced apoptosis. Apoptosis was detected by DNA fragmentation and annexin V staining. The prosurvival function of v-raf was, in part, mitogen-activated protein kinase/ERK kinase (MEK)-dependent, as shown by pharmacological MEK inhibition. The MEK-dependent antiapoptotic effect of v-raf was exerted despite a lower level of P-ERK1/2 in EIA + raf cells with respect to EIA + Py/EIA cells, which was dependent on a high tyrosine phosphatase activity, as shown by orthovanadate blockade. An ERK1/2 tyrosine phosphatase was likely involved. The high tyrosine phosphatase activity was instrumental to the complete suppression of senescence, detected by β-galactosidase activity, because tyrosine phosphatase blockade induced senescence in EIA + raf but not in EIA + Py cells. High tyrosine phosphatase activity and evasion from senescence were confirmed in an anaplastic thyroid cancer cell line. Therefore, besides EIA, EIA + raf cells suppress senescence through a new mechanism, namely, phosphatase-mediated P-ERK1/2 inhibition, but, paradoxically, retain the oncogenic effects of the Raf-ERK pathway. We propose that the survival effect of Raf is not a function of absolute P-ERK1/2 levels at a given time but is rather dynamically dependent on greater variations after an apoptotic stimulus.

HDAC Inhibition Induces Increased Choline Uptake and Elevated Phosphocholine Levels in MCF7 Breast Cancer Cells

PubMed Central

Ward, Christopher S.; Eriksson, Pia; Izquierdo-Garcia, Jose L.; Brandes, Alissa H.; Ronen, Sabrina M.

2013-01-01

Histone deacetylase (HDAC) inhibitors have emerged as effective antineoplastic agents in the clinic. Studies from our lab and others have reported that magnetic resonance spectroscopy (MRS)-detectable phosphocholine (PC) is elevated following SAHA treatment, providing a potential noninvasive biomarker of response. Typically, elevated PC is associated with cancer while a decrease in PC accompanies response to antineoplastic treatment. The goal of this study was therefore to elucidate the underlying biochemical mechanism by which HDAC inhibition leads to elevated PC. We investigated the effect of SAHA on MCF-7 breast cancer cells using 13C MRS to monitor [1,2-13C] choline uptake and phosphorylation to PC. We found that PC synthesis was significantly higher in treated cells, representing 154±19% of control. This was within standard deviation of the increase in total PC levels detected by 31P MRS (129±7% of control). Furthermore, cellular choline kinase activity was elevated (177±31%), while cytidylyltransferase activity was unchanged. Expression of the intermediate-affinity choline transporter SLC44A1 and choline kinase α increased (144% and 161%, respectively) relative to control, as determined by mRNA microarray analysis with protein-level confirmation by Western blotting. Taken together, our findings indicate that the increase in PC levels following SAHA treatment results from its elevated synthesis. Additionally, the concentration of glycerophosphocholine (GPC) increased significantly with treatment to 210±45%. This is likely due to the upregulated expression of several phospholipase A2 (PLA2) isoforms, resulting in increased PLA2 activity (162±18%) in SAHA-treated cells. Importantly, the levels of total choline (tCho)-containing metabolites, comprised of choline, PC and GPC, are readily detectable clinically using 1H MRS. Our findings thus provide an important step in validating clinically translatable non-invasive imaging methods for follow-up diagnostics of HDAC inhibitor treatment. PMID:23626839

Reciprocal signals between microglia and neurons regulate α-synuclein secretion by exophagy through a neuronal cJUN-N-terminal kinase-signaling axis.

PubMed

Christensen, Dan Ploug; Ejlerskov, Patrick; Rasmussen, Izabela; Vilhardt, Frederik

2016-03-08

Secretion of proteopathic α-synuclein (α-SNC) species from neurons is a suspected driving force in the propagation of Parkinson's disease (PD). We have previously implicated exophagy, the exocytosis of autophagosomes, as a dominant mechanism of α-SNC secretion in differentiated PC12 or SH-SY5Y nerve cells. Here we have examined the regulation of exophagy associated with different forms of nerve cell stress relevant to PD. We identify cJUN-N-terminal kinase (JNK) activity as pivotal in the secretory fate of autophagosomes containing α-SNC. Pharmacological inhibition or genetic (shRNA) knockdown of JNK2 or JNK3 decreases α-SNC secretion in differentiated PC12 and SH-SY5Y cells, respectively. Conversely, expression of constitutively active mitogen-activated protein kinase kinase 7 (MKK7)-JNK2 and -JNK3 constructs augment secretion. The transcriptional activity of cJUN was not required for the observed effects. We establish a causal relationship between increased α-SNC release by exophagy and JNK activation subsequent to lysosomal fusion deficiency (overexpression of Lewy body-localized protein p25α or bafilomycin A1). JNK activation following neuronal ER or oxidative stress was not correlated with exophagy, but of note, we demonstrate that reciprocal signaling between microglia and neurons modulates α-SNC secretion. NADPH oxidase activity of microglia cell lines was upregulated by direct co-culture with α-SNC-expressing PC12 neurons or by passive transfer of nerve cell-conditioned medium. Conversely, inflammatory factors secreted from activated microglia increased JNK activation and α-SNC secretion several-fold in PC12 cells. While we do not identify these factors, we extend our observations by showing that exposure of neurons in monoculture to TNFα, a classical pro-inflammatory mediator of activated microglia, is sufficient to increase α-SNC secretion in a mechanism dependent on JNK2 or JNK3. In continuation hereof, we show that also IFNβ and TGFβ increase the release of α-SNC from PC12 neurons. We implicate stress kinases of the JNK family in the regulation of exophagy and release of α-SNC following endogenous or exogenous stimulation. In a wider scope, our results imply that microglia not only inflict bystander damage to neurons in late phases of inflammatory brain disease but may also be active mediators of disease propagation.

Cellular prion protein controls stem cell-like properties of human glioblastoma tumor-initiating cells.

PubMed

Corsaro, Alessandro; Bajetto, Adriana; Thellung, Stefano; Begani, Giulia; Villa, Valentina; Nizzari, Mario; Pattarozzi, Alessandra; Solari, Agnese; Gatti, Monica; Pagano, Aldo; Würth, Roberto; Daga, Antonio; Barbieri, Federica; Florio, Tullio

2016-06-21

Prion protein (PrPC) is a cell surface glycoprotein whose misfolding is responsible for prion diseases. Although its physiological role is not completely defined, several lines of evidence propose that PrPC is involved in self-renewal, pluripotency gene expression, proliferation and differentiation of neural stem cells. Moreover, PrPC regulates different biological functions in human tumors, including glioblastoma (GBM). We analyzed the role of PrPC in GBM cell pathogenicity focusing on tumor-initiating cells (TICs, or cancer stem cells, CSCs), the subpopulation responsible for development, progression and recurrence of most malignancies. Analyzing four GBM CSC-enriched cultures, we show that PrPC expression is directly correlated with the proliferation rate of the cells. To better define its role in CSC biology, we knocked-down PrPC expression in two of these GBM-derived CSC cultures by specific lentiviral-delivered shRNAs. We provide evidence that CSC proliferation rate, spherogenesis and in vivo tumorigenicity are significantly inhibited in PrPC down-regulated cells. Moreover, PrPC down-regulation caused loss of expression of the stemness and self-renewal markers (NANOG, Sox2) and the activation of differentiation pathways (i.e. increased GFAP expression). Our results suggest that PrPC controls the stemness properties of human GBM CSCs and that its down-regulation induces the acquisition of a more differentiated and less oncogenic phenotype.

Cellular prion protein controls stem cell-like properties of human glioblastoma tumor-initiating cells

PubMed Central

Corsaro, Alessandro; Bajetto, Adriana; Thellung, Stefano; Begani, Giulia; Villa, Valentina; Nizzari, Mario; Pattarozzi, Alessandra; Solari, Agnese; Gatti, Monica; Pagano, Aldo; Würth, Roberto; Daga, Antonio; Barbieri, Federica; Florio, Tullio

2016-01-01

Prion protein (PrPC) is a cell surface glycoprotein whose misfolding is responsible for prion diseases. Although its physiological role is not completely defined, several lines of evidence propose that PrPC is involved in self-renewal, pluripotency gene expression, proliferation and differentiation of neural stem cells. Moreover, PrPC regulates different biological functions in human tumors, including glioblastoma (GBM). We analyzed the role of PrPC in GBM cell pathogenicity focusing on tumor-initiating cells (TICs, or cancer stem cells, CSCs), the subpopulation responsible for development, progression and recurrence of most malignancies. Analyzing four GBM CSC-enriched cultures, we show that PrPC expression is directly correlated with the proliferation rate of the cells. To better define its role in CSC biology, we knocked-down PrPC expression in two of these GBM-derived CSC cultures by specific lentiviral-delivered shRNAs. We provide evidence that CSC proliferation rate, spherogenesis and in vivo tumorigenicity are significantly inhibited in PrPC down-regulated cells. Moreover, PrPC down-regulation caused loss of expression of the stemness and self-renewal markers (NANOG, Sox2) and the activation of differentiation pathways (i.e. increased GFAP expression). Our results suggest that PrPC controls the stemness properties of human GBM CSCs and that its down-regulation induces the acquisition of a more differentiated and less oncogenic phenotype. PMID:27229535

Radiation-induced cyclooxygenase 2 up-regulation is dependent on redox status in prostate cancer cells.

PubMed

Li, Lingyun; Steinauer, Kirsten K; Dirks, Amie J; Husbeck, Bryan; Gibbs, Iris; Knox, Susan J

2003-12-01

Cyclooxygenase 2 (COX2) is the inducible isozyme of COX, a key enzyme in arachidonate metabolism and the conversion of arachidonic acid (AA) to prostaglandins (PGs) and other eicosanoids. Previous studies have demonstrated that the COX2 protein is up-regulated in prostate cancer cells after irradiation and that this results in elevated levels of PGE(2). In the present study, we further investigated whether radiation-induced COX2 up-regulation is dependent on the redox status of cells from the prostate cancer cell line PC-3. l-Buthionine sulfoximine (BSO), which inhibits gamma glutamyl cysteine synthetase (gammaGCS), and the antioxidants alpha-lipoic acid and N-acetyl-l-cysteine (NAC) were used to modulate the cellular redox status. BSO decreased the cellular GSH level and increased cellular reactive oxygen species (ROS) in PC-3 cells, whereas alpha-lipoic acid and NAC increased the GSH level and decreased cellular ROS. Both radiation and the oxidant H(2)O(2) had similar effects on COX2 up-regulation and PGE(2) production in PC-3 cells, suggesting that radiation-induced COX2 up-regulation is secondary to the production of ROS. The relative increases in COX2 expression and PGE(2) production induced by radiation and H(2)O(2) were even greater when PC-3 cells were pretreated with BSO. When the cells were pretreated with alpha-lipoic acid or NAC for 24 h, both radiation- and H(2)O(2)-induced COX2 up-regulation and PGE(2) production were markedly inhibited. These results demonstrate that radiation-induced COX2 up-regulation in prostate cancer cells is modulated by the cellular redox status. Radiation-induced increases in ROS levels contribute to the adaptive response of PC-3 cells, resulting in elevated levels of COX2.

Extracellular Alpha-Synuclein Oligomers Induce Parkin S-Nitrosylation: Relevance to Sporadic Parkinson's Disease Etiopathology.

PubMed

Wilkaniec, Anna; Lenkiewicz, Anna M; Czapski, Grzegorz A; Jęśko, Henryk M; Hilgier, Wojciech; Brodzik, Robert; Gąssowska-Dobrowolska, Magdalena; Culmsee, Carsten; Adamczyk, Agata

2018-04-21

α-Synuclein (ASN) and parkin, a multifunctional E3 ubiquitin ligase, are two proteins that are associated with the pathophysiology of Parkinson's disease (PD). Excessive release of ASN, its oligomerization, aggregation, and deposition in the cytoplasm contribute to neuronal injury and cell death through oxidative-nitrosative stress induction, mitochondrial impairment, and synaptic dysfunction. In contrast, overexpression of parkin provides protection against cellular stresses and prevents dopaminergic neural cell loss in several animal models of PD. However, the influence of ASN on the function of parkin is largely unknown. Therefore, the aim of this study was to investigate the effect of extracellular ASN oligomers on parkin expression, S-nitrosylation, as well as its activity. For these investigations, we used rat pheochromocytoma (PC12) cell line treated with exogenous oligomeric ASN as well as PC12 cells with parkin overexpression and parkin knock-down. The experiments were performed using spectrophotometric, spectrofluorometric, and immunochemical methods. We found that exogenous ASN oligomers induce oxidative/nitrosative stress leading to parkin S-nitrosylation. Moreover, this posttranslational modification induced the elevation of parkin autoubiquitination and degradation of the protein. The decreased parkin levels resulted in significant cell death, whereas parkin overexpression protected against toxicity induced by extracellular ASN oligomers. We conclude that lowering parkin levels by extracellular ASN may significantly contribute to the propagation of neurodegeneration in PD pathology through accumulation of defective proteins as a consequence of parkin degradation.

Memory Impairment in Transgenic Alzheimer Mice Requires Cellular Prion Protein

PubMed Central

Gimbel, David A.; Nygaard, Haakon B.; Coffey, Erin E.; Gunther, Erik C.; Laurén, Juha; Gimbel, Zachary A.; Strittmatter, Stephen M.

2012-01-01

Soluble oligomers of the amyloid-β (Aβ) peptide are thought to play a key role in the pathophysiology of Alzheimer’s disease (AD). Recently, we reported that synthetic Aβ oligomers bind to cellular prion protein (PrPC) and that this interaction is required for suppression of synaptic plasticity in hippocampal slices by oligomeric Aβ peptide. We hypothesized that PrPC is essential for the ability of brain-derived Aβ to suppress cognitive function. Here, we crossed familial AD transgenes encoding APPswe and PSen1ΔE9 into Prnp−/− mice to examine the necessity of PrPC for AD-related phenotypes. Neither APP expression nor Aβ level is altered by PrPC absence in this transgenic AD model, and astrogliosis is unchanged. However, deletion of PrPC expression rescues 5-HT axonal degeneration, loss of synaptic markers, and early death in APPswe/PSen1ΔE9 transgenic mice. The AD transgenic mice with intact PrPC expression exhibit deficits in spatial learning and memory. Mice lacking PrPC, but containing Aβ plaque derived from APPswe/PSen1ΔE9 transgenes, show no detectable impairment of spatial learning and memory. Thus, deletion of PrPC expression dissociates Aβ accumulation from behavioral impairment in these AD mice, with the cognitive deficits selectively requiring PrPC. PMID:20445063

G3-C12 Peptide Reverses Galectin-3 from Foe to Friend for Active Targeting Cancer Treatment.

PubMed

Sun, Wei; Li, Lian; Yang, Qingqing; Shan, Wei; Zhang, Zhirong; Huang, Yuan

2015-11-02

Galectin-3 is overexpressed by numerous carcinomas and is a potential target for active tumor treatments. On the other hand, galectin-3 also plays a key role in cancer progression and prevents cells from undergoing apoptosis, thereby offsetting the benefits of active targeting drugs. However, the relative contribution of the protective antiapoptotic effects of galectin-3 and the proapoptotic effects of galectin-3-targeted therapies has remained yet unrevealed. Here, we show that a galectin-3-binding peptide G3-C12 could reverse galectin-3 from foe to friend for active targeting delivery system. Results showed G3-C12 modified N-(2-hydroxypropyl)methacrylamide copolymer doxorubicin conjugates (G3-C12-HPMA-Dox) could internalize into galectin-3 overexpressed PC-3 cells via a highly specific ligand-receptor pathway (2.2 times higher cellular internalization than HPMA-Dox). The internalized Dox stimulated the translocation of galectin-3 to the mitochondria to prevent from apoptosis. In turn, this caused G3-C12-HPMA-Dox to concentrate into the mitochondria after binding to galectin-3 intracellularly. Initially, mitochondrial galectin-3 weakened Dox-induced mitochondrial damage; however, as time progressed, G3-C12 active-mediation allowed increasing amounts of Dox to be delivered to the mitochondria, which eventually induced higher level of apoptosis than nontargeted copolymers. In addition, G3-C12 downregulates galectin-3 expression, 0.43 times lower than control cells, which could possibly be responsible for the suppressed cell migration. Thus, G3-C12 peptide exerts sequential targeting to both cell membrane and mitochondria via regulating galectin-3, and eventually reverses and overcomes the protective effects of galectin-3; therefore, it could be a promising agent for the treatment of galectin-3-overexpressing cancers.

Improved performance by morphology control via fullerenes in PBDT-TBT-alkoBT based organic solar cells

DOE PAGES

Khatiwada, Devendra; Venkatesan, Swaminathan; Chen, QIliang; ...

2015-07-03

In this work, we report improved performance by controlling morphology using different fullerene derivatives in poly{2-octyldodecyloxy-benzo[1,2-b;3,4-b]dithiophene-alt-5,6-bis(dodecyloxy)-4,7- di(thieno[3,2-b]thiophen-2-yl)-benzo[c][1,2,5]thiadiazole} (PBDT-TBT-alkoBT) based organic solar cells. PC60BM and PC70BM fullerenes were used to investigate the characteristic change in morphology and device performance. Fullerene affects device efficiency by changing active layer morphology. PC70BM with broader absorption than PC 60BM resulted in reduced device performance which was elucidated by the intermixed granular morphology separating each larger grain in the PC70BM/polymer composite layer which created higher density of traps. However after adding additive 1,8-diiodooctane (DIO), the fibrous morphology was observed due to reduced solubility of polymer andmore » increased solubility of PC 70BM in chloroform. The fibrous morphology improved charge transport leading to increase in overall device performance. Atomic force microscopies (AFM), photo induced charge extraction by linearly increasing voltage (photo-CELIV), and Kelvin prove force microscope (KPFM) were used to investigate nanoscale morphology of active layer with different fullerene derivatives. For PC 60BM based active layer, AFM images revealed dense fibrous morphology and more distinct fibrous morphology was observed by adding DIO. The PC 70BM based active layer only exhibited intermixed granular morphology instead of fibrous morphology observed in PC60BM based active layer. However, addition of DIO in PC 70BM based active layer led to fibrous morphology. When additive DIO was not used, a wider distribution of surface potential was observed for PC 70BM than PC 60BM based active layer by KPFM measurements, indicating 2 polymer and fullerene domains are separated. When DIO was used, narrower distribution of surface potential for both PC 70BM and PC 60BM based active layers was observed. Photo-CELIV experiment showed larger extracted charge carrier density and mobility in PC 70BM/DIO film.« less

Improved performance by morphology control via fullerenes in PBDT-TBT-alkoBT based organic solar cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Khatiwada, Devendra; Venkatesan, Swaminathan; Chen, QIliang

In this work, we report improved performance by controlling morphology using different fullerene derivatives in poly{2-octyldodecyloxy-benzo[1,2-b;3,4-b]dithiophene-alt-5,6-bis(dodecyloxy)-4,7- di(thieno[3,2-b]thiophen-2-yl)-benzo[c][1,2,5]thiadiazole} (PBDT-TBT-alkoBT) based organic solar cells. PC60BM and PC70BM fullerenes were used to investigate the characteristic change in morphology and device performance. Fullerene affects device efficiency by changing active layer morphology. PC70BM with broader absorption than PC 60BM resulted in reduced device performance which was elucidated by the intermixed granular morphology separating each larger grain in the PC70BM/polymer composite layer which created higher density of traps. However after adding additive 1,8-diiodooctane (DIO), the fibrous morphology was observed due to reduced solubility of polymer andmore » increased solubility of PC 70BM in chloroform. The fibrous morphology improved charge transport leading to increase in overall device performance. Atomic force microscopies (AFM), photo induced charge extraction by linearly increasing voltage (photo-CELIV), and Kelvin prove force microscope (KPFM) were used to investigate nanoscale morphology of active layer with different fullerene derivatives. For PC 60BM based active layer, AFM images revealed dense fibrous morphology and more distinct fibrous morphology was observed by adding DIO. The PC 70BM based active layer only exhibited intermixed granular morphology instead of fibrous morphology observed in PC60BM based active layer. However, addition of DIO in PC 70BM based active layer led to fibrous morphology. When additive DIO was not used, a wider distribution of surface potential was observed for PC 70BM than PC 60BM based active layer by KPFM measurements, indicating 2 polymer and fullerene domains are separated. When DIO was used, narrower distribution of surface potential for both PC 70BM and PC 60BM based active layers was observed. Photo-CELIV experiment showed larger extracted charge carrier density and mobility in PC 70BM/DIO film.« less

Economic-Analysis Program for a Communication System

NASA Technical Reports Server (NTRS)

Chamberlain, R. G.

1986-01-01

Prices and profits of alternative designs compared. Objective of Land Mobile Satellite Service Finance Report (LMSS) program is to provide means for comparing alternative designs of LMSS systems. Program is Multiplan worksheet program. Labels used in worksheet chosen for satellite-based cellular communication service, but analysis not restricted to such cases. LMSS written for interactive execution with Multiplan (version 1.2) and implemented on IBM PC series computer operating under DOS (version 2.11).

Molecular Cloning of Human Gene(s) Directing the Synthesis of Nervous System Cholinesterases

DTIC Science & Technology

1987-09-01

and shed light on the unknown physiological function of these serine hydrolases in proliferating and differentiating cells. In pheochromocytoma cells...Reiness, C.G., Reichardt, L.F. and Hall, Z.W. (1981) Cellular localization of the molecular forms of acetylcholinesterase in rat pheochromocytoma PC12...melanogaster: Structural gene for acetylcholinesterase with an unusual 5’ leader. The EMBO J., 2949-2954. 99. Merken, L., Simons, M.J., Swillens, S

Induction of cyclo-oxygenase-2 mRNA by prostaglandin E2 in human prostatic carcinoma cells

NASA Technical Reports Server (NTRS)

Tjandrawinata, R. R.; Dahiya, R.; Hughes-Fulford, M.

1997-01-01

Prostaglandins are synthesized from arachidonic acid by the enzyme cyclo-oxygenase. There are two isoforms of cyclooxygenases: COX-1 (a constitutive form) and COX-2 (an inducible form). COX-2 has recently been categorized as an immediate-early gene and is associated with cellular growth and differentiation. The purpose of this study was to investigate the effects of exogenous dimethylprostaglandin E2 (dmPGE2) on prostate cancer cell growth. Results of these experiments demonstrate that administration of dmPGE2 to growing PC-3 cells significantly increased cellular proliferation (as measured by the cell number), total DNA content and endogenous PGE2 concentration. DmPGE2 also increased the steady-state mRNA levels of its own inducible synthesizing enzyme, COX-2, as well as cellular growth to levels similar to those seen with fetal calf serum and phorbol ester. The same results were observed in other human cancer cell types, such as the androgen-dependent LNCaP cells, breast cancer MDA-MB-134 cells and human colorectal carcinoma DiFi cells. In PC-3 cells, the dmPGE2 regulation of the COX-2 mRNA levels was both time dependent, with maximum stimulation seen 2 h after addition, and dose dependent on dmPGE2 concentration, with maximum stimulation seen at 5 microg ml(-1). The non-steroidal anti-inflammatory drug flurbiprofen (5 microM), in the presence of exogenous dmPGE2, inhibited the up-regulation of COX-2 mRNA and PC-3 cell growth. Taken together, these data suggest that PGE2 has a specific role in the maintenance of human cancer cell growth and that the activation of COX-2 expression depends primarily upon newly synthesized PGE2, perhaps resulting from changes in local cellular PGE2 concentrations.

The chemistry of prions: small molecules, protein conformers and mass spectrometry

USDA-ARS?s Scientific Manuscript database

Background/Introduction. Prions propagate by converting a normal cellular isoform (PrPC) into the prion isoform (PrPSc) in a template-driven process. The lysines in PrPC are highly conserved and strongly influence prion propagation, based on studies using natural polymorphisms of PrPC and transg...

Toxicity of bovicin HC5 against mammalian cell lines and the role of cholesterol in bacteriocin activity.

PubMed

Paiva, Aline Dias; de Oliveira, Michelle Dias; de Paula, Sérgio Oliveira; Baracat-Pereira, Maria Cristina; Breukink, Eefjan; Mantovani, Hilário Cuquetto

2012-11-01

Bacteriocins are ribosomally synthesized antimicrobial peptides produced by Bacteria and some Archaea. The assessment of the toxic potential of antimicrobial peptides is important in order to apply these peptides on an industrial scale. The aim of the present study was to investigate the in vitro cytotoxic and haemolytic potential of bovicin HC5, as well as to determine whether cholesterol influences bacteriocin activity on model membranes. Nisin, for which the mechanism of action is well described, was used as a reference peptide in our assays. The viability of three distinct eukaryotic cell lines treated with bovicin HC5 or nisin was analysed by using the MTT assay and cellular morphological changes were determined by light microscopy. The haemolytic potential was evaluated by using the haemoglobin liberation assay and the role of cholesterol on bacteriocin activity was examined by using model membranes composed of DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) and DPoPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine). The IC(50) of bovicin HC5 and nisin against Vero cells was 65.42 and 13.48 µM, respectively. When the MTT assay was performed with MCF-7 and HepG2 cells, the IC(50) obtained for bovicin HC5 was 279.39 and 289.30 µM, respectively, while for nisin these values were 105.46 and 112.25 µM. The haemolytic activity of bovicin HC5 against eukaryotic cells was always lower than that determined for nisin. The presence of cholesterol did not influence the activity of either bacteriocin on DOPC model membranes, but nisin showed reduced carboxyfluorescein leakage in DPoPC membranes containing cholesterol. In conclusion, bovicin HC5 only exerted cytotoxic effects at concentrations that were greater than the concentration needed for its biological activity, and the presence of cholesterol did not affect its interaction with model membranes.

Ascorbyl coumarates as multifunctional cosmeceutical agents that inhibit melanogenesis and enhance collagen synthesis.

PubMed

Kwak, Jun Yup; Park, Soojin; Seok, Jin Kyung; Liu, Kwang-Hyeon; Boo, Yong Chool

2015-09-01

L-Ascorbic acid (AA) and p-coumaric acid (p-CA) are naturally occurring antioxidants that are known to enhance collagen synthesis and inhibit melanin synthesis, respectively. The purpose of this study was to examine hybrid compounds between AA and p-CA as multifunctional cosmeceutical agents. Ascorbyl 3-p-coumarate (A-3-p-C), ascorbyl 2-p-coumarate (A-2-p-C), and their parent compounds were tested for their effects on cellular melanin synthesis and collagen synthesis. At 100 μM, A-3-p-C and A-2-p-C decreased melanin content of human dermal melanocytes stimulated by L-tyrosine, by 65 and 59%, respectively, compared to 11% inhibition of AA and 70% inhibition of p-CA. A-3-p-C and A-2-p-C were less effective than p-CA but more effective than AA at inhibiting tyrosinase activity. A-3-p-C and A-2-p-C were more effective than p-CA at inhibiting the autoxidation of L-3,4-dihydroxyphenylalanine. At 100-300 μM, A-3-p-C and A-2-p-C augmented collagen release from human dermal fibroblasts by 120-144% and 125-191%, respectively, compared to 126-133% increase of AA and 120-146% increase of p-CA. They increased procollagen type I C-peptide release (A-3-p-C, and A-2-p-C) like AA, and decreased matrix metalloproteinase 1 level (A-2-p-C) like p-CA, implicating that they might regulate collagen metabolism by multiple mechanisms. This study suggests that A-3-p-C and A-2-p-C could be used as multifunctional cosmeceutical agents for the attenuation of certain aspects of skin aging.

Biogenic selenium nanoparticles induce ROS-mediated necroptosis in PC-3 cancer cells through TNF activation.

PubMed

Sonkusre, Praveen; Cameotra, Swaranjit Singh

2017-06-07

Selenium is well documented to inhibit cancer at higher doses; however, the mechanism behind this inhibition varies widely depending on the cell type and selenium species. Previously, we have demonstrated that Bacillus licheniformis JS2 derived biogenic selenium nanoparticles (SeNPs) induce non-apoptotic cell death in prostate adenocarcinoma cell line, PC-3, at a minimal concentration of 2 µg Se/ml, without causing toxicity to the primary cells. However, the mechanism behind its anticancer activity was elusive. Our results have shown that these SeNPs at a concentration of 2 µg Se/ml were able to induce reactive oxygen species (ROS) mediated necroptosis in PC-3 cells by gaining cellular internalization. Real-time qPCR analysis showed increased expression of necroptosis associated tumor necrotic factor (TNF) and interferon regulatory factor 1 (IRF1). An increased expression of RIP1 protein was also observed at the translational level upon SeNP treatment. Moreover, the cell viability was significantly increased in the presence of necroptosis inhibitor, Necrostatin-1. Data suggest that our biogenic SeNPs induce cell death in PC-3 cells by the ROS-mediated activation of necroptosis, independent to RIP3 and MLKL, regulated by a RIP1 kinase.

Spirulina maxima extract prevents cell death through BDNF activation against amyloid beta 1-42 (Aβ1-42) induced neurotoxicity in PC12 cells.

PubMed

Koh, Eun-Jeong; Kim, Kui-Jin; Choi, Jia; Kang, Do-Hyung; Lee, Boo-Yong

2018-04-23

Spirulina maxima is a blue-green micro alga that contains abundant amounts of proteins (60-70%), vitamins, chlorophyll a, and C-phycocyanin (C-PC). It has been shown to reduce oxidative stress, and prevent diabetes and non-alcoholic fatty liver disease. However, it is unclear whether Spirulina maxima 70% ethanol extract (SM70EE), chlorophyll a, and C-PC prevent Aβ 1-42 -induced neurotoxicity in PC12 cells. The aim of this study was to investigate whether SM70EE, chlorophyll a, and C-PC prevent Aβ 1-42 -induced cell death. SM70EE, chlorophyll a, and C-PC suppressed the Aβ 1-42 -induced increase in poly-ADP ribose polymerase-1 (PARP-1) cleavage and reduced Aβ 1-42 -induced decreases in glutathione and its associated factors. The level of brain-derived neurotrophic factor (BDNF), which plays a critical role in neuronal survival and neuroprotection, was increased by SM70EE, chlorophyll a, and C-PC in Aβ 1-42 -treated cells. SM70EE treatment decreased oxidative stress and cell death in response to Aβ 1-42 treatment, while simultaneously suppressing PARP cleavage and increasing the levels of glutathione (GSH) and its associated factors. Moreover, SM70EE lowered the levels of APP and BACE1, two major factors involved in APP processing, and increased BDNF expression during Aβ 1-42 -induced neurotoxicity in PC12 cells. We suggest that SM70EE prevents cell death caused by Aβ 1-42 -induced neurotoxicity via the activation of BDNF signaling. Copyright © 2018 Elsevier B.V. All rights reserved.

Geraniol induces cooperative interaction of apoptosis and autophagy to elicit cell death in PC-3 prostate cancer cells.

PubMed

Kim, Su-Hwa; Park, Eun-Jung; Lee, Chae Ryun; Chun, Jung Nyeo; Cho, Nam-Hyuk; Kim, In-Gyu; Lee, Sanghoon; Kim, Tae Woo; Park, Hyun Ho; So, Insuk; Jeon, Ju-Hong

2012-05-01

Geraniol, an acyclic dietary monoterpene, suppresses prostate cancer growth and enhances docetaxel chemosensitivity in cultured cell or xenograft tumor models. However, the mechanisms of the geraniol action against prostate cancer are largely unknown. In this study, we investigated the cellular and molecular mechanisms of geraniol-induced cell death in PC-3 prostate cancer cells. Among the examined structurally and functionally similar monoterpenes, geraniol potently induced apoptosis and autophagy. Although independent processes, apoptosis and autophagy acted as cooperative partners to elicit geraniol-induced cell death in PC-3 cells. At a molecular level, geraniol inhibited AKT signaling and activated AMPK signaling, resulting in mTOR inhibition. Combined treatment of AKT inhibitor and AMPK activator markedly suppressed cell growth compared to either treatment alone. Our findings provide insight into future investigations that are aimed at elucidating the role of apoptosis and autophagy in prostate cancer therapy and at developing anticancer strategies co-targeting AKT and AMPK.

Artemisinin conferred ERK mediated neuroprotection to PC12 cells and cortical neurons exposed to sodium nitroprusside-induced oxidative insult.

PubMed

Zheng, Wenhua; Chong, Cheong-Meng; Wang, Haitao; Zhou, Xuanhe; Zhang, Lang; Wang, Rikang; Meng, Qian; Lazarovici, Philip; Fang, Jiankang

2016-08-01

The production of nitric oxide (NO) is one of the primary mediators of ischemic damage, glutamate neurotoxicity and neurodegeneration and therefore inhibition of NO-induced neurotoxicity may be considered a therapeutic target for reducing neuronal cell death (neuroprotection). In this study, artemisinin, a well-known anti-malaria drug was found to suppress sodium nitroprusside (SNP, a nitric oxide donor)-induced cell death in the PC12 cells and brain primary cortical neuronal cultures. Pretreatment of PC12 cells with artemisinin significantly suppressed SNP-induced cell death by decreasing the extent of oxidation, preventing the decline of mitochondrial membrane potential, restoring abnormal changes in nuclear morphology and reducing lactate dehydrogenase release and inhibiting caspase 3/7 activities. Western blotting analysis revealed that artemisinin was able to activate extracellular regulated protein kinases (ERK) pathway. Furthermore, the ERK inhibitor PD98059 blocked the neuroprotective effect of artemisinin whereas the PI3K inhibitor LY294002 had no effect. Cumulatively these findings support the notion that artemisinin confers neuroprotection from SNP-induce neuronal cell death insult, a phenomenon coincidentally related to activation of ERK phosphorylation. This SNP-induced oxidative insult in PC12 cell culture model may be useful to investigate molecular mechanisms of NO-induced neurotoxicity and drug-induced neuroprotection, and to generate novel therapeutic concepts for ischemic disease treatment. Copyright © 2016 Elsevier Inc. All rights reserved.

Dexmedetomidine inhibits activation of the MAPK pathway and protects PC12 and NG108-15 cells from lidocaine-induced cytotoxicity at its maximum safe dose.

PubMed

Wang, Qiong; Tan, Yonghong; Zhang, Na; Xu, Yingyi; Wei, Wei; She, Yingjun; Bi, Xiaobao; Zhao, Baisong; Ruan, Xiangcai

2017-07-01

The developing brains of pediatric patients are highly vulnerable to anesthetic regimen (e.g., lidocaine), potentially causing neurological impairment. Recently, dexmedetomidine (DEX) has been used as an adjunct for sedation, and was shown to exert dose-dependent neuroprotective effects during brain injury. However, the maximum safe dose of DEX is unclear, and its protective effects against lidocaine-related neurotoxicity need to be confirmed. In this study, PC12 and NG108-15 cells were used to estimate safe, non-cytotoxic doses of DEX. We found that 100 and 60μM are the maximum safe dose of DEX for PC12 and NG108-15 cells, respectively, with no significant cytotoxicity. Lidocaine was found to remarkably inhibit cell vitality, but could be reversed by different doses of DEX, especially its maximum safe dose. Furthermore, the apoptosis induced by lidocaine was also assessed, and 100 and 60μM DEX showed optimal protective effects in PC12 and NG108-15 cells, respectively. Mechanistically, DEX activated the mitogen-activated protein kinase (MAPK) pathway, impaired caspase-3 expression, and enhanced anti-apoptotic factor Bcl-2 to resist lidocaine-induced apoptosis, indicating that the optimal dose of DEX alleviates lidocaine-induced cytotoxicity and should be considered in clinical application. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Alzheimer's Disease and Prion Protein

PubMed Central

Zhou, Jiayi; Liu, Bingqian

2013-01-01

Summary Alzheimer's disease (AD) is a devastating neurodegenerative disease with progressive loss of memory and cognitive function, pathologically hallmarked by aggregates of the amyloid-beta (Aβ) peptide and hyperphosphorylated tau in the brain. Aggregation of Aβ under the form of amyloid fibrils has long been considered central to the pathogenesis of AD. However, recent evidence has indicated that soluble Aβ oligomers, rather than insoluble fibrils, are the main neurotoxic species in AD. The cellular prion protein (PrPC) has newly been identified as a cell surface receptor for Aβ oligomers. PrPC is a cell surface glycoprotein that plays a key role in the propagation of prions, proteinaceous infectious agents that replicate by imposing their abnormal conformation to PrPC molecules. In AD, PrPC acts to transduce the neurotoxic signals arising from Aβ oligomers, leading to synaptic failure and cognitive impairment. Interestingly, accumulating evidence has also shown that aggregated Aβ or tau possesses prion-like activity, a property that would allow them to spread throughout the brain. In this article, we review recent findings regarding the function of PrPC and its role in AD, and discuss potential therapeutic implications of PrPC-based approaches in the treatment of AD. PMID:25343100

The association between impaired proinsulin processing and type 2 diabetes mellitus in non-obese Japanese individuals.

PubMed

Katsuta, Hidenori; Ozawa, Sachihiko; Suzuki, Kiyoshi; Takahashi, Kazuto; Tanaka, Toshiaki; Sumitani, Yoshikazu; Nishida, Susumu; Kondo, Takuma; Hosaka, Toshio; Inukai, Kouichi; Ishida, Hitoshi

2015-01-01

We aimed to examine the association between impaired proinsulin processing in pancreatic beta cells and type 2 diabetes mellitus in non-obese Japanese patients. Participants were divided into groups for normal glucose tolerance, prediabetes, and type 2 diabetes based on the oral glucose tolerance test (OGTT). Activities of prohormone convertase (PC) 1/3 and PC2 in fasting states were estimated. Multiple regression analysis was undertaken to ascertain if alteration of the activities of these enzymes contributes to the development of impaired glucose tolerance by comparison with HOMA-β and the oral disposition index (DI(O)). Overall, 452 subjects were included. PC1/3 activity tended to decrease in type 2 diabetes compared with normal glucose tolerance. PC2 activity showed no difference among the three groups. Decreased estimated PC1/3 activity was significantly associated with type 2 diabetes after adjustment for sex, age, creatinine, triglycerides, HOMA-β and DI(O). Odds ratios (95% CI) of PC1/3, HOMA-β, and DI(O) were 2.16 (1.12-4.19), 3.44 (1.82-6.52) and 14.60 (7.87-27.11), respectively. Furthermore, decreased PC1/3(≤1.7) combined with decreased HOMA-β (≤30) had a sensitivity of 73% and specificity of 62%. Decreased PC1/3 activity may be a useful measurement of beta-cell function alongside decreased HOMA-β or DI(O). A combined decrease in estimated fasting PC1/3 activity and HOMA-β measurement led to suspicion of type 2 diabetes in the non-obese Japanese population studied.

Lipid Rafts and Clathrin Cooperate in the Internalization of PrPC in Epithelial FRT Cells

PubMed Central

Casanova, Philippe; Puri, Claudia; Paladino, Simona; Tivodar, Simona S.; Campana, Vincenza; Tacchetti, Carlo; Zurzolo, Chiara

2009-01-01

Background The cellular prion protein (PrPC) plays a key role in the pathogenesis of Transmissible Spongiform Encephalopathies in which the protein undergoes post-translational conversion to the infectious form (PrPSc). Although endocytosis appears to be required for this conversion, the mechanism of PrPC internalization is still debated, as caveolae/raft- and clathrin-dependent processes have all been reported to be involved. Methodology/Principal Findings We have investigated the mechanism of PrPC endocytosis in Fischer Rat Thyroid (FRT) cells, which lack caveolin-1 (cav-1) and caveolae, and in FRT/cav-1 cells which form functional caveolae. We show that PrPC internalization requires activated Cdc-42 and is sensitive to cholesterol depletion but not to cav-1 expression suggesting a role for rafts but not for caveolae in PrPC endocytosis. PrPC internalization is also affected by knock down of clathrin and by the expression of dominant negative Eps15 and Dynamin 2 mutants, indicating the involvement of a clathrin-dependent pathway. Notably, PrPC co-immunoprecipitates with clathrin and remains associated with detergent-insoluble microdomains during internalization thus indicating that PrPC can enter the cell via multiple pathways and that rafts and clathrin cooperate in its internalization. Conclusions/Significance These findings are of particular interest if we consider that the internalization route/s undertaken by PrPC can be crucial for the ability of different prion strains to infect and to replicate in different cell lines. PMID:19503793

Quercetin increased the antiproliferative activity of green tea polyphenol (−)-epigallocatechin gallate in prostate cancer cells

PubMed Central

Wang, Piwen; Heber, David; Henning, Susanne M.

2012-01-01

We previously demonstrated that 50% of (−)-epigallocatechin gallate (EGCG) was present in methylated form (4″-MeEGCG) in human prostate tissue, which is less bioactive. We therefore investigated whether quercetin, a natural inhibitor of catechol-O-methyl transferase (COMT), will inhibit EGCG methylation leading to enhanced antiproliferative activity of EGCG in prostate cancer cells. Incubation with both, quercetin and EGCG, for 2 hr increased the cellular concentrations of EGCG by 4 to 8-fold and 6 to 10-fold in androgen-independent PC-3 cells and androgen-dependent LNCaP cells, respectively. Concurrently, the percent of 4″-MeEGCG in the total EGCG was decreased from 39% to 15% in PC-3 cells and from 61% to 38% in LNCaP cells. Quercetin and EGCG in combination synergistically inhibited cell proliferation, caused cell cycle arrest and induced apoptosis in PC-3 cells. In LNCaP cells EGCG and quercetin exhibited a stronger antiproliferative activity leading to an additive effect. The synergistic effect of these two agents in PC-3 cells could be based on the fact that EGCG primarily inhibited COMT activity while quercetin reduced the amount of COMT protein. In summary, quercetin combined with EGCG in vitro demonstrated enhanced inhibition of cell proliferation by increasing the intracellular concentration of EGCG and decreasing EGCG methylation. PMID:22452782

Transcriptional regulation of α1H T-type calcium channel under hypoxia

PubMed Central

Sellak, Hassan; Zhou, Chun; Liu, Bainan; Chen, Hairu; Lincoln, Thomas M.

2014-01-01

The low-voltage-activated T-type Ca2+ channels play an important role in mediating the cellular responses to altered oxygen tension. Among three T-type channel isoforms, α1G, α1H, and α1I, only α1H was found to be upregulated under hypoxia. However, mechanisms underlying such hypoxia-dependent isoform-specific gene regulation remain incompletely understood. We, therefore, studied the hypoxia-dependent transcriptional regulation of α1G and α1H gene promoters with the aim to identify the functional hypoxia-response elements (HREs). In rat pulmonary artery smooth muscle cells (PASMCs) and pheochromocytoma (PC12) cells after hypoxia (3% O2) exposure, we observed a prominent increase in α1H mRNA at 12 h along with a significant rise in α1H-mediated T-type current at 24 and 48 h. We then cloned two promoter fragments from the 5′-flanking regions of rat α1G and α1H gene, 2,000 and 3,076 bp, respectively, and inserted these fragments into a luciferase reporter vector. Transient transfection of PASMCs and PC12 cells with these recombinant constructs and subsequent luciferase assay revealed a significant increase in luciferase activity from the reporter containing the α1H, but not α1G, promoter fragment under hypoxia. Using serial deletion and point mutation analysis strategies, we identified a functional HRE at site −1,173cacgc−1,169 within the α1H promoter region. Furthermore, an electrophoretic mobility shift assay using this site as a DNA probe demonstrated an increased binding activity to nuclear protein extracts from the cells after hypoxia exposure. Taken together, these findings indicate that hypoxia-induced α1H upregulation involves binding of hypoxia-inducible factor to an HRE within the α1H promoter region. PMID:25099734

Development of a liquid chromatography-mass spectrometry based enzyme activity assay for phosphatidylcholine-specific phospholipase C.

PubMed

Murakami, Chiaki; Mizuno, Satoru; Kado, Sayaka; Sakane, Fumio

2017-06-01

Phosphatidylcholine (PC)-specific phospholipase C (PC-PLC) hydrolyzes PC to generate the second messenger 1,2-diacylglycerol (DG) and phosphocholine. PC-PLC plays pivotal roles in inflammation, carcinogenesis, tumor progression, atherogenesis, and subarachnoid hemorrhage. Although the activity of PC-PLC in mammalian tissues was discovered approximately 40 years ago, neither the protein nor its gene has been identified. In the present study, we developed a non-radioactive enzyme activity assay for PC-PLC based on mass spectrometric detection of DG following HPLC separation. This new liquid chromatography-mass spectrometry (LC-MS) assay directly determines a specific reaction product, 1-palmitoyl-2-oleoyl-DG, that is generated from 1-palmitoyl-2-oleoyl-PC by purified Bacillus cereus PC-PLC. The LC-MS assay offers several advantages including a lower background (0.02% versus 91%), higher signal background ratio (4242 versus 1.06)/signal noise ratio (7494 versus 4.4), higher sensitivity (≥32-fold), and lower limit of quantitation (0.04 pmol versus 0.69 pmol of PC-PLC), than a conventional fluorometric assay, which indirectly detects phosphocholine produced in the reaction. In addition to Bacillus cereus PC-PLC, the LC-MS assay was applicable to the measurement of mammalian PC-PLC prepared from the mouse brain. The radioisotope-free, highly sensitive and precise LC-MS assay for PC-PLC would be useful for the purification and identification of PC-PLC protein. Copyright © 2017 Elsevier Inc. All rights reserved.

Antioxidant and anti-inflammatory properties of an aqueous cyanophyta extract derived from Arthrospira platensis: contribution to bioactivities by the non-phycocyanin aqueous fraction.

PubMed

Jensen, Gitte S; Attridge, Victoria L; Beaman, Joni L; Guthrie, Jesse; Ehmann, Axel; Benson, Kathleen F

2015-05-01

The goal for this work was to characterize basic biological properties of a novel Arthrospira platensis-based aqueous cyanophyta extract (ACE), enriched in the known anti-inflammatory cyclooxygenase-2 (COX-2) inhibitor phycocyanin (PC), but also containing a high level of non-PC bioactive compounds. Antioxidant properties were tested in parallel in the Folin-Ciocalteu assay (chemical antioxidant capacity) and in the cellular antioxidant protection (CAP-e) bioassay, where both the PC and the non-PC fractions contributed to the antioxidant capacity and CAP of ACE. In contrast to the COX-2 inhibition seen in the presence of PC, the inhibition of enzymatic activity of the inflammatory mediator Lipoxygenase was associated specifically with the non-PC fraction of ACE. Inhibition of formation of reactive oxygen species (ROS) was evaluated using polymorphonuclear cells from healthy human donors. The inhibition of ROS formation was seen for both the PC and non-PC fractions, with ACE showing the most robust effect. The effects of PC, non-PC, and ACE on clotting and clot lysing was tested using a modified Euglobulin fibrinolytic assay in vitro. In the presence of PC, non-PC, and ACE, the time for clot formation and lysis was not affected; however, the clots were significantly more robust. This effect was statistically significant (p<.05) at doses between 125-500 μg/mL, and returned to baseline at lower doses. Both PC and the non-PC fraction contributed to the antioxidant properties and anti-inflammatory effects, without a negative impact on blood clotting in vitro. This suggests a potential benefit for the consumable ACE extract in assisting the reduction of inflammatory conditions.

The p21-activated kinase 4-Slug transcription factor axis promotes epithelial-mesenchymal transition and worsens prognosis in prostate cancer.

PubMed

Park, Jung-Jin; Park, Mee-Hee; Oh, Eun Hye; Soung, Nak-Kyun; Lee, Soo Jae; Jung, Jae-Kyung; Lee, Ok-Jun; Yun, Seok Joong; Kim, Wun-Jae; Shin, Eun-Young; Kim, Eung-Gook

2018-05-30

Epithelial-mesenchymal transition (EMT) facilitates cancer invasion and metastasis and thus accelerates cancer progression. p21-activated kinase 4 (PAK4) is a critical regulator of prostate cancer (PC) progression. Here, we report that PAK4 activation promotes PC progression through the EMT regulator Slug. We find that phosphorylated PAK4 S474 (pPAK4) levels, an index of PAK4 activation, were tightly associated with Gleason score (p < 0.001), a clinical indicator of PC progression, but not with prostate serum antigen levels or tumor stage. Stable silencing of PAK4 in PC cells reduced their potential for EMT, cellular invasion, and metastasis in vivo. PAK4 bound and directly phosphorylated Slug at two previously unknown sites, S158 and S254, which resulted in its stabilization. The non-phosphorylatable form Slug S158A/S254A upregulated transcription of CDH1, which encodes E-cadherin, and thus suppressed EMT and invasion, to a greater extent than did wild-type Slug. The strong EMT inducer TGF-β elevated pPAK4 and pSlug S158 levels; PAK4 knockdown or introduction of a dominant-negative form of PAK4 inhibited both TGF-β-stimulated EMT and an increase in pSlug S158 levels. Finally, immunohistochemistry revealed a positive correlation between pPAK4 and pSlug S158 but an inverse correlation between pSlug S158 and E-cadherin. The results suggest that the PAK4-Slug axis represents a novel pathway that promotes PC progression.

Structure and biological function of ENPP6, a choline-specific glycerophosphodiester-phosphodiesterase

PubMed Central

Morita, Junko; Kano, Kuniyuki; Kato, Kazuki; Takita, Hiroyuki; Sakagami, Hideki; Yamamoto, Yasuo; Mihara, Emiko; Ueda, Hirofumi; Sato, Takanao; Tokuyama, Hidetoshi; Arai, Hiroyuki; Asou, Hiroaki; Takagi, Junichi; Ishitani, Ryuichiro; Nishimasu, Hiroshi; Nureki, Osamu; Aoki, Junken

2016-01-01

Choline is an essential nutrient for all living cells and is produced extracellularly by sequential degradation of phosphatidylcholine (PC). However, little is known about how choline is produced extracellularly. Here, we report that ENPP6, a choline-specific phosphodiesterase, hydrolyzes glycerophosphocholine (GPC), a degradation product of PC, as a physiological substrate and participates in choline metabolism. ENPP6 is highly expressed in liver sinusoidal endothelial cells and developing oligodendrocytes, which actively incorporate choline and synthesize PC. ENPP6-deficient mice exhibited fatty liver and hypomyelination, well known choline-deficient phenotypes. The choline moiety of GPC was incorporated into PC in an ENPP6-dependent manner both in vivo and in vitro. The crystal structure of ENPP6 in complex with phosphocholine revealed that the choline moiety of the phosphocholine is recognized by a choline-binding pocket formed by conserved aromatic and acidic residues. The present study provides the molecular basis for ENPP6-mediated choline metabolism at atomic, cellular and tissue levels. PMID:26888014

Distinctive effect on nerve growth factor-induced PC12 cell neurite outgrowth by two unique neolignan enantiomers from Illicium merrillianum

PubMed Central

Tian, Xinhui; Yue, Rongcai; Zeng, Huawu; Li, Honglin; Shan, Lei; He, Weiwei; Shen, Yunheng; Zhang, Weidong

2015-01-01

Merrillianoid (1), a racemic neolignan possessing the characteristic benzo-2,7-dioxabicyclo[3.2.1]octane moiety, was isolated from the branches and leaves of Illicium merrillianum. Chiral separation of 1 gave two enantiomers (+)−1 and (−)−1. The structure of 1 was established by comprehensive spectroscopic analysis and single crystal X-ray diffraction. The absolute configurations of enantiomers were determined by quantum mechanical calculation. Compound (+)−1 exhibited a better neurotrophic activity than racemate 1 by promoting nerve growth factor (NGF) induced PC12 cell neurite outgrowth, while (−)−1 showed a distinctive inhibitory effect. Furthermore, a mechanism study indicated that the two enantiomers influenced NGF-induced neurite outgrowth of PC12 cells possibly by interacting with the trkA receptor, and extracellular signal regulated kinases 1/2 (ERK1/2) and mitogen-activated protein kinase (MEK) in Ras/ERK signal cascade. But the phosphorylation level of serine/threonine kinase Akt1 and Akt2 in PI3K/Akt signal pathway showed no significant difference between (+)−1 and (−)−1. PMID:26585042

Protective effect of lavender oil on scopolamine induced cognitive deficits in mice and H2O2 induced cytotoxicity in PC12 cells.

PubMed

Xu, Pan; Wang, Kezhu; Lu, Cong; Dong, Liming; Gao, Li; Yan, Ming; Aibai, Silafu; Liu, Xinmin

2016-12-04

Lavender essential oil (LO), an aromatic liquid extracted from Lavandula angustifolia Mill., has been traditionally used in the treatments of many nervous system diseases, and recently LO also reported to be effective for the Alzheimer's disease (AD). The improvement effect of lavender oil (LO) on the scopolamine-induced cognitive deficits in mice and H 2 O 2 induced cytotoxicity in PC12 cells have been evaluated. The relevant mechanism was also researched from the perspective of antioxidant effect and cholinergic system modulation. Cognitive deficits were induced in C57BL/6J mice treated with scopolamine (1mg/kg, i.p.) and were assessed by Morris water maze (MWM) and step-through passive avoidance tests. Then their hippocampus were removed for biochemical assays (acetylcholinesterase (AChE), superoxide dismutase (SOD), glutathione peroxidase (GPX) and malondialdehyde (MDA)). In vitro, the cytotoxicity were induced by 4h exposure to H 2 O 2 in PC12 and evaluated by cell viability (MTT), lactate dehydrogenase (LDH) level, nitric oxide (NO) release, reactive oxygen species (ROS) production and mitochondrial membrane potential (MMP). The results demonstrated that LO (100mg/kg) could improve the cognitive performance of scopolamine induced mice in behavioral tests. Meanwhile, it significantly decreased the AChE activity, MDA level, and increase SOD and GPX activities of the model. Moreover, LO (12μg/mL) protected PC12 cells from H 2 O 2 induced cytotoxicity by reducing LDH, NO release, intracellular ROS accumulation and MMP loss. It was suggested that LO could show neuroprotective effect in AD model in vivo (scopolamine-treated mice) and in vitro (H 2 O 2 induced PC12 cells) via modulating oxidative stress and AChE activity. Copyright © 2016. Published by Elsevier Ireland Ltd.

Secretome of Differentiated PC12 Cells Enhances Neuronal Differentiation in Human Mesenchymal Stem Cells Via NGF-Like Mechanism.

PubMed

Srivastava, A; Singh, S; Pandey, A; Kumar, D; Rajpurohit, C S; Khanna, V K; Pant, A B

2018-03-12

The secretome-mediated responses over cellular physiology are well documented. Stem cells have been ruling the field of secretomics and its role in regenerative medicine since the past few years. However, the mechanistic aspects of secretome-mediated responses and the role of other cells in this area remain somewhat elusive. Here, we investigate the effects of secretome-enriched conditioned medium (CM) of neuronally differentiated PC12 cells on the neuronal differentiation of human mesenchymal stem cells (hMSCs). The exposure to CM at a ratio of 1:1 (CM: conditioned medium of PC12 cells) led to neuronal induction in hMSCs. This neuronal induction was compared with a parallel group of cells exposed to nerve growth factor (NGF). There was a marked increase in neurite length and expression of neuronal markers (β-III tubulin, neurofilament-M (NF-M), synaptophysin, NeuN in exposed hMSCs). Experimental group co-exposed to NGF and CM showed an additive response via MAPK signaling and directed the cells particularly towards cholinergic lineage. The ability of CM to enhance the neuronal properties of stem cells could aid in their rapid differentiation into neuronal subtypes in case of stem cell transplantation for neuronal injuries, thus broadening the scope of non-stem cell-based applications in the area of secretomics.

Plant Flavone Apigenin Binds to Nucleic Acid Bases and Reduces Oxidative DNA Damage in Prostate Epithelial Cells

PubMed Central

Bhaskaran, Natarajan; Gupta, Sanjay

2014-01-01

Oxidative stress has been linked to prostate carcinogenesis as human prostate tissue is vulnerable to oxidative DNA damage. Apigenin, a dietary plant flavone, possesses anti-proliferative and anticancer effects; however, its antioxidant properties have not been fully elucidated. We investigated sub-cellular distribution of apigenin, it’s binding to DNA and protective effects against H2O2-induced DNA damage using transformed human prostate epithelial RWPE-1 cells and prostate cancer LNCaP, PC-3 and DU145 cells. Exposure of cells to apigenin exhibited higher accumulation in RWPE-1 and LNCaP cells, compared to PC-3 and DU145 cells. The kinetics of apigenin uptake in LNCaP cells was estimated with a Km value of 5 µmole/L and Vmax of 190 pmoles/million cells/h. Sub-cellular fractionation demonstrated that nuclear matrix retains the highest concentration of apigenin (45.3%), followed by cytosol (23.9%), nuclear membranes (17.9%) and microsomes (12.9%), respectively. Spectroscopic analysis of apigenin with calf-thymus DNA exhibited intercalation as the dominant binding mode to DNA duplex. Apigenin exposure resulted in significant genoprotective effects in H2O2-stressed RWPE-1 cells by reduction in reactive oxygen species levels. In addition, apigenin exposure suppressed the formation of 8-hydroxy-2′ deoxyguanosine and protected exposed cells from apoptosis. Our studies demonstrate that apigenin is readily taken up by normal prostatic epithelial cells and prostate cancer cells, and is incorporated into their nuclei, where its intercalation with nucleic acid bases may account for its antioxidant and chemopreventive activities. PMID:24614817

Plant flavone apigenin binds to nucleic acid bases and reduces oxidative DNA damage in prostate epithelial cells.

PubMed

Sharma, Haripaul; Kanwal, Rajnee; Bhaskaran, Natarajan; Gupta, Sanjay

2014-01-01

Oxidative stress has been linked to prostate carcinogenesis as human prostate tissue is vulnerable to oxidative DNA damage. Apigenin, a dietary plant flavone, possesses anti-proliferative and anticancer effects; however, its antioxidant properties have not been fully elucidated. We investigated sub-cellular distribution of apigenin, it's binding to DNA and protective effects against H2O2-induced DNA damage using transformed human prostate epithelial RWPE-1 cells and prostate cancer LNCaP, PC-3 and DU145 cells. Exposure of cells to apigenin exhibited higher accumulation in RWPE-1 and LNCaP cells, compared to PC-3 and DU145 cells. The kinetics of apigenin uptake in LNCaP cells was estimated with a Km value of 5 µmole/L and Vmax of 190 pmoles/million cells/h. Sub-cellular fractionation demonstrated that nuclear matrix retains the highest concentration of apigenin (45.3%), followed by cytosol (23.9%), nuclear membranes (17.9%) and microsomes (12.9%), respectively. Spectroscopic analysis of apigenin with calf-thymus DNA exhibited intercalation as the dominant binding mode to DNA duplex. Apigenin exposure resulted in significant genoprotective effects in H2O2-stressed RWPE-1 cells by reduction in reactive oxygen species levels. In addition, apigenin exposure suppressed the formation of 8-hydroxy-2' deoxyguanosine and protected exposed cells from apoptosis. Our studies demonstrate that apigenin is readily taken up by normal prostatic epithelial cells and prostate cancer cells, and is incorporated into their nuclei, where its intercalation with nucleic acid bases may account for its antioxidant and chemopreventive activities.

Low concentrations of alendronate increase the local invasive potential of osteoblastic sarcoma cell lines via connexin 43 activation.

PubMed

Yoshitani, Kazuhiro; Kido, Akira; Honoki, Kanya; Akahane, Manabu; Fujii, Hiromasa; Tanaka, Yasuhito

2011-07-15

Bisphosphonates (BPs) are agents used for treating disorders of excessive bone resorption. In addition, due to their cell-killing activity, BPs were potent candidates for adjuvant cancer therapy. On the other hand, low-concentrations of BPs have been reported to increase cellular viability in several types of tumor cells. Therefore, we focused on the effect of BPs on cellular aggressiveness of malignant bone tumors at low concentrations. MTS assay was performed using osteosarcoma cell lines MG63 and HOS, fibrosarcoma cell line HT1080, and prostate cancer cell line PC3. All the cell lines showed toxicity at high concentrations. On the other hand, at lower concentrations, the cellular viabilities of HOS and MG63 were rather higher than those of untreated controls. Since this tendency was most evident, HOS was used for further assays, including cellular motility, bone resorption activity, and cathepsin K activity. The low-concentration of alendronate enhanced cellular viability and motility, which correlated with the expression of connexin 43 at the mRNA and protein levels. Interestingly, oleamide, a potent connexin 43 inhibitor, had an inhibitory effect on the enhanced proliferation. Our data suggest that alendronate may enhance the proliferation of osteoblastic cell line through connexin 43 activation. Copyright © 2011 Elsevier GmbH. All rights reserved.

Does methionine oxidation influence the progression of classical or atypical scrapie

USDA-ARS?s Scientific Manuscript database

Introduction Prions are pathological proteins that propagate by converting a normal cellular prion protein (PrPC) into a prion (PrPSc). PrPC and PrPSc possess identical covalent structures and only differ in their conformations. The conversion of the PrPC conformation to the PrPSc one is template ...

Trithorax monomethylates histone H3K4 and interacts directly with CBP to promote H3K27 acetylation and antagonize Polycomb silencing

PubMed Central

Tie, Feng; Banerjee, Rakhee; Saiakhova, Alina R.; Howard, Benny; Monteith, Kelsey E.; Scacheri, Peter C.; Cosgrove, Michael S.; Harte, Peter J.

2014-01-01

Trithorax (TRX) antagonizes epigenetic silencing by Polycomb group (PcG) proteins, stimulates enhancer-dependent transcription, and establishes a ‘cellular memory’ of active transcription of PcG-regulated genes. The mechanisms underlying these TRX functions remain largely unknown, but are presumed to involve its histone H3K4 methyltransferase activity. We report that the SET domains of TRX and TRX-related (TRR) have robust histone H3K4 monomethyltransferase activity in vitro and that Tyr3701 of TRX and Tyr2404 of TRR prevent them from being trimethyltransferases. The trxZ11 missense mutation (G3601S), which abolishes H3K4 methyltransferase activity in vitro, reduces the H3K4me1 but not the H3K4me3 level in vivo. trxZ11 also suppresses the impaired silencing phenotypes of the Pc3 mutant, suggesting that H3K4me1 is involved in antagonizing Polycomb silencing. Polycomb silencing is also antagonized by TRX-dependent H3K27 acetylation by CREB-binding protein (CBP). We show that perturbation of Polycomb silencing by TRX overexpression requires CBP. We also show that TRX and TRR are each physically associated with CBP in vivo, that TRX binds directly to the CBP KIX domain, and that the chromatin binding patterns of TRX and TRR are highly correlated with CBP and H3K4me1 genome-wide. In vitro acetylation of H3K27 by CBP is enhanced on K4me1-containing H3 substrates, and independently altering the H3K4me1 level in vivo, via the H3K4 demethylase LSD1, produces concordant changes in H3K27ac. These data indicate that the catalytic activities of TRX and CBP are physically coupled and suggest that both activities play roles in antagonizing Polycomb silencing, stimulating enhancer activity and cellular memory. PMID:24550119

Photochemical internalization (PCI) of bleomycin is equally effective in two dissimilar leiomyosarcoma xenografts in athymic mice.

PubMed

Sellevold, Simen; Peng, Qian; Fremstedal, Ane Sofie Viset; Berg, Kristian

2017-12-01

Photochemical internalization (PCI) is a novel technique for delivery of active macromolecules into cancerous cells, via light activation of a specific photosensitizer and a low dose systemic drug. Numerous pre-clinical studies and one clinical trial have confirmed the treatment potential in carcinomas. Soft tissue sarcomas are rare and generally resistant to radio- and chemotherapy. Due to treatment resistance and surgical morbidity in sarcoma care, we seek to increase knowledge on PCI effects in sarcomas by studying two different, but closely related leiomyosarcomas. MES-SA and SK-LMS-1 tumours were established in the leg muscles of athymic mice. Treatment effects after AlPcS 2a -PCI of bleomycin, PCI with no drug (photodynamic therapy, PDT) and control groups were evaluated by: 1) assessment of tumour growth, 2) uptake of contrast agent during MRI and 3) histopathology. PCI of bleomycin induced a similar and significant increase in time to reach the end point in both tumour models, while neither responded to AlPcS 2a -PDT. In the MES-SA tumours PCI reduced the growth rate, while in the SK-LMS-1 tumours the growth was blocked for 12days followed by exponential growth close to that of untreated tumours. SK-LMS-1 tumours were more homogenously and better vascularized than MES-SA. After PCI the vascular shutdown was more complete in the SK-LMS-1 tumours than in the MES-SA tumours. AlPcS2a-based PCI, but not PDT, induced significant tumour growth delay in the evaluated sarcomas. Cellular responsiveness to bleomycin and tumour vascularity are identified as predictive markers for PCI treatment effects. Copyright © 2017 Elsevier B.V. All rights reserved.

Ligand-independent activation of EphA2 by arachidonic acid induces metastasis-like behaviour in prostate cancer cells

PubMed Central

Tawadros, T; Brown, M D; Hart, C A; Clarke, N W

2012-01-01

Background: High intake of omega-6 polyunsaturated fatty acids (PUFA) has been associated with clinical progression in prostate cancer (CaP). This study investigates the signalling mechanism by which the omega-6 PUFA arachidonic acid (AA) induces prostatic cellular migration to bone marrow stroma. Methods: Western blot analysis of the PC-3, PC3-GFP, DU 145 and LNCaP cells or their lipid raft (LR) components post AA stimulation was conducted in association with assays for adhesion and invasion through the bone marrow endothelial monolayers. Results: Arachidonic acid increased transendothelial migration of PC3-GFP cells (adhesion 37%±0.08, P=0.0124; transmigration 270%±0.145, P=0.0008). Akt, Src and focal adhesion kinase (FAK) pathways were induced by AA and integrally involved in transendothelial migration. LR were critical in AA uptake and induced Akt activity. Ephrin receptor A2 (EphA2), localised in LR, is expressed in DU 145 and PC-3 cells. Arachidonic acid induced a rapid increase of EphA2 Akt-dependent/ligand-independent activation, while knockdown of the EphrinA1 ligand decreased AA induced transendothelial migration, with an associated decrease in Src and FAK activity. Arachidonic acid activated Akt in EphA2− LNCaP cells but failed to induce BMEC transendothelial invasion. Conclusion: Arachidonic acid induced stimulation of EphA2 in vitro is associated fundamentally with CaP epithelial migration across the endothelial barrier. PMID:23037715

Procyanidins from apples (Malus pumila Mill.) extend the lifespan of Caenorhabditis elegans.

PubMed

Sunagawa, Tadahiro; Shimizu, Takahiko; Kanda, Tomomasa; Tagashira, Motoyuki; Sami, Manabu; Shirasawa, Takuji

2011-01-01

Apple polyphenols (AP) mainly consist of procyanidins (PC), which are composed of (-)-epicatechins and (+)-catechins. In order to investigate the antiageing effects of PC, we measured the lifespan of CAENORHABDITIS ELEGANS worms treated with PC. Treatment with 65 µg/mL PC extended the mean lifespan of wild-type N2 and FEM-1 worms by 12.1 % and 8.4 %, respectively, i.e., to a similar extent as resveratrol. In addition, treatment with 100 µg/mL AP also significantly prolonged the mean lifespan of the same worms by 12.0 % and 5.3 %, respectively, i.e., to a similar extent as PC. In contrast, treatment with (-)-epicatechin did not extend the lifespan of the worms. PC did not modify the growth, food intake, or fecundity of C. elegans. Treatment with PC did not extend the lifespan of MEV-1 worms, which show excessive oxidative stress, indicating that PC had no antioxidant ability in the MEV-1 mutant. Moreover, treatment with PC had no effect on the longevity of SIR-2.1 worms, which lack the activity of SIR-2, a member of the sirtuin family of NAD (+)-dependent protein deacetylases. These results indicated that PC has SIR-2.1-dependent antiageing effects on C. elegans. © Georg Thieme Verlag KG Stuttgart · New York.

Hyperforin attenuates aluminum-induced Aβ production and Tau phosphorylation via regulating Akt/GSK-3β signaling pathway in PC12 cells.

PubMed

Huang, Wanyue; Cheng, Ping; Yu, Kaiyuan; Han, Yanfei; Song, Miao; Li, Yanfei

2017-12-01

Aluminum (Al) is a neurotoxicant and cause β-amyloid (Aβ) peptides aggregation and tau hyperphosphorylation. Hyperforin (HF) is one of the major active constituents of the extracts of St. John's Wort (Hypericum perforatum), can treat Alzheimer's disease (AD) and other diseases involving peptide accumulation and cognition impairment. To determine the effects of HF on Al-induced Aβ formation and tau hyperphosphorylation, PC12 cells were cultured and treated with Al-malt (500μM) and/or HF (1μM). The results showed that HF treatment significantly attenuated Al-malt-induced Aβ 1-42 production by reducing the expressions of APP, BACE1 and PS1, while increasing the expressions of sAPPα, ADAM9/10/17, and tau phosphorylation in PC12 cells. In addition, HF treatment also increased phosphorylation of AKT (Ser473) and inhibited GSK-3β activity by increasing phosphorylation of GSK-3β (Ser9). These results indicated that HF may exert the protection via regulating the AKT/GSK-3β signaling to reduce Aβ production and tau phosphorylation in PC12 cells. Furthermore, these results could lead a possible therapeutics for the management of Al neurotoxicity. Copyright © 2017. Published by Elsevier Masson SAS.

Pleiotrophin prevents cocaine-induced toxicity in vitro.

PubMed

Gramage, Esther; Alguacil, Luis F; Herradon, Gonzalo

2008-10-24

Pleiotrophin is a cytokine involved in differentiation, survival and repair processes in the central nervous system. Pleiotrophin is upregulated in the brain after administration of different drugs of abuse, thus suggesting a protective role of this cytokine on drug-induced toxicity. We have tested this hypothesis in vitro using NG108-15 cells, a line widely used for neurotoxicity studies. It was found that pleiotrophin (3 and 6 microM) significantly prevents cocaine (5 mM)-induced cytotoxicity as measured by the neutral red test. Similar results were obtained in PC12 cells, which were found to endogenously express both pleiotrophin and its main target, receptor protein tyrosine phosphatase (RPTP) beta/zeta. Blockade of pleiotrophin signaling using anti-pleiotrophin antibodies (2 microg/ml) did not potentiate cocaine-induced toxicity; interestingly, incubation of PC12 cells only with anti-pleiotrophin antibodies significantly reduced cellular viability, thus suggesting an important role of endogenous pleiotrophin signaling in cell survival. The data suggest that pleiotrophin overexpression in response to drugs of abuse may be relevant to prevent drug-induced toxicity.

Effects of phenolic constituents of daylily flowers on corticosterone- and glutamate-treated PC12 cells.

PubMed

Tian, Huan; Yang, Fei-Fei; Liu, Chun-Yu; Liu, Xin-Min; Pan, Rui-Le; Chang, Qi; Zhang, Ze-Sheng; Liao, Yong-Hong

2017-01-21

Daylily flowers, the flower and bud parts of Hemerocallis citrina or H. fulva, are well known as Wang-You-Cao in Chinese, meaning forget-one's sadness plant. However, the major types of active constituents responsible for the neurological effects remain unclear. This study was to examine the protective effects of hydroalcoholic extract and fractions and to identify the active fractions. The extract of daylily flowers was separated with AB-8 resin into different fractions containing non-phenolic compounds, phenolic acid derivatives and flavonoids as determined using UPLC-DAD chromatograms. The neuroprotective activity was measured by evaluating the cell viability and lactate dehydrogenase release using PC12 cell damage models induced by corticosterone and glutamate. The neurological mechanisms were explored by determining their effect on the levels of dopamine (DA), 5-hydroxy tryptamine (5-HT), γ-aminobutyric acid (GABA), noradrenaline (NE) and acetylcholine (ACh) in the cell culture medium measured using an LC-MS/MS method. Pretreatment of PC12 cells with the extract and phenolic fractions of daylily flowers at concentrations ranging from 0.63 to 5 mg raw material/mL significantly reversed corticosterone- and glutamate-induced neurotoxicity in a dose-dependent manner. The fractions containing phenolic acid derivatives (0.59% w/w in the flowers) and/or flavonoids (0.60% w/w) exerted similar dose-dependent neuroprotective effect whereas the fractions with non-phenolic compounds exhibited no activity. The presence of phenolic acid derivatives in the corticosterone- and glutamate-treated PC12 cells elevated the DA level in the cell culture medium whereas flavonoids resulted in increased ACH and 5-HT levels. Phenolic acid derivatives and flavonoids were likely the active constituents of daylily flowers and they conferred a similar extent of neuroprotection, but affected the release of neurotransmitters in a different manner.

Release of Full-Length PrPC from Cultured Neurons Following Neurotoxic Challenge

PubMed Central

Wang, Kevin K. W.; Zoltewicz, J. Susie; Chiu, Allen; Zhang, Zhiqun; Rubenstein, Richard

2012-01-01

The susceptibility of the normal cellular prion protein isoform, cellular prion protein (PrPC), to proteolytic digestion has been well documented. In addition, a link between PrPC and the cytosolic protease, calpain, has been reported although the specifics of the interaction remain unclear. We performed in vitro and in cell-based studies to examine this relationship. We observed that human recombinant PrP (HrPrP) was readily cleaved by calpain-1 and -2, and we have identified and defined the targeted cleavage sites. In contrast, HrPrP was resistant to caspase-3 digestion. Unexpectedly, when brain lysates from PrPC-expressing mice were treated with calpain, no appreciable loss of the intact PrPC, nor the appearance of PrPC breakdown products (BDPs) were observed, even though alpha II-spectrin was converted to its signature calpain-induced BDPs. In addition, when rat cerebrocortical neuronal cultures (RtCNC) were subjected to the two neurotoxins at subacute levels, maitotoxin (MTX) and N-methyl-d-aspartate (NMDA), PrPC-BDPs were also not detectable. However, a novel finding from these cell-based studies is that apparently full-length, mature PrPC is released into culture media from RtCNC challenged with subacute doses of MTX and NMDA. Calpain inhibitor SNJ-1945 and caspase inhibitor IDN-6556 did not attenuate the release of PrPC. Similarly, the lysosomal protease inhibitor, NH4Cl, and the proteasome inhibitor, lactacystin, did not significantly alter the integrity of PrPC or its release from the RtCNC. In conclusion, rat neuronal PrPC is not a significant target for proteolytic modifications during MTX and NMDA neurotoxic challenges. However, the robust neurotoxin-mediated release of full-length PrPC into the cell culture media suggests an unidentified neuroprotective mechanism for PrPC. PMID:23093947

Chitooligosaccharides suppress the level of protein expression and acetylcholinesterase activity induced by Abeta25-35 in PC12 cells.

PubMed

Lee, Sang-Hoon; Park, Jin-Sook; Kim, Se-Kwon; Ahn, Chang-Bum; Je, Jae-Young

2009-02-01

Clinical applications of acetylcholinesterase (AChE) inhibitors are widespread in Alzheimer's sufferers in order to activate central cholinergic system and alleviate cognitive deficits by inhibiting the hydrolysis of acetylcholine. In this study, six kinds of chitooligosaccharides (COSs) with different molecular weight and degree of deacetylation were examined for their inhibitory effects against AChE. The 90-COSs exhibited potent AChE inhibitory activities compared to 50-COSs, while 90-MMWCOS (1000-5000 Da) in the 90-COSs showed the highest activity. Cell culture experiment revealed that 90-MMWCOS suppressed the level of AChE protein expression and AChE activity induced by Abeta(25-35) in PC12 cell lines.

Behavioral abnormalities in prion protein knockout mice and the potential relevance of PrPc for the cytoskeleton

USDA-ARS?s Scientific Manuscript database

The cellular prion protein (PrPC) is a highly conserved protein, which is anchored to the outer surface of the plasma membrane. Even though its physiological function has already been investigated in different cell or mouse models where PrPC expression is either up-regulated or depleted, its exact p...

Modulation of PC12 cell viability by forskolin-induced cyclic AMP levels through ERK and JNK pathways: an implication for L-DOPA-induced cytotoxicity in nigrostriatal dopamine neurons.

PubMed

Park, Keun Hong; Park, Hyun Jin; Shin, Keon Sung; Choi, Hyun Sook; Kai, Masaaki; Lee, Myung Koo

2012-07-01

The intracellular levels of cyclic AMP (cAMP) increase in response to cytotoxic concentrations of L-DOPA in PC12 cells, and forskolin that induces intracellular cAMP levels either protects PC12 cells from L-DOPA-induced cytotoxicity or enhances cytotoxicity in a concentration-dependent manner. This study investigated the effects of cAMP induced by forskolin on cell viability of PC12 cells, relevant to L-DOPA-induced cytotoxicity in Parkinson's disease therapy. The low levels of forskolin (0.01 and 0.1 μM)-induced cAMP increased dopamine biosynthesis and tyrosine hydroxylase (TH) phosphorylation, and induced transient phosphorylation of ERK1/2 within 1 h. However, at the high levels of forskolin (1.0 and 10 μM)-induced cAMP, dopamine biosynthesis and TH phosphorylation did not increase, but rapid differentiation in neurite-like formation was observed with a steady state. The high levels of forskolin-induced cAMP also induced sustained increase in ERK1/2 phosphorylation within 0.25-6 h and then led to apoptosis, which was apparently mediated by JNK1/2 and caspase-3 activation. Multiple treatment of PC12 cells with nontoxic L-DOPA (20 μM) for 4-6 days induced neurite-like formation and decreased intracellular dopamine levels by reducing TH phosphorylation. These results suggest that the low levels of forskolin-induced cAMP increased dopamine biosynthesis in cell survival via transient ERK1/2 phosphorylation. In contrast, the high levels of forskolin-induced cAMP induced differentiation via sustained ERK1/2 phosphorylation and then led to apoptosis. Taken together, the intracellular levels of cAMP play a dual role in cell survival and death through the ERK1/2 and JNK1/2 pathways in PC12 cells.

Use of Topical PC-NSAIDs to Treat Burn Injury and Pain

DTIC Science & Technology

2015-08-01

1R41CA171408 (PI: Lichtenberger) 05/03/2013-04/30/2014 NIH/NCI 1.2 calendar mo. $85,000/yr Aspirin -PC for Chemoprevention of Colorectal Cancer The...major goal of this Phase I STTR grant is to test aspirin -PC for chemopreventive activity in an animal model of colon cancer. Role: Principal...calendar months $155,000/yr Effects of anti-platelet drugs on colon cancer in the elderly The major goal of this R21 grant is to test Aspirin -PC and

Resveratrol-Activated AMPK/SIRT1/Autophagy in Cellular Models of Parkinson's Disease

PubMed Central

Wu, Yuncheng; Li, Xinqun; Zhu, Julie Xiaohong; Xie, Wenjie; Le, Weidong; Fan, Zhen; Jankovic, Joseph; Pan, Tianhong

2011-01-01

Excessive misfolded proteins and/or dysfunctional mitochondria, which may cause energy deficiency, have been implicated in the etiopathogenesis of Parkinson's disease (PD). Enhanced clearance of misfolded proteins or injured mitochondria via autophagy has been reported to have neuroprotective roles in PD models. The fact that resveratrol is a known compound with multiple beneficial effects similar to those associated with energy metabolism led us to explore whether neuroprotective effects of resveratrol are related to its role in autophagy regulation. We tested whether modulation of mammalian silent information regulator 2 (SIRT1) and/or metabolic energy sensor AMP-activated protein kinase (AMPK) are involved in autophagy induction by resveratrol, leading to neuronal survival. Our results showed that resveratrol protected against rotenone-induced apoptosis in SH-SY5Y cells and enhanced degradation of α-synucleins in α-synuclein-expressing PC12 cell lines via autophagy induction. We found that suppression of AMPK and/or SIRT1 caused decrease of protein level of LC3-II, indicating that AMPK and/or SIRT1 are required in resveratrol-mediated autophagy induction. Moreover, suppression of AMPK caused inhibition of SIRT1 activity and attenuated protective effects of resveratrol on rotenone-induced apoptosis, further suggesting that AMPK-SIRT1-autophagy pathway plays an important role in the neuroprotection by resveratrol on PD cellular models. PMID:21778691

Static Magnetic Field Exposure Reproduces Cellular Effects of the Parkinson's Disease Drug Candidate ZM241385

PubMed Central

Wang, Zhiyun; Che, Pao-Lin; Du, Jian; Ha, Barbara; Yarema, Kevin J.

2010-01-01

Background This study was inspired by coalescing evidence that magnetic therapy may be a viable treatment option for certain diseases. This premise is based on the ability of moderate strength fields (i.e., 0.1 to 1 Tesla) to alter the biophysical properties of lipid bilayers and in turn modulate cellular signaling pathways. In particular, previous results from our laboratory (Wang et al., BMC Genomics, 10, 356 (2009)) established that moderate strength static magnetic field (SMF) exposure altered cellular endpoints associated with neuronal function and differentiation. Building on this background, the current paper investigated SMF by focusing on the adenosine A2A receptor (A2AR) in the PC12 rat adrenal pheochromocytoma cell line that displays metabolic features of Parkinson's disease (PD). Methodology and Principal Findings SMF reproduced several responses elicited by ZM241385, a selective A2AR antagonist, in PC12 cells including altered calcium flux, increased ATP levels, reduced cAMP levels, reduced nitric oxide production, reduced p44/42 MAPK phosphorylation, inhibited proliferation, and reduced iron uptake. SMF also counteracted several PD-relevant endpoints exacerbated by A2AR agonist CGS21680 in a manner similar to ZM241385; these include reduction of increased expression of A2AR, reversal of altered calcium efflux, dampening of increased adenosine production, reduction of enhanced proliferation and associated p44/42 MAPK phosphorylation, and inhibition of neurite outgrowth. Conclusions and Significance When measured against multiple endpoints, SMF elicited qualitatively similar responses as ZM241385, a PD drug candidate. Provided that the in vitro results presented in this paper apply in vivo, SMF holds promise as an intriguing non-invasive approach to treat PD and potentially other neurological disorders. PMID:21079735

First evidence for the anti-inflammatory activity of fucoxanthin in high-fat-diet-induced obesity in mice and the antioxidant functions in PC12 cells.

PubMed

Tan, Cong-ping; Hou, Yun-hua

2014-04-01

Obesity, characterized as a state of low-level inflammation, is a powerful determinant influencing the development of insulin resistance and progression to type 2 diabetes. The purpose of the present study was to investigate the anti-inflammatory activity of fucoxanthin in experimental high-fat-diet-induced obesity in mice and antioxidant activity in PC12 cells under oxidative stress situation. The anti-inflammatory potential of fucoxanthin in the regulation of maleic dialdehyde (MDA), polymorphonuclear cells (PMNs), interleukin-1β (IL-1β), inducible nitric oxide synthase (iNOS), tumor necrosis factor alpha (TNF-α), and cyclooxygenase-2 (COX-2) was determined by ELISA. Fucoxanthin significantly inhibited obesity-induced upregulation of the production of IL-1β, TNF-α, iNOS, and COX-2. Moreover, fucoxanthin suppressed MDA and infiltration of PMNs. The protective effects were associated with lack of hypertrophy and crown-like structures in mammary gland. At the same time, fucoxanthin showed an advantage of antioxidant activity in PC12 cells under oxidative stress situation. These results suggest that supplementation of fucoxanthin is a promising strategy for blocking macrophage-mediated inflammation and inflammation-induced obesity and its associated complications.

Analysis of nucleic acid chaperoning by the prion protein and its inhibition by oligonucleotides

PubMed Central

Guichard, Cécile; Ivanyi-Nagy, Roland; Sharma, Kamal Kant; Gabus, Caroline; Marc, Daniel; Mély, Yves; Darlix, Jean-Luc

2011-01-01

Prion diseases are unique neurodegenerative illnesses associated with the conversion of the cellular prion protein (PrPC) into the aggregated misfolded scrapie isoform, named PrPSc. Recent studies on the physiological role of PrPC revealed that this protein has probably multiple functions, notably in cell–cell adhesion and signal transduction, and in assisting nucleic acid folding. In fact, in vitro findings indicated that the human PrP (huPrP) possesses nucleic acid binding and annealing activities, similarly to nucleic acid chaperone proteins that play essential roles in cellular DNA and RNA metabolism. Here, we show that a peptide, representing the N-terminal domain of huPrP, facilitates nucleic acid annealing by two parallel pathways nucleated through the stem termini. We also show that PrP of human or ovine origin facilitates DNA strand exchange, ribozyme-directed cleavage of an RNA template and RNA trans-splicing in a manner similar to the nucleocapsid protein of HIV-1. In an attempt to characterize inhibitors of PrP-chaperoning in vitro we discovered that the thioaptamer 5′-GACACAAGCCGA-3′ was extensively inhibiting the PrP chaperoning activities. At the same time a recently characterized methylated oligoribonucleotide inhibiting the chaperoning activity of the HIV-1 nucleocapsid protein was poorly impairing the PrP chaperoning activities. PMID:21737432

Phospholipid-esterified eicosanoids are generated in agonist-activated human platelets and enhance tissue factor-dependent thrombin generation.

PubMed

Thomas, Christopher P; Morgan, Lloyd T; Maskrey, Benjamin H; Murphy, Robert C; Kühn, Hartmut; Hazen, Stanley L; Goodall, Alison H; Hamali, Hassan A; Collins, Peter W; O'Donnell, Valerie B

2010-03-05

Here, a group of specific lipids, comprising phosphatidylethanolamine (PE)- or phosphatidylcholine (PC)-esterified 12S-hydroxyeicosatetraenoic acid (12S-HETE), generated by 12-lipoxygenase was identified and characterized. 12S-HETE-PE/PCs were formed within 5 min of activation by thrombin, ionophore, or collagen. Esterified HETE levels generated in response to thrombin were 5.85 +/- 1.42 (PE) or 18.35 +/- 4.61 (PC), whereas free was 65.5 +/- 17.6 ng/4 x 10(7) cells (n = 5 separate donors, mean +/- S.E.). Their generation was stimulated by triggering protease-activated receptors-1 and -4 and signaling via Ca(2+) mobilization secretory phospholipase A2, platelet-activating factor-acetylhydrolase, src tyrosine kinases, and protein kinase C. Stable isotope labeling showed that they form predominantly by esterification that occurs on the same time scale as free acid generation. Unlike free 12S-HETE that is secreted, esterified HETEs remain cell-associated, with HETE-PEs migrating to the outside of the plasma membrane. 12-Lipoxygenase inhibition attenuated externalization of native PE and phosphatidylserine and HETE-PEs. Platelets from a patient with the bleeding disorder, Scott syndrome, did not externalize HETE-PEs, and liposomes supplemented with HETE-PC dose-dependently enhanced tissue factor-dependent thrombin generation in vitro. This suggests a role for these novel lipids in promoting coagulation. Thus, oxidized phospholipids form by receptor/agonist mechanisms, not merely as an undesirable consequence of vascular and inflammatory disease.

Bioassay-guided Isolation of Neuroprotective Fatty Acids from Nigella sativa against 1-methyl-4-phenylpyridinium-induced Neurotoxicity

PubMed Central

Hosseinzadeh, Leila; Monaghash, Hoda; Ahmadi, Farahnaz; Ghiasvand, Nastaran; Shokoohinia, Yalda

2017-01-01

Objective: Parkinson's disease, a slowly progressive neurological disease, is associated with degeneration of the basal ganglia of the brain and a deficiency of the neurotransmitter dopamine. The main aspects of researches are the protection of normal neurons against degeneration. Fatty acids (FAs), the key structural elements of dietary lipids, are carboxylic straight chains and notable parameters in nutritional and industrial usefulness of a plant. Materials and Methods: Black cumin, a popular anti-inflammatory and antioxidant food seasoning, contains nonpolar constituents such as FAs which were extracted using hexane. Different fractions and subfractions were apt to cytoprotection against apoptosis and inflammation induced by 1-methyl-4-phenylpyridinium (MPP+) in rat pheochromocytoma cell line (PC12) as a neural cell death model. The experiment consisted of examination of cell viability assessment, mitochondrial membrane potential (MMP), caspase-3 and -9 activity, and measurement of cyclooxygenase (COX) activity. Results: MPP+ induced neurotoxicity in PC12 cells. Pretreatment with subfractions containing FA mixtures attenuated MPP+-mediated apoptosis partially dependent on the inhibition of caspase-3 and -9 activity and increasing the MMP. A mixture of linoleic acid, oleic acid, and palmitic acid also decreased the COX activity induced by MPP+ in PC12 cells. Conclusion: Our observation indicated that subtoxic concentration of FA from Nigella sativa may exert cytoprotective effects through their anti-apoptotic and anti-inflammation actions and could be regarded as a dietary supplement. SUMMARY MPP+ induced neurotoxicity in PC12 cellsNigella sativa contains bioactive fatty acidsPretreatment with fatty acids attenuated MPP+ mediated apoptosis through inhibition of caspase 3 and 9 activityA mixture of linoleic acid, oleic acid, and palmitic acid decreased the COX activity induced by MPP+ in PC12 cellsDue to cytoprotective, anti apoptotic and anti inflammation actions of N. sativa, it could be regarded as a dietary supplement. Abbreviations used: ANOVA: Analysis of variance; Ca: Calcium; CDCl3: Chloroform; COX: Cyclooxygenase; DMSO: Dimethyl sulfoxide; EA: Elidic acid; EDTA: Ethylene diamine tetraacetic acid; ELISA: Enzyme Linked Immunosorbent Assay; ESI-MS: Electron spray mass spectroscopy; FAs: Fatty acids; FBS: Fetal bovine serum; GC: Gas chromatography; 1HNMR: Hydrogen nuclear magnetic resonance; LA: Linoleic acid; MPP+: 1-Methyl-4-phenylpyridinium; MPTP: 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine; MTT: 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide; N. sativa: Nigella sativa; OA: Oleic acid; PA: Palmitic acid; PBS: Phosphate buffer saline; PC12: Rat pheochromocytoma cell line; PD: Parkinson's disease; PDA: Photo diode array detector; PGE2: Prostaglandin E2; TLC: Thin layer chromatography; TMPD: N,N,N’,N’-tetramethyl-p-phenylenediamine; USA: United states of America. PMID:29200724

Structural elucidation of novel phosphocholine-containing glycosylinositol-phosphoceramides in filamentous fungi and their induction of cell death of cultured rice cells.

PubMed Central

Aoki, Kazuhiro; Uchiyama, Ryosuke; Itonori, Saki; Sugita, Mutsumi; Che, Fang-Sik; Isogai, Akira; Hada, Noriyasu; Hada, Junko; Takeda, Tadahiro; Kumagai, Hidehiko; Yamamoto, Kenji

2004-01-01

Novel ZGLs (zwitterionic glycosphingolipids) have been found in and extracted from the mycelia of filamentous fungi ( Acremonium sp.) isolated from soil. Five ZGLs (ZGL1-ZGL5) were structurally elucidated by sugar compositional analysis, methylation analysis, periodate oxidation, matrix-assisted laser-desorption ionization-time-of-flight MS, (1)H-NMR spectroscopy and fast-atom bombardment MS. Their chemical structures were as follows: GlcN(alpha1-2)Ins1-P-1Cer (ZGL1), Man(alpha1-6)GlcN(alpha1-2)Ins1-P-1Cer (ZGL2), Man(alpha1-6)Man(alpha1-6)GlcN(alpha1-2)Ins1-P-1Cer (ZGL3), PC-->6Man(alpha1-6)GlcN(alpha1-2)Ins1- P -1Cer (ZGL4), and PC-->6Man(alpha1-6)Man(alpha1-6)GlcN(alpha1-2)Ins1-P-1Cer (ZGL5) (where Cer is ceramide and PC is phosphocholine). In addition, one acidic glycosphingolipid, which was the precursor of ZGLs, was also characterized as inositol-phosphoceramide. The core structure of the ZGLs, GlcN(alpha1-2)Ins1- P, is rather different from those found in other fungi, such as Man(alpha1-2)Ins1- P and Man(alpha1-6)Ins1- P. Interestingly, the terminal mannose residue of ZGL4 and ZGL5 was modified further with a PC group. The presence of PC-containing glycosylinositol-phosphoceramides has not been reported previously in any organism. The ceramide constituents of both ZGLs and acidic glycosphingolipid were essentially the same, and consisted of a 4-hydroxyoctadecasphinganine (phytosphingosine) as the sole sphingoid base and 2-hydroxytetracosanoic acid (>90%) as the major fatty acid. ZGLs were found to cause cell death in suspensions of cultured rice cells. The cell death-inducing activity of ZGLs is probably due to the characteristic glycan moiety of Man(alpha1-6)GlcN, and PC-containing ZGLs had high activity. This study is the first to demonstrate that fungal glycosylinositol-phosphoceramides induce cell death in cultured rice cells. PMID:14583095

Prion Protein-mediated Toxicity of Amyloid-β Oligomers Requires Lipid Rafts and the Transmembrane LRP1*

PubMed Central

Rushworth, Jo V.; Griffiths, Heledd H.; Watt, Nicole T.; Hooper, Nigel M.

2013-01-01

Soluble oligomers of the amyloid-β (Aβ) peptide cause neurotoxicity, synaptic dysfunction, and memory impairments that underlie Alzheimer disease (AD). The cellular prion protein (PrPC) was recently identified as a high affinity neuronal receptor for Aβ oligomers. We report that fibrillar Aβ oligomers recognized by the OC antibody, which have been shown to correlate with the onset and severity of AD, bind preferentially to cells and neurons expressing PrPC. The binding of Aβ oligomers to cell surface PrPC, as well as their downstream activation of Fyn kinase, was dependent on the integrity of cholesterol-rich lipid rafts. In SH-SY5Y cells, fluorescence microscopy and co-localization with subcellular markers revealed that the Aβ oligomers co-internalized with PrPC, accumulated in endosomes, and subsequently trafficked to lysosomes. The cell surface binding, internalization, and downstream toxicity of Aβ oligomers was dependent on the transmembrane low density lipoprotein receptor-related protein-1 (LRP1). The binding of Aβ oligomers to cell surface PrPC impaired its ability to inhibit the activity of the β-secretase BACE1, which cleaves the amyloid precursor protein to produce Aβ. The green tea polyphenol (−)-epigallocatechin gallate and the red wine extract resveratrol both remodeled the fibrillar conformation of Aβ oligomers. The resulting nonfibrillar oligomers displayed significantly reduced binding to PrPC-expressing cells and were no longer cytotoxic. These data indicate that soluble, fibrillar Aβ oligomers bind to PrPC in a conformation-dependent manner and require the integrity of lipid rafts and the transmembrane LRP1 for their cytotoxicity, thus revealing potential targets to alleviate the neurotoxic properties of Aβ oligomers in AD. PMID:23386614

Cellular Prion Protein Combined with Galectin-3 and -6 Affects the Infectivity Titer of an Endogenous Retrovirus Assayed in Hippocampal Neuronal Cells

PubMed Central

Shin, Hae-Young; Goto, Joy J.; Carp, Richard I.; Choi, Eun-Kyoung; Kim, Yong-Sun

2016-01-01

Prion diseases are infectious and fatal neurodegenerative diseases which require the cellular prion protein, PrPC, for development of diseases. The current study shows that the PrPC augments infectivity and plaque formation of a mouse endogenous retrovirus, MuLV. We have established four neuronal cell lines expressing mouse PrPC, PrP+/+; two express wild type PrPC (MoPrPwild) and the other two express mutant PrPC (MoPrPmut). Infection of neuronal cells from various PrP+/+ and PrP-/- (MoPrPKO) lines with MuLV yielded at least three times as many plaques in PrP+/+ than in PrP-/-. Furthermore, among the four PrP+/+ lines, one mutant line, P101L, had at least 2.5 times as many plaques as the other three PrP+/+ lines. Plaques in P101L were four times larger than those in other PrP+/+ lines. Colocalization of PrP and CAgag was seen in MuLV-infected PrP+/+ cells. In the PrP-MuLV interaction, the involvement of galectin-3 and -6 was observed by immunoprecipitation with antibody to PrPC. These results suggest that PrPC combined with galectin-3 and -6 can act as a receptor for MuLV. P101L, the disease form of mutant PrPC results suggest the genetic mutant form of PrPC may be more susceptible to viral infection. PMID:27936017

Neurocytoprotective Effects of Aliphatic Hydroxamates from Lovastatin, a Secondary Metabolite from Monascus-Fermented Red Mold Rice, in 6-Hydroxydopamine (6-OHDA)-Treated Nerve Growth Factor (NGF)-Differentiated PC12 Cells.

PubMed

Lin, Chien-Min; Lin, Yi-Tzu; Lin, Rong-Dih; Huang, Wei-Jan; Lee, Mei-Hsien

2015-05-20

Lovastatin, a secondary metabolite isolated from Monascus-fermented red rice mold, has neuroprotective activity and permeates the blood-brain barrier. The aim of this study was to enhance the activity of lovastatin for potential use as a treatment for neuronal degeneration in Parkinson's disease. Six lovastatin-derived compounds were semisynthesized and screened for neurocytoprotective activity against 6-hydroxydopamine (6-OHDA)-induced toxicity in human neuroblastoma PC12 cells. Four compounds, designated as 3a, 3d, 3e, and 3f, significantly enhanced cell viability. In particular, compound 3f showed excellent neurocytoprotective activity (97.0 ± 2.7%). Annexin V-FITC and propidium iodide double staining and 4',6-diamidino-2-phenylindole staining indicated that compound 3f reduced 6-OHDA-induced apoptosis in PC12 cells. Compound 3f also reduced caspase-3, -8, and -9 activities, and intracellular calcium concentrations elevated by 6-OHDA in a concentration-dependent manner, without inhibiting reactive oxygen species generation. JC-1 staining indicated that compound 3f also stabilized mitochondrial membrane potential. Thus, compound 3f may be used as a neurocytoprotective agent. Future studies should investigate its potential application as a treatment for Parkinson's disease.

[Studies on bioactive constituents of whole herbs of Vernonia cinerea].

PubMed

Zhu, Hua-xu; Tang, Yu-ping; Pan, Lin-mei; Min, Zhi-da

2008-08-01

To study the constituents of the whole herbs of Vernonia cinerea. by bio-activity guided isolation with PC-12 model. The constituents were separated by column chromatography and the structures were elucidated by spectroscopic methods. Four compounds were identified to be (+)-lirioresinol B (1), stigmasterol (2), stigmasterol-3-O-beta-D-glucoside (3), 4-sulfo-benzocyclobutene (4), and their NGF inducing activity were also investigated. Compounds 1, 3, 4 were isolated from this genus for the first time, and compound 4 was identified as a new natural product. Compounds 1, 3, 4 showed cytotoxicity on PC-12, and compounds 2, 3, 4 showed inhibition activity. Compound 4 showed a specific effect on the survival of TrkA fibroblasts, and resulted in the inducing NGF activity.

Aminophthalocyanine-Mediated Photodynamic Inactivation of Leishmania tropica

PubMed Central

Al-Qahtani, Ahmed; Alkahtani, Saad; Kolli, Bala; Tripathi, Pankaj; Dutta, Sujoy; Al-Kahtane, Abdullah A.; Jiang, Xiong-Jie; Ng, Dennis K. P.

2016-01-01

Photodynamic inactivation of Leishmania spp. requires the cellular uptake of photosensitizers, e.g., endocytosis of silicon(IV)-phthalocyanines (PC) axially substituted with bulky ligands. We report here that when substituted with amino-containing ligands, the PCs (PC1 and PC2) were endocytosed and displayed improved potency against Leishmania tropica promastigotes and axenic amastigotes in vitro. The uptake of these PCs by both Leishmania stages followed saturation kinetics, as expected. Sensitive assays were developed for assessing the photodynamic inactivation of Leishmania spp. by rendering them fluorescent in two ways: transfecting promastigotes to express green fluorescent protein (GFP) and loading them with carboxyfluorescein succinimidyl ester (CFSE). PC-sensitized Leishmania tropica strains were seen microscopically to lose their motility, structural integrity, and GFP/CFSE fluorescence after exposure to red light (wavelength, ∼650 nm) at a fluence of 1 to 2 J cm−2. Quantitative fluorescence assays based on the loss of GFP/CFSE from live Leishmania tropica showed that PC1 and PC2 dose dependently sensitized both stages for photoinactivation, consistent with the results of a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell viability assay. Leishmania tropica strains are >100 times more sensitive than their host cells or macrophages to PC1- and PC2-mediated photoinactivation, judging from the estimated 50% effective concentrations (EC50s) of these cells. Axial substitution of the PC with amino groups instead of other ligands appears to increase its leishmanial photolytic activity by up to 40-fold. PC1 and PC2 are thus potentially useful for photodynamic therapy of leishmaniasis and for oxidative photoinactivation of Leishmania spp. for use as vaccines or vaccine carriers. PMID:26824938

Neuroprotective Activities of Saffron and Crocin.

PubMed

Soeda, Shinji; Aritake, Kosuke; Urade, Yoshihiro; Sato, Hiroshi; Shoyama, Yukihiro

2016-01-01

We first considered that saffron is really safety food because it has a long-use history. The neuroprotective activities of saffron and its major constituent, crocin, are separately discussed in vitro and in vivo. We reviewed the inhibitory activities of crocin against PC-12 cell apoptosis. The oxidative stress decreased the cellular levels of glutathione (GSH) which is an inhibitor of neutral sphingomyelinase (N-SMase). Therefore, the level of GSH was assayed by the addition of crocin resulted in the activation of glutathione reductase (GR). It became evident that crocin treatment prevents the N-SMase activation resulting in the decrease of ceramide release. From these evidences we summarized the role of crocin for neuronal cell death. We used the ethanol-blocking assay system for learning and memory activities. The effect of saffron and crocin on improving ethanol-induced impairment of learning behaviors of mice in passive avoidance tasks has been clear. Further, we did make clear that saffron and crocin prevent the inhibitory effect of ethanol on long-term potentiation (LTP) in the dentate gyrus. Finally we found that 100 mg/kg of crocin gave non-rapid eye movement sleep (non-REM sleep) although mice were started to be active during night time.

Synthesis, characterization and cellular location of cytotoxic constitutional organometallic isomers of rhenium delivered on a cyanocobalmin scaffold.

PubMed

Santoro, Giuseppe; Zlateva, Theodora; Ruggi, Albert; Quaroni, Luca; Zobi, Fabio

2015-04-21

Constitutional isomers of cyanocobalamin adducts based on a fluorescent rhenium tris-carbonyl diimine complex were prepared, characterized and tested against PC-3 cancer cells. The adducts differ only in the relative binding position of the organometallic species which is either bound at the cyano or the 5'-hydroxo group of vitamin B12. When tested for their cytotoxic potency, the species showed IC50 values in the low μM rage. Upon conjugation to the vitamin an energy transfer process causes an extremely low quantum yield of fluorescence emission, making the conjugates unsuitable for fluorescence imaging. However, by exploiting the vibrational signature of the fac-[Re(CO)3](+) core, their cellular distribution was evaluated via FTIR spectromicroscopy.

Activin A prevents neuron-like PC12 cell apoptosis after oxygen-glucose deprivation☆

PubMed Central

Xu, Guihua; He, Jinting; Guo, Hongliang; Mei, Chunli; Wang, Jiaoqi; Li, Zhongshu; Chen, Han; Mang, Jing; Yang, Hong; Xu, Zhongxin

2013-01-01

In this study, PC12 cells were induced to differentiate into neuron-like cells using nerve growth factor, and were subjected to oxygen-glucose deprivation. Cells were treated with 0, 10, 20, 30, 50, 100 ng/mL exogenous Activin A. The 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide assay and Hoechst 33324 staining showed that the survival percentage of PC12 cells significantly decreased and the rate of apoptosis significantly increased after oxygen-glucose deprivation. Exogenous Activin A significantly increased the survival percentage of PC12 cells in a dose-dependent manner. Reverse transcription-PCR results revealed a significant increase in Activin receptor IIA, Smad3 and Smad4 mRNA levels, which are key sites in the Activin A/Smads signaling pathway, in neuron-like cells subjected to oxygen-glucose deprivation, while mRNA expression of the apoptosis-regulation gene caspase-3 decreased. Our experimental findings indicate that exogenous Activin A plays an anti-apoptotic role and protects neurons by means of activating the Activin A/Smads signaling pathway. PMID:25206395

Neurotensin is metabolized by endogenous proteases in prostate cancer cell lines.

PubMed

Moody, T W; Mayr, C A; Gillespie, T J; Davis, T P

1998-01-01

The formation and processing of neurotensin (NT) by three prostate cancer cell lines was investigated. Neurotensin (NT) immunoreactivity was detected in conditioned media and extracts of LNCaP cells. Using HPLC techniques, the immunoreactivity extracted from LNCaP cells coeluted with synthetic NT standard. Metalloendopeptidase 3.4.24.15 activity was detected in PC-3, DU-145 and LNCaP cells, whereas high levels of neutral endopeptidase 3.4.24.1 1 activity was detected only in LNCaP cells. NT was relatively stable when incubated with PC-3 or D-145 cells but was rapidly degraded by LNCaP cells to NT1-11 and NT1-10. Phosphoramidon inhibited the metabolism of NT by LNCaP cells. These data suggest that NT is present in and metabolized by LNCaP cellular enzymes.

Tyrosine hydroxylase is activated and phosphorylated at different sites in rat pheochromocytoma PC 12 cells treated with phorbol ester and forskolin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tachikawa, E.; Tank, A.W.; Weiner, D.H.

1986-03-01

The effects of phorbol ester (4..beta..-phorbol, 12..beta..-myristate, 13..cap alpha..-acetate; TPA), an activator of Ca/sup + +//phospholipid-dependent protein kinase (PK-C), and forskolin, which stimulates adenylate cyclase and cyclic AMP-dependent protein kinase (cAMP-PK), on the activation and phosphorylation of tyrosine hydroxylase (TH) in rat pheochromocytoma (PC 12) cells were examined. Incubation of the cells with TPA (0.01-1 ..mu..M) or forskolin (0.01-0.1 ..mu..M) produces increases in activation and phosphorylation of TH in a concentration-dependent manner. The stimulatory effects of TPA are dependent on extracellular Ca/sup + +/ and are inhibited by pretreatment of the cells with trifluoperazine (TFP). The effects of forskolin aremore » independent of Ca/sup + +/ and are not inhibited by TFP. In cells treated with forskolin, the time course of the increase in cAMP correlates with the increases in TH activity and phosphorylation. cAMP levels do not increase in cells treated with TPA. There is an increase in the phosphorylation of only one tryptic phosphopeptide derived from TH in cells treated with either forskolin or TPA. The peptide phosphorylated in TPA-treated cells exhibits different elution characteristics on HPLC from that in forskolin-treated cells. The authors conclude that TH in PC 12 cells is phosphorylated on different sites by cAMP-PK and PK-C. Phosphorylation of either of these sites is associated with enzyme activation.« less

The orally active pterocarpanquinone LQB-118 exhibits cytotoxicity in prostate cancer cell and tumor models through cellular redox stress.

PubMed

Martino, Thiago; Kudrolli, Tarana A; Kumar, Binod; Salviano, Isis; Mencalha, André; Coelho, Marsen Garcia P; Justo, Graça; Costa, Paulo R Ribeiro; Sabino, Kátia C Carvalho; Lupold, Shawn E

2018-02-01

The targeted induction of reactive oxygen species (ROS) is a developing mechanism for cancer therapy. LQB-118 is a pterocarpanquinone and ROS-inducing agent with proven antineoplastic activity. Here, LQB-118 efficacy and mechanism of activity, were examined in Prostate Cancer (PCa) cell and tumor models. PC3, LNCaP, and LAPC4 PCa cells were applied. Dicoumarol treatment was used to inhibit quinone reductase activity. N-acetylcysteine (NAC) was applied as a ROS scavenger. ROS production was quantified by H 2 DCFDA flow cytometry. LQB-118 treated cells were evaluated for changes in lipid peroxidation, viability, and apoptosis. Treatment-induced gene expression was measured by RT-qPCR and Western Blot. SOD1 knockdown was achieved with siRNA or miRNA mimic transfection. MicroRNA specificity was determined by 3'UTR reporter assay. Oral LQB-118 treatment (10 mg/kg/day) efficacy was determined in athymic male nude mice bearing subcutaneous PC3 xenograft tumors. LQB-118 treatment triggered PCa cell death and apoptosis. Therapeutic activity was at least partially dependent upon quinone reduction and ROS generation. LQB-118 treatment caused an increase in cellular ROS and lipid peroxidation. Treated cells exhibited elevated levels of NQO1, Nrf2, and SOD1. The miRNAs miR-206, miR-1, and miR-101 targeted and reduced SOD1 expression. The knockdown of SOD1, by siRNA or miRNA, enhanced LQB-118 cytotoxicity. Orally administered LQB-118 treatment significantly reduced the growth of established PCa xenograft tumors. LQB-118 is a developing and orally active pterocarpanquinone agent that effectively kills PCa cells through quinone reduction and ROS generation. The inhibition SOD1 expression enhances LQB-118 activity, presumably by impairing the cellular antioxidant response. © 2017 Wiley Periodicals, Inc.

miR-143 decreases COX-2 mRNA stability and expression in pancreatic cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pham, Hung; Department of Medicine, Veterans Affair Greater Los Angeles Healthcare System, Los Angeles, CA 90073; Ekaterina Rodriguez, C.

2013-09-13

Highlights: •Pancreatic cancer cells express low miR-143 levels and elevated p-MEK, p-MAPK and RREB1. •MEK inhibitors U0126 and PD98059 increase miR-143 expression. •miR-143 decreases COX-2 mRNA stability and expression and PGE{sub 2}. •miR-143 decreases p-p38MAPK, p-MEK, p-MAPK and RREB1 expression. -- Abstract: Small non-coding RNAs, microRNAs (miRNA), inhibit the translation or accelerate the degradation of message RNA (mRNA) by targeting the 3′-untranslated region (3′-UTR) in regulating growth and survival through gene suppression. Deregulated miRNA expression contributes to disease progression in several cancers types, including pancreatic cancers (PaCa). PaCa tissues and cells exhibit decreased miRNA, elevated cyclooxygenase (COX)-2 and increased prostaglandinmore » E{sub 2} (PGE{sub 2}) resulting in increased cancer growth and metastases. Human PaCa cell lines were used to demonstrate that restoration of miRNA-143 (miR-143) regulates COX-2 and inhibits cell proliferation. miR-143 were detected at fold levels of 0.41 ± 0.06 in AsPC-1, 0.20 ± 0.05 in Capan-2 and 0.10 ± 0.02 in MIA PaCa-2. miR-143 was not detected in BxPC-3, HPAF-II and Panc-1 which correlated with elevated mitogen-activated kinase (MAPK) and MAPK kinase (MEK) activation. Treatment with 10 μM of MEK inhibitor U0126 or PD98059 increased miR-143, respectively, by 187 ± 18 and 152 ± 26-fold in BxPC-3 and 182 ± 7 and 136 ± 9-fold in HPAF-II. miR-143 transfection diminished COX-2 mRNA stability at 60 min by 2.6 ± 0.3-fold in BxPC-3 and 2.5 ± 0.2-fold in HPAF-II. COX-2 expression and cellular proliferation in BxPC-3 and HPAF-II inversely correlated with increasing miR-143. PGE{sub 2} levels decreased by 39.3 ± 5.0% in BxPC-3 and 48.0 ± 3.0% in HPAF-II transfected with miR-143. Restoration of miR-143 in PaCa cells suppressed of COX-2, PGE{sub 2}, cellular proliferation and MEK/MAPK activation, implicating this pathway in regulating miR-143 expression.« less

Cell Growth Arrest Mediated by STAT Proteins in Breast Cancer Cells

DTIC Science & Technology

1998-07-01

serum, PC 12 rat adrenal pheochromocytoma cells (ATCC, CRL-1721) in DMEM containing 10 % fetal bovine serum, 10 % horse serum, and 1 raM L-glutamine... unusual . Additionally, we have mostly presented the biochemical data showing this inhibitor activity. What is in vivo implication of the STAT...PC12 rat adrenal pheochromocytoma cells (ATCC, CRL-1721) in Dulbecco’s mod- ified Eagle’s medium containing 10% fetal bovine serum, 10% horse

Evidence that PC2 is the endogenous pro-neurotensin convertase in rMTC 6-23 cells and that PC1- and PC2-transfected PC12 cells differentially process pro-neurotensin.

PubMed

Rovère, C; Barbero, P; Kitabgi, P

1996-05-10

The neuropeptide precursor proneurotensin/neuromedin N (pro-NT/NN) is mainly expressed and differentially processed in the brain and in the small intestine. We showed previously that rMTC 6-23 cells process pro-NT/NN with a pattern similar to brain tissue and increase pro-NT/NN expression in response to dexamethasone, and that PC12 cells also produce pro-NT/NN but are virtually unable to process it. In addition, PC12 cells were reported to be devoid of the prohormone convertases PC1 and PC2. The present study was designed to identify the proprotein convertase(s) (PC) involved in pro-NT/NN processing in rMTC 6-23 cells and to compare PC1- and PC2-transfected PC12 cells for their ability to process pro-NT/NN. rMTC 6-23 cells were devoid of PC1, PC4, and PC5 but expressed furin and PC2. Stable expression of antisense PC2 RNA in rMTC 6-23 cells led to a 90% decrease in PC2 protein levels that correlated with a > 80% reduction of pro-NT/NN processing. PC2 expression was stimulated by dexamethasone in a time- and concentration-dependent manner. Stable PC12/PC2 transfectants processed pro-NT/NN with a pattern similar to that observed in the brain and in rMTC 6-23 cells. In contrast, stable PC12/PC1 transfectants reproduced the pro-NT/NN processing pattern seen in the gut. We conclude that (i) PC2 is the major pro-NT/NN convertase in rMTC 6-23 cells; (ii) its expression is coregulated with that of pro-NT/NN in this cell line; and (iii) PC2 and PC1 differentially process pro-NT/NN with brain and intestinal phenotype, respectively.

Prostate-Specific Membrane Antigen Targeted Gold Nanoparticles for Theranostics of Prostate Cancer.

PubMed

Mangadlao, Joey Dacula; Wang, Xinning; McCleese, Christopher; Escamilla, Maria; Ramamurthy, Gopalakrishnan; Wang, Ziying; Govande, Mukul; Basilion, James P; Burda, Clemens

2018-04-24

Prostate cancer is one of the most common cancers and among the leading causes of cancer deaths in the United States. Men diagnosed with the disease typically undergo radical prostatectomy, which often results in incontinence and impotence. Recurrence of the disease is often experienced by most patients with incomplete prostatectomy during surgery. Hence, the development of a technique that will enable surgeons to achieve a more precise prostatectomy remains an open challenge. In this contribution, we report a theranostic agent (AuNP-5kPEG-PSMA-1-Pc4) based on prostate-specific membrane antigen (PSMA-1)-targeted gold nanoparticles (AuNPs) loaded with a fluorescent photodynamic therapy (PDT) drug, Pc4. The fabricated nanoparticles are well-characterized by spectroscopic and imaging techniques and are found to be stable over a wide range of solvents, buffers, and media. In vitro cellular uptake experiments demonstrated significantly higher nanoparticle uptake in PSMA-positive PC3pip cells than in PSMA-negative PC3flu cells. Further, more complete cell killing was observed in Pc3pip than in PC3flu cells upon exposure to light at different doses, demonstrating active targeting followed by Pc4 delivery. Likewise, in vivo studies showed remission on PSMA-expressing tumors 14 days post-PDT. Atomic absorption spectroscopy revealed that targeted AuNPs accumulate 4-fold higher in PC3pip than in PC3flu tumors. The nanoparticle system described herein is envisioned to provide surgical guidance for prostate tumor resection and therapeutic intervention when surgery is insufficient.

Revisiting PC1/3 Mutants: Dominant-Negative Effect of Endoplasmic Reticulum-Retained Mutants.

PubMed

Blanco, Elias H; Ramos-Molina, Bruno; Lindberg, Iris

2015-10-01

Prohormone convertase 1/3 (PC1/3), encoded by the gene PCSK1, is critical for peptide hormone synthesis. An increasing number of studies have shown that inactivating mutations in PCSK1 are correlated with endocrine pathologies ranging from intestinal dysfunction to morbid obesity, whereas the common nonsynonymous polymorphisms rs6232 (N221D) and rs6234-rs6235 (Q665E-S690T) are highly associated with obesity risk. In this report, we revisited the biochemical and cellular properties of PC1/3 variants in the context of a wild-type PC1/3 background instead of the S357G hypermorph background used for all previous studies. In the wild-type background the PC1/3 N221D variant exhibited 30% lower enzymatic activity in a fluorogenic assay than wild-type PC1/3; this inhibition was greater than that detected in an equivalent experiment using the PC1/3 S357G background. A PC1/3 variant with the linked carboxyl-terminal polymorphisms Q665E-S690T did not show this difference. We also analyzed the biochemical properties of 2 PC1/3 mutants, G209R and G593R, which are retained in the endoplasmic reticulum (ER), and studied their effects on wild-type PC1/3. The expression of ER-retained mutants induced ER stress markers and also resulted in dominant-negative blockade of wild-type PC1/3 prodomain cleavage and decreased expression of wild-type PC1/3, suggesting facilitation of the entry of wild-type protein to a degradative proteasomal pathway. Dominant-negative effects of PC1/3 mutations on the expression and maturation of wild-type protein, with consequential effects on PC1/3 availability, add a new element which must be considered in population and clinical studies of this gene.

Identification of JAK2 as a Mediator of FIP1L1-PDGFRA-Induced Eosinophil Growth and Function in CEL

PubMed Central

Li, Bin; Zhang, Guangsen; Li, Cui; He, Dan; Li, Xinying; Zhang, Chunfang; Tang, Faqing; Deng, Xiyun; Lu, Jingchen; Tang, Youhong; Li, Ruijuan; Chen, Zhuchu; Duan, Chaojun

2012-01-01

The Fip1-like1 (FIP1L1)-platelet-derived growth factor receptor alpha fusion gene (F/P) arising in the pluripotent hematopoietic stem cell (HSC),causes 14% to 60% of patients with hypereosinophilia syndrome (HES). These patients, classified as having F/P (+) chronic eosinophilic leukemia (CEL), present with clonal eosinophilia and display a more aggressive disease phenotype than patients with F/P (–) HES patients. The mechanisms underlying predominant eosinophil lineage targeting and the cytotoxicity of eosinophils in this leukemia remain unclear. Given that the Janus tyrosine kinase (JAK)/signal transducers and activators of transcription (Stat) signaling pathway is key to cytokine receptor-mediated eosinophil development and activated Stat3 and Stat5 regulate the expression of genes involved in F/P malignant transformation, we investigated whether and how JAK proteins were involved in the pathogenesis of F/P-induced CEL. F/P activation of JAK2, Stat3 and Stat5, were confirmed in all the 11 F/P (+) CEL patients examined. In vitro inhibition of JAK2 in EOL-1, primary F/P(+) CEL cells (PC) and T674I F/P Imatinib resistant cells(IR) by either JAK2-specific short interfering RNA (siRNA) or the tryphostin derivative AG490(AG490), significantly reduced cellular proliferation and induced cellular apoptosis. The F/P can enhance the IL-5-induced JAK2 activation, and further results indicated that JAK2 inhibition blocked IL-5-induced cellular migration and activation of the EOL-1 and PC cells in vitro. F/P-stimulation of the JAK2 suppressed cells led to a significantly reduction in Stat3 activation, but relatively normal induction of Stat5 activation. Interestingly, JAK2 inhibition also reduced PI3K, Akt and NF-κB activity in a dose-dependent manner, and suppressed expression levels of c-Myc and Survivin. These results strongly suggest that JAK2 is activated by F/P and is required for F/P stimulation of cellular proliferation and infiltration, possibly through induction of c-Myc and Survivin expression via activation of multiple signaling pathways, including NF-κB, Stat3, and PI3K/Akt. PMID:22523564

Long noncoding RNA MALAT1 enhances the docetaxel resistance of prostate cancer cells via miR-145-5p-mediated regulation of AKAP12.

PubMed

Xue, Dong; Lu, Hao; Xu, Han-Yan; Zhou, Cui-Xing; He, Xiao-Zhou

2018-06-01

Our present work was aimed to study on the regulatory role of MALAT1/miR-145-5p/AKAP12 axis on docetaxel (DTX) sensitivity of prostate cancer (PCa) cells. The microarray data (GSE33455) to identify differentially expressed lncRNAs and mRNAs in DTX-resistant PCa cell lines (DU-145-DTX and PC-3-DTX) was retrieved from the Gene Expression Omnibus (GEO) database. QRT-PCR analysis was performed to measure MALAT1 expression in DTX-sensitive and DTX-resistant tissues/cells. The human DTX-resistant cell lines DU145-PTX and PC3-DTX were established as in vitro cell models, and the expression of MALAT1, miR-145-5p and AKAP12 was manipulated in DTX-sensitive and DTX-resistant cells. Cell viability was examined using MTT assay and colony formation methods. Cell apoptosis was assessed by TUNEL staining. Cell migration and invasion was determined by scratch test (wound healing) and Transwell assay, respectively. Dual-luciferase assay was applied to analyse the target relationship between lncRNA MALAT1 and miR-145-5p, as well as between miR-145-5p and AKAP12. Tumour xenograft study was undertaken to confirm the correlation of MALAT1/miR-145-5p/AKAP12 axis and DTX sensitivity of PCa cells in vivo. In this study, we firstly notified that the MALAT1 expression levels were up-regulated in clinical DTX-resistant PCa samples. Overexpressed MALAT1 promoted cell proliferation, migration and invasion but decreased cell apoptosis rate of PCa cells in spite of DTX treatment. We identified miR-145-5p as a target of MALAT1. MiR-145-5p overexpression in PC3-DTX led to inhibited cell proliferation, migration and invasion as well as reduced chemoresistance to DTX, which was attenuated by MALAT1. Moreover, we determined that AKAP12 was a target of miR-145-5p, which significantly induced chemoresistance of PCa cells to DTX. Besides, it was proved that MALAT1 promoted tumour cell proliferation and enhanced DTX-chemoresistance in vivo. There was an lncRNA MALAT1/miR-145-5p/AKAP12 axis involved in DTX resistance of PCa cells and provided a new thought for PCa therapy. © 2018 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Polycomb group (PcG) proteins and Pax6 cooperate to inhibit in vivo reprogramming of the developing Drosophila eye.

PubMed

Zhu, Jinjin; Ordway, Alison J; Weber, Lena; Buddika, Kasun; Kumar, Justin P

2018-04-04

How different cells and tissues commit to and determine their fates has been a central question in developmental biology since the seminal embryological experiments conducted by Wilhelm Roux and Hans Driesch in sea urchins and frogs. Here, we demonstrate that Polycomb group (PcG) proteins maintain Drosophila eye specification by suppressing the activation of alternative fate choices. The loss of PcG in the developing eye results in a cellular reprogramming event in which the eye is redirected to a wing fate. This fate transformation occurs with either the individual loss of Polycomb proteins or the simultaneous reduction of the Pleiohomeotic repressive complex and Pax6. Interestingly, the requirement for retinal selector genes is limited to Pax6, as the removal of more downstream members does not lead to the eye-wing transformation. We also show that distinct PcG complexes are required during different developmental windows throughout eye formation. These findings build on earlier observations that the eye can be reprogrammed to initiate head epidermis, antennal and leg development. © 2018. Published by The Company of Biologists Ltd.

Using mass spectrometry and small molecule reagents to detect distinctive structural features of different prion conformations (strains)

USDA-ARS?s Scientific Manuscript database

A prion (PrPSc) is a conformer of a normal cellular prion protein (PrPC). Although they are isosequential, PrPSc is an infectious protein able to convert PrPC into the prion conformation and thereby propagate an infection. PrPC is monomeric while PrPSc is a multimer. PrPSc can adopt more than one co...

Polycaprolactone/maltodextrin nanocarrier for intracellular drug delivery: formulation, uptake mechanism, internalization kinetics, and subcellular localization.

PubMed

Korang-Yeboah, Maxwell; Gorantla, Yamini; Paulos, Simon A; Sharma, Pankaj; Chaudhary, Jaideep; Palaniappan, Ravi

2015-01-01

Prostate cancer (PCa) disease progression is associated with significant changes in intracellular and extracellular proteins, intracellular signaling mechanism, and cancer cell phenotype. These changes may have direct impact on the cellular interactions with nanocarriers; hence, there is the need for a much-detailed understanding, as nanocarrier cellular internalization and intracellular sorting mechanism correlate directly with bioavailability and clinical efficacy. In this study, we report the differences in the rate and mechanism of cellular internalization of a biocompatible polycaprolactone (PCL)/maltodextrin (MD) nanocarrier system for intracellular drug delivery in LNCaP, PC3, and DU145 PCa cell lines. PCL/MD nanocarriers were designed and characterized. PCL/MD nanocarriers significantly increased the intracellular concentration of coumarin-6 and fluorescein isothiocyanate-labeled bovine serum albumin, a model hydrophobic and large molecule, respectively. Fluorescence microscopy and flow cytometry analysis revealed rapid internalization of the nanocarrier. The extent of nanocarrier cellular internalization correlated directly with cell line aggressiveness. PCL/MD internalization was highest in PC3 followed by DU145 and LNCaP, respectively. Uptake in all PCa cell lines was metabolically dependent. Extraction of endogenous cholesterol by methyl-β-cyclodextrin reduced uptake by 75%±4.53% in PC3, 64%±6.01% in LNCaP, and 50%±4.50% in DU145, indicating the involvement of endogenous cholesterol in cellular internalization. Internalization of the nanocarrier in LNCaP was mediated mainly by macropinocytosis and clathrin-independent pathways, while internalization in PC3 and DU145 involved clathrin-mediated endocytosis, clathrin-independent pathways, and macropinocytosis. Fluorescence microscopy showed a very diffused and non-compartmentalized subcellular localization of the PCL/MD nanocarriers with possible intranuclear localization and minor colocalization in the lysosomes with time.

pcDNA-IL-12 vaccination blocks eosinophilic inflammation but not airway hyperresponsiveness following murine Toxocara canis infection.

PubMed

Malheiro, Adriana; Aníbal, Fernanda F; Martins-Filho, Olindo Assis; Teixeira-Carvalho, Andréa; Perini, Adenir; Martins, Milton A; Medeiros, Alexandra I; Turato, Walter M; Acencio, Milene P M; Brandão, Izaíra T; Nomizo, Auro; Silva, Célio L; Faccioli, Lúcia H

2008-01-17

We have investigated the effect of pcDNA3-CpG and pcDNA-IL-12, delivered by intradermal gene gun administration, on the blood/lung eosinophilia, airway hyperresponsiveness as well as the immune response in a murine model of toxocariasis. Our results demonstrated that pcDNA-IL-12 but not pcDNA3-CpG vaccination led to a persistent lower blood/bronchoalveolar eosinophilia following Toxocara canis infection, as pcDNA3-CpG led only to an early transient blockage of eosinophil transmigration into bronchoalveolar fluid following T. canis infection. Prominent Type-1 immune response was pointed out as the hallmark of T. canis infection following pcDNA-IL-12 vaccination. Outstanding IFN-gamma/IL-4 ratio besides low levels of IgG1 with subsequent high IgG2a/IgG1 ratio further characterized a Type-1 polarized immunological profile in pcDNA-IL-12-vaccinated animals. Nevertheless, only pcDNA3-CpG was able to prevent airway hyperresponsiveness induced by T. canis infection. The persistent airway hyperresponsiveness observed in pcDNA-IL-12-vaccinated animals demonstrated that the airway constriction involved other immunological mediator than those blocked by pcDNA-IL-12. Together, these data indicated that pcDNA-IL-12 and pcDNA3-CpG vaccines have distinct therapeutic benefits regarding the eosinophilic inflammation/airway hyperresponsiveness triggered by T. canis infection, suggesting their possible use in further combined therapeutic interventions.

Puerarin protects differentiated PC12 cells from H₂O₂-induced apoptosis through the PI3K/Akt signalling pathway.

PubMed

Zhang, Qin; Huang, Wei-Dong; Lv, Xue-Ying; Yang, Yun-Mei

2012-05-01

Oxidative stress has been implicated as a major mechanism underlying the pathogenesis of neurodegenerative disorders. ROS (reactive oxygen species) can cause cell death via apoptosis. NGF (nerve growth factor) differentiated rat PC12 cells have been extensively used to study the differentiation and apoptosis of neurons. This study has investigated the protective effects of puerarin in H2O2-induced apoptosis of differentiated PC12 cells, and the possible molecular mechanisms involved. Differentiated PC12 cells were incubated with 700 μM H2O2 in the absence or presence of different doses of puerarin (4, 8 and 16 μM). Apoptosis was assessed by MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay, TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling) analysis and Annexin V-PI (propidium iodide) double staining flow cytometry. Protein levels of phospho-Akt and phospho-BAD (Bcl-2/Bcl-XL-antagonist, causing cell death) were assayed by Western blotting. After stimulation with H2O2 for 18 h, the viability of differentiated PC12 cells decreased significantly and a large number of cells underwent apoptosis. Differentiated PC12 cells were rescued from H2O2-induced apoptosis at different concentrations of puerarin in a dose-dependent manner. This was through increased production of phospho-Akt and phospho-BAD, an effect that could be reversed by wortmannin, an inhibitor of PI3K (phosphoinositide 3-kinase). The results suggest that puerarin may have neuroprotective effect through activation of the PI3K/Akt signalling pathway.

Acetylated Chitosan Oligosaccharides Act as Antagonists against Glutamate-Induced PC12 Cell Death via Bcl-2/Bax Signal Pathway

PubMed Central

Hao, Cui; Gao, Lixia; Zhang, Yiran; Wang, Wei; Yu, Guangli; Guan, Huashi; Zhang, Lijuan; Li, Chunxia

2015-01-01

Chitosan oligosaccharides (COSs), depolymerized products of chitosan composed of β-(1→4) d-glucosamine units, have broad range of biological activities such as antitumour, antifungal, and antioxidant activities. In this study, peracetylated chitosan oligosaccharides (PACOs) and N-acetylated chitosan oligosaccharides (NACOs) were prepared from the COSs by chemcal modification. The structures of these monomers were identified using NMR and ESI-MS spectra. Their antagonist effects against glutamate-induced PC12 cell death were investigated. The results showed that pretreatment of PC12 cells with the PACOs markedly inhibited glutamate-induced cell death in a concentration-dependent manner. The PACOs were better glutamate antagonists compared to the COSs and the NACOs, suggesting the peracetylation is essential for the neuroprotective effects of chitosan oligosaccharides. In addition, the PACOs pretreatment significantly reduced lactate dehydrogenase release and reactive oxygen species production. It also attenuated the loss of mitochondrial membrane potential. Further studies indicated that the PACOs inhibited glutamate-induced cell death by preventing apoptosis through depressing the elevation of Bax/Bcl-2 ratio and caspase-3 activation. These results suggest that PACOs might be promising antagonists against glutamate-induced neural cell death. PMID:25775423

Effect of fluoride exposure on mRNA expression of cav1.2 and calcium signal pathway apoptosis regulators in PC12 cells.

PubMed

Liao, Qiuxia; Zhang, Rui; Wang, Xiaoyu; Nian, Weiwei; Ke, Lulu; Ouyang, Wei; Zhang, Zigui

2017-09-01

This study investigated the effects of fluoride exposure on the mRNA expression of Cav1.2 calcium signaling pathway and apoptosis regulatory molecules in PC12 cells. The viability of PC12 cell receiving high fluoride (5.0mM) and low fluoride (0.5mM) alone or fluoride combined with L-type calcium channel (LTCC) agonist/inhibitor (5umol/L FPL6417/2umol/L nifedipine) was detected using cell counting kit-8 at different time points (2, 4, 6, 8, 12, 10, and 24h). Changes in the cell configuration were observed after exposing the cells to fluoride for 24h. The expression levels of molecules related to the LTCC were examined, particularly, Cav1.2, c-fos, CAMK II, Bax, and Bcl-2. Fluoride poisoning induced severe cell injuries, such as decreased PC12 cell activity, enhanced cell apoptosis, high c-fos, CAMKII, and Bax mRNA expression levels. Bcl-2 expression level was also reduced. Meanwhile, high fluoride, high fluoride with FPL64176, and low fluoride with FPL64176 enhanced the Cav1.2 expression level. In contrast, low fluoride, high fluoride with nifedipine, and low fluoride with nifedipine reduced the Cav1.2 expression level. Thus, Cav1.2 may be an important molecular target for the fluorosis treatment, and the LTCC inhibitor nifedipine may be an effective drug for fluorosis. Copyright © 2017 Elsevier B.V. All rights reserved.

Cellular prion protein protects from inflammatory and neuropathic pain

PubMed Central

2011-01-01

Cellular prion protein (PrPC) inhibits N-Methyl-D-Aspartate (NMDA) receptors. Since NMDA receptors play an important role in the transmission of pain signals in the dorsal horn of spinal cord, we thus wanted to determine if PrPC null mice show a reduced threshold for various pain behaviours. We compared nociceptive thresholds between wild type and PrPC null mice in models of inflammatory and neuropathic pain, in the presence and the absence of a NMDA receptor antagonist. 2-3 months old male PrPC null mice exhibited an MK-801 sensitive decrease in the paw withdrawal threshold in response both mechanical and thermal stimuli. PrPC null mice also exhibited significantly longer licking/biting time during both the first and second phases of formalin-induced inflammation of the paw, which was again prevented by treatment of the mice with MK-801, and responded more strongly to glutamate injection into the paw. Compared to wild type animals, PrPC null mice also exhibited a significantly greater nociceptive response (licking/biting) after intrathecal injection of NMDA. Sciatic nerve ligation resulted in MK-801 sensitive neuropathic pain in wild-type mice, but did not further augment the basal increase in pain behaviour observed in the null mice, suggesting that mice lacking PrPC may already be in a state of tonic central sensitization. Altogether, our data indicate that PrPC exerts a critical role in modulating nociceptive transmission at the spinal cord level, and fit with the concept of NMDA receptor hyperfunction in the absence of PrPC. PMID:21843375

Platycodin D induced apoptosis and autophagy in PC-12 cells through mitochondrial dysfunction pathway

NASA Astrophysics Data System (ADS)

Zeng, Chuan-Chuan; Zhang, Cheng; Yao, Jun-Hua; Lai, Shang-Hai; Han, Bing-Jie; Li, Wei; Tang, Bing; Wan, Dan; Liu, Yun-Jun

2016-11-01

In this article, the in vitro cytotoxicity of platycodin D was evaluated in human PC-12, SGC-7901, BEL-7402, HeLa and A549 cancer cell lines. PC-12 cells were sensitive to platycodin D treatment, with an IC50 value of 13.5 ± 1.2 μM. Morphological and comet assays showed that platycodin D effectively induced apoptosis in PC-12 cells. Platycodin D increased the levels of reactive oxygen species (ROS) and induced a decrease in mitochondrial membrane potential. Platycodin D induced cell cycle arrest at the G0/G1 phase in the PC-12 cell line. Platycodin D can induce autophagy. In addition, platycodin D can down-regulate the expression of Bcl-2 and Bcl-x, and up-regulate the levels of Bid protein in the PC-12 cells. The results demonstrated that platycodin D induced PC-12 cell apoptosis through a ROS-mediated mitochondrial dysfunction pathway.

Silymarin versus Silibinin: Differential Antioxidant and Neuroprotective Effects against H2O2-induced Oxidative Stress in PC12 Cells.

PubMed

Jiang, Hui-Hui; Yan, Fa-Shun; Shen, Liang; Ji, Hong-Fang

2016-05-01

The present study assessed comparatively the antioxidant activities of silymarin and its major active component silibinin and their neuroprotective effects against hydrogen peroxide (H2O2)-induced oxidative stress in rat pheochromocytoma PC12 cells. It was found that despite newly prepared silymarin and silibinin solution possessing comparable superoxide anion (O2*-)-scavenging activities, with time the activity of silymarin lowered slightly, but that of silibinin decreased dramatically. Both silymarin and silibinin suppressed H2O2-induced oxidative stress and apoptosis, and the neuroprotective effect of silymarin was overall relatively stronger than that of silibinin. The findings provided clues for future studies on therapeutic potentials of the whole silymarin or purified silibinin for neurodegenerative diseases.

Enhanced activity and stability of binuclear iron (III) phthalocyanine on graphene nanosheets for electrocatalytic oxygen reduction in acid

NASA Astrophysics Data System (ADS)

Li, Tengfei; Peng, Yingxiang; Li, Kai; Zhang, Rui; Zheng, Lirong; Xia, Dingguo; Zuo, Xia

2015-10-01

Binuclear iron (III) phthalocyanine (bi-FePc) and iron (III) phthalocyanine (FePc) are synthesized in situ on graphene nanosheets (GNS) by a microwave-assisted method. TEM, ultraviolet-visible spectroscopy and Raman spectroscopy confirm that bi-FePc is supported on GNS through π-π interactions. The catalytic activity of the bi-FePc/GNS and FePc/GNS composites in the oxygen reduction reaction (ORR) is investigated by CV and RDE measurements. The bi-FePc/GNS composite shows a more positive onset potential (0.12 V vs. Hg/Hg2SO4) for the ORR than FePc/GNS (-0.02 V vs. Hg/Hg2SO4), and a four-electron mechanism similar to commercial Pt/C (0.22 V vs. Hg/Hg2SO4). Moreover, bi-FePc/GNS exhibits good stability with 100% retention after 36,000 s, while Pt/C has a retention of only 50% after the same period. Additionally, bi-FePc/GNS shows higher tolerance toward methanol than the Pt/C catalyst. XPS and X-ray absorption fine structure spectroscopy demonstrate that compared with FePc/GNS, bi-FePc/GNS possesses a higher concentration of Fe3+ and smaller skeleton radius of the phthalocyanine ring, which has a square-planar structure that evidently favors the ORR. Thus, bi-FePc/GNS is a promising candidate as a cathode catalyst in direct methanol fuel cells.

Snail regulates cell survival and inhibits cellular senescence in human metastatic prostate cancer cell lines.

PubMed

Emadi Baygi, Modjtaba; Soheili, Zahra Soheila; Schmitz, Ingo; Sameie, Shahram; Schulz, Wolfgang A

2010-12-01

The epithelial-mesenchymal transition (EMT) is regarded as an important step in cancer metastasis. Snail, a master regulator of EMT, has been recently proposed to act additionally as a cell survival factor and inducer of motility. We have investigated the function of Snail (SNAI1) in prostate cancer cells by downregulating its expression via short (21-mer) interfering RNA (siRNA) and measuring the consequences on EMT markers, cell viability, death, cell cycle, senescence, attachment, and invasivity. Of eight carcinoma cell lines, the prostate carcinoma cell lines LNCaP and PC-3 showed the highest and moderate expression of SNAI1 mRNA, respectively, as measured by quantitative RT-PCR. Long-term knockdown of Snail induced a severe decline in cell numbers in LNCaP and PC-3 and caspase activity was accordingly enhanced in both cell lines. In addition, suppression of Snail expression induced senescence in LNCaP cells. SNAI1-siRNA-treated cells did not tolerate detachment from the extracellular matrix, probably due to downregulation of integrin α6. Expression of E-cadherin, vimentin, and fibronectin was also affected. Invasiveness of PC-3 cells was not significantly diminished by Snail knockdown. Our data suggest that Snail acts primarily as a survival factor and inhibitor of cellular senescence in prostate cancer cell lines. We therefore propose that Snail can act as early driver of prostate cancer progression.

Single-Molecule Imaging Reveals that Small Amyloid-β1–42 Oligomers Interact with the Cellular Prion Protein (PrPC)

PubMed Central

Ganzinger, Kristina A; Narayan, Priyanka; Qamar, Seema S; Weimann, Laura; Ranasinghe, Rohan T; Aguzzi, Adriano; Dobson, Christopher M; McColl, James; St George-Hyslop, Peter; Klenerman, David

2014-01-01

Oligomers of the amyloid-β peptide (Aβ) play a central role in the pathogenesis of Alzheimer’s disease and have been suggested to induce neurotoxicity by binding to a plethora of cell-surface receptors. However, the heterogeneous mixtures of oligomers of varying sizes and conformations formed by Aβ42 have obscured the nature of the oligomeric species that bind to a given receptor. Here, we have used single-molecule imaging to characterize Aβ42 oligomers (oAβ42) and to confirm the controversial interaction of oAβ42 with the cellular prion protein (PrPC) on live neuronal cells. Our results show that, at nanomolar concentrations, oAβ42 interacts with PrPC and that the species bound to PrPC are predominantly small oligomers (dimers and trimers). Single-molecule biophysical studies can thus aid in deciphering the mechanisms that underlie receptor-mediated oAβ-induced neurotoxicity, and ultimately facilitate the discovery of novel inhibitors of these pathways. PMID:25294384

Differential contribution of key metabolic substrates and cellular oxygen in HIF signalling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhdanov, Alexander V., E-mail: a.zhdanov@ucc.ie; Waters, Alicia H.C.; Golubeva, Anna V.

2015-01-01

Changes in availability and utilisation of O{sub 2} and metabolic substrates are common in ischemia and cancer. We examined effects of substrate deprivation on HIF signalling in PC12 cells exposed to different atmospheric O{sub 2}. Upon 2–4 h moderate hypoxia, HIF-α protein levels were dictated by the availability of glutamine and glucose, essential for deep cell deoxygenation and glycolytic ATP flux. Nuclear accumulation of HIF-1α dramatically decreased upon inhibition of glutaminolysis or glutamine deprivation. Elevation of HIF-2α levels was transcription-independent and associated with the activation of Akt and Erk1/2. Upon 2 h anoxia, HIF-2α levels strongly correlated with cellular ATP,more » produced exclusively via glycolysis. Without glucose, HIF signalling was suppressed, giving way to other regulators of cell adaptation to energy crisis, e.g. AMPK. Consequently, viability of cells deprived of O{sub 2} and glucose decreased upon inhibition of AMPK with dorsomorphin. The capacity of cells to accumulate HIF-2α decreased after 24 h glucose deprivation. This effect, associated with increased AMPKα phosphorylation, was sensitive to dorsomorphin. In chronically hypoxic cells, glutamine played no major role in HIF-2α accumulation, which became mainly glucose-dependent. Overall, the availability of O{sub 2} and metabolic substrates intricately regulates HIF signalling by affecting cell oxygenation, ATP levels and pathways involved in production of HIF-α. - Highlights: • Gln and Glc regulate HIF levels in hypoxic cells by maintaining low O{sub 2} and high ATP. • HIF-α levels under anoxia correlate with cellular ATP and critically depend on Glc. • Gln and Glc modulate activity of Akt, Erk and AMPK, regulating HIF production. • HIF signalling is differentially inhibited by prolonged Glc and Gln deprivation. • Unlike Glc, Gln plays no major role in HIF signalling in chronically hypoxic cells.« less

Cost-Utility Analysis of Pharmaceutical Care Intervention Versus Usual Care in Management of Nigerian Patients with Type 2 Diabetes.

PubMed

Adibe, Maxwell O; Aguwa, Cletus N; Ukwe, Chinwe V

To assess the cost-effectiveness of pharmaceutical care (PC) intervention versus usual care (UC) in the management of type 2 diabetes. This study was a randomized, controlled study with a 12-month patient follow-up in two Nigerian tertiary hospitals. One hundred and ten patients were randomly assigned to each of the "intervention" (PC) and the "control" (UC) groups. Patients in the UC group received the usual/conventional care offered by the hospitals. Patients in the PC group received UC and PC in the form of structural self-care education and training for 12 months. The economic evaluation was based on patients' perspective. Costs of management of individual complications were calculated from activities involved in their management by using activity-based costing. The impact of the interventions on quality of life was estimated by using the HUI23S4EN.40Q (Mark index 3) questionnaire. The primary outcomes were incremental cost-utility ratio and net monetary benefit. An intention-to-treat approach was used. Two-sample comparisons were made by using Student's t tests for normally distributed variables data at baseline, 6 months, and 12 months. Comparisons of proportions were done by using the chi-square test. The PC intervention led to incremental cost and effect of Nigerian naira (NGN) 10,623 ($69) and 0.12 quality-adjusted life-year (QALY) gained, respectively, with an associated incremental cost-utility ratio of NGN 88,525 ($571) per QALY gained. In the cost-effectiveness acceptability curve, the probability that PC was more cost-effective than UC was 95% at the NGN 250,000 ($1613) per QALY gained threshold and 52% at the NGN 88,600 ($572) per QALY gained threshold. The PC intervention was very cost-effective among patients with type 2 diabetes at the NGN 88,525 ($571.13) per QALY gained threshold, although considerable uncertainty surrounds these estimates. Copyright © 2013, International Society for Pharmacoeconomics and Outcomes Research (ISPOR). Published by Elsevier Inc.

Identification of a novel K311 ubiquitination site critical for androgen receptor transcriptional activity

PubMed Central

Cork, David M.W.; Darby, Steven; Ryan-Munden, Claudia A.; Nakjang, Sirintra; Mendes Côrtes, Leticia; Treumann, Achim; Gaughan, Luke

2017-01-01

Abstract The androgen receptor (AR) is the main driver of prostate cancer (PC) development and progression, and the primary therapeutic target in PC. To date, two functional ubiquitination sites have been identified on AR, both located in its C-terminal ligand binding domain (LBD). Recent reports highlight the emergence of AR splice variants lacking the LBD that can arise during disease progression and contribute to castrate resistance. Here, we report a novel N-terminal ubiquitination site at lysine 311. Ubiquitination of this site plays a role in AR stability and is critical for its transcriptional activity. Inactivation of this site causes AR to accumulate on chromatin and inactivates its transcriptional function as a consequence of inability to bind to p300. Additionally, mutation at lysine 311 affects cellular transcriptome altering the expression of genes involved in chromatin organization, signaling, adhesion, motility, development and metabolism. Even though this site is present in clinically relevant AR-variants it can only be ubiquitinated in cells when AR retains LBD suggesting a role for AR C-terminus in E2/E3 substrate recognition. We report that as a consequence AR variants lacking the LBD cannot be ubiquitinated in the cellular environment and their protein turnover must be regulated via an alternate pathway. PMID:27903893

Impaired ERAD and ER stress are early and specific events in polyglutamine toxicity

PubMed Central

Duennwald, Martin L.; Lindquist, Susan

2008-01-01

Protein misfolding, whether caused by aging, environmental factors, or genetic mutations, is a common basis for neurodegenerative diseases. The misfolding of proteins with abnormally long polyglutamine (polyQ) expansions causes several neurodegenerative disorders, such as Huntington’s disease (HD). Although many cellular pathways have been documented to be impaired in HD, the primary triggers of polyQ toxicity remain elusive. We report that yeast cells and neuron-like PC12 cells expressing polyQ-expanded huntingtin (htt) fragments display a surprisingly specific, immediate, and drastic defect in endoplasmic reticulum (ER)-associated degradation (ERAD). We further decipher the mechanistic basis for this defect in ERAD: the entrapment of the essential ERAD proteins Npl4, Ufd1, and p97 by polyQ-expanded htt fragments. In both yeast and mammalian neuron-like cells, overexpression of Npl4 and Ufd1 ameliorates polyQ toxicity. Our results establish that impaired ER protein homeostasis is a broad and highly conserved contributor to polyQ toxicity in yeast, in PC12 cells, and, importantly, in striatal cells expressing full-length polyQ-expanded huntingtin. PMID:19015277

Ultrasound-mediated piezoelectric differentiation of neuron-like PC12 cells on PVDF membranes.

PubMed

Hoop, Marcus; Chen, Xiang-Zhong; Ferrari, Aldo; Mushtaq, Fajer; Ghazaryan, Gagik; Tervoort, Theo; Poulikakos, Dimos; Nelson, Bradley; Pané, Salvador

2017-06-22

Electrical and/or electromechanical stimulation has been shown to play a significant role in regenerating various functionalities in soft tissues, such as tendons, muscles, and nerves. In this work, we investigate the piezoelectric polymer polyvinylidene fluoride (PVDF) as a potential substrate for wireless neuronal differentiation. Piezoelectric PVDF enables generation of electrical charges on its surface upon acoustic stimulation, inducing neuritogenesis of PC12 cells. We demonstrate that the effect of pure piezoelectric stimulation on neurite generation in PC12 cells is comparable to the ones induced by neuronal growth factor (NGF). In inhibitor experiments, our results indicate that dynamic stimulation of PVDF by ultrasonic (US) waves activates calcium channels, thus inducing the generation of neurites via a cyclic adenosine monophosphate (cAMP)-dependent pathway. This mechanism is independent from the well-studied NGF induced mitogen-activated protein kinases/extracellular signal-regulated kinases (MAPK/ERK) pathway. The use of US, in combination with piezoelectric polymers, is advantageous since focused power transmission can occur deep into biological tissues, which holds great promise for the development of non-invasive neuroregenerative devices.

Retinoid Signaling in Pancreatic Cancer, Injury and Regeneration

PubMed Central

Colvin, Emily K.; Susanto, Johana M.; Kench, James G.; Ong, Vivienna N.; Mawson, Amanda; Pinese, Mark; Chang, David K.; Rooman, Ilse; O'Toole, Sandra A.; Segara, Davendra; Musgrove, Elizabeth A.; Sutherland, Robert L.; Apte, Minoti V.; Scarlett, Christopher J.; Biankin, Andrew V.

2011-01-01

Background Activation of embryonic signaling pathways quiescent in the adult pancreas is a feature of pancreatic cancer (PC). These discoveries have led to the development of novel inhibitors of pathways such as Notch and Hedgehog signaling that are currently in early phase clinical trials in the treatment of several cancer types. Retinoid signaling is also essential for pancreatic development, and retinoid therapy is used successfully in other malignancies such as leukemia, but little is known concerning retinoid signaling in PC. Methodology/Principal Findings We investigated the role of retinoid signaling in vitro and in vivo in normal pancreas, pancreatic injury, regeneration and cancer. Retinoid signaling is active in occasional cells in the adult pancreas but is markedly augmented throughout the parenchyma during injury and regeneration. Both chemically induced and genetically engineered mouse models of PC exhibit a lack of retinoid signaling activity compared to normal pancreas. As a consequence, we investigated Cellular Retinoid Binding Protein 1 (CRBP1), a key regulator of retinoid signaling known to play a role in breast cancer development, as a potential therapeutic target. Loss, or significant downregulation of CRBP1 was present in 70% of human PC, and was evident in the very earliest precursor lesions (PanIN-1A). However, in vitro gain and loss of function studies and CRBP1 knockout mice suggested that loss of CRBP1 expression alone was not sufficient to induce carcinogenesis or to alter PC sensitivity to retinoid based therapies. Conclusions/Significance In conclusion, retinoid signalling appears to play a role in pancreatic regeneration and carcinogenesis, but unlike breast cancer, it is not mediated directly by CRBP1. PMID:22220202

NF-κB-dependent transcriptional upregulation of cyclin D1 exerts cytoprotection against hypoxic injury upon EGFR activation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Zhi-Dong; Xu, Liang; Tang, Kan-Kai

Apoptosis of neural cells is one of the main pathological features in hypoxic/ischemic brain injury. Nuclear factor-κB (NF-κB) might be a potential therapeutic target for hypoxic/ischemic brain injury since NF-κB has been found to be inactivated after hypoxia exposure, yet the underlying molecular mechanisms of NF-κB inactivation are largely unknown. Here we report that epidermal growth factor receptor (EGFR) activation prevents neuron-like PC12 cells apoptosis in response to hypoxia via restoring NF-κB-dependent transcriptional upregulation of cyclin D1. Functionally, EGFR activation by EGF stimulation mitigates hypoxia-induced PC12 cells apoptosis in both dose- and time-dependent manner. Of note, EGFR activation elevates IKKβmore » phosphorylation, increases IκBα ubiquitination, promotes P65 nuclear translocation and recruitment at cyclin D1 gene promoter as well as upregulates cyclin D1 expression. EGFR activation also abrogates the decrease of IKKβ phosphorylation, reduction of IκBα ubiquitination, blockade of P65 nuclear translocation and recruitment at cyclin D1 gene promoter as well as downregulation of cyclin D1 expression induced by hypoxia. Furthermore, NF-κB-dependent upregulation of cyclin D1 is instrumental for the EGFR-mediated cytoprotection against hypoxic apoptosis. In addition, the dephosphorylation of EGFR induced by either EGF siRNA transfection or anti-HB-EGF neutralization antibody treatment enhances hypoxic cytotoxicity, which are attenuated by EGF administration. Our results highlight the essential role of NF-κB-dependent transcriptional upregulation of cyclin D1 in EGFR-mediated cytoprotective effects under hypoxic preconditioning and support further investigation of EGF in clinical trials of patients with hypoxic/ischemic brain injury. - Highlights: • EGFR activation significantly decreases hypoxia-induced PC12 cells injury. • EGFR activation abrogates the transcriptional repression of cyclin D1 induced by hypoxia in a NF-κB-dependent manner. • NF-κB-dependent cyclin D1 upregulation is required for the EGFR-mediated cytoprotection against hypoxia-induced injury. • Endogenous EGFR activity antagonizes hypoxia-induced PC12 cells injury.« less

Maslinic acid promotes autophagy by disrupting the interaction between Bcl2 and Beclin1 in rat pheochromocytoma PC12 cells

PubMed Central

Dong, Xiaoli; Zhang, Jiaxiao; Zhou, Zhilin; Ye, Zhennan; Chen, Jiahao; Yuan, Jifan; Cao, Fengjun; Wang, Xuanbin; Liu, Wenchao; Yu, Wenxuan; Li, Xiaohua

2017-01-01

Maslinic acid (2α, 3β-dihydroxyolean-12-en-28-oic acid, MA) was isolated from natural plants and showed anti-cancer activity in rat Pheochromocytoma PC12 cells in our previous studies. We now discover that MA disrupts the interaction between Bcl2 and autophagy scaffold protein Beclin1 in the above cell line, leading to the up-regulation of autophagy. We investigated the effect of MA on the interaction between Bcl2 and Beclin1 by biochemical and biophysical methods in combination with autophagy characterization in the above cell line. Our results suggest that MA may serve as an autophagy activator by directly blocking the Bcl2-Beclin1 interaction to release free Beclin1 required for the recruitment of autophagy positive regulators, implying MA may exert its anti-cancer activity by regulating autophagy. PMID:29088805

The cellular transcription factor CREB corresponds to activating transcription factor 47 (ATF-47) and forms complexes with a group of polypeptides related to ATF-43.

PubMed

Hurst, H C; Masson, N; Jones, N C; Lee, K A

1990-12-01

Promoter elements containing the sequence motif CGTCA are important for a variety of inducible responses at the transcriptional level. Multiple cellular factors specifically bind to these elements and are encoded by a multigene family. Among these factors, polypeptides termed activating transcription factor 43 (ATF-43) and ATF-47 have been purified from HeLa cells and a factor referred to as cyclic AMP response element-binding protein (CREB) has been isolated from PC12 cells and rat brain. We demonstrated that CREB and ATF-47 are identical and that CREB and ATF-43 form protein-protein complexes. We also found that the cis requirements for stable DNA binding by ATF-43 and CREB are different. Using antibodies to ATF-43 we have identified a group of polypeptides (ATF-43) in the size range from 40 to 43 kDa. ATF-43 polypeptides are related by their reactivity with anti-ATF-43, DNA-binding specificity, complex formation with CREB, heat stability, and phosphorylation by protein kinase A. Certain cell types vary in their ATF-43 complement, suggesting that CREB activity is modulated in a cell-type-specific manner through interaction with ATF-43. ATF-43 polypeptides do not appear simply to correspond to the gene products of the ATF multigene family, suggesting that the size of the ATF family at the protein level is even larger than predicted from cDNA-cloning studies.

PC12 Cells Differentiate into Chromaffin Cell-Like Phenotype in Coculture with Adrenal Medullary Endothelial Cells

NASA Astrophysics Data System (ADS)

Mizrachi, Yaffa; Naranjo, Jose R.; Levi, Ben-Zion; Pollard, Harvey B.; Lelkes, Peter I.

1990-08-01

Previously we described specific in vitro interactions between PC12 cells, a cloned, catecholamine-secreting pheochromocytoma cell line derived from the rat adrenal medulla, and bovine adrenal medullary endothelial cells. We now demonstrate that these interactions induce the PC12 cells to acquire physical and biochemical characteristics reminiscent of chromaffin cells. Under coculture conditions involving direct cell-cell contact, the endothelial cells and the PC12 cells reduced their rates of proliferation; upon prolonged coculture PC12 cells clustered into nests of cells similar to the organization of chromaffin cells seen in vivo. Within 3 days in coculture with endothelial cells, but not with unrelated control cells, PC12 cells synthesized increased levels of [Met]enkephalin. In addition, PC12 cells, growing on confluent endothelial monolayers, failed to extend neurites in response to nerve growth factor. Neither medium conditioned by endothelial cells nor fixed endothelial cells could by themselves induce all of these different phenomena in the PC12 cells. These results suggest that under coculture conditions PC12 cells change their state of differentiation toward a chromaffin cell-like phenotype. The rapid, transient increase in the expression of the protooncogene c-fos suggests that the mechanism(s) inducing the change in the state of differentiation in PC12 cells in coculture with the endothelial cells may be distinct from that described for the differentiation of PC12 cells--e.g., by glucocorticoids. We propose that similar interactions between endothelial cells and chromaffin cell precursors may occur during embryonic development and that these interactions might be instrumental for the organ-specific differentiation of the adrenal medulla in vivo.

Composting of 4-nonylphenol-contaminated river sediment with inocula of Phanerochaete chrysosporium.

PubMed

Huang, Danlian; Qin, Xingmeng; Xu, Piao; Zeng, Guangming; Peng, Zhiwei; Wang, Rongzhong; Wan, Jia; Gong, Xiaomin; Xue, Wenjing

2016-12-01

A composting study was performed to investigate the degradation of 4-nonylphenol (4-NP) in river sediment by inoculating Phanerochaete chrysosporium (Pc). Pc was inoculated into composting Reactor A, C and D, while Reactor B without inocula was used as control. The results showed that composting with Pc accelerated the degradation of 4-NP, increased the catalase and polyphenol oxidase enzyme activities in contaminated sediment. The dissipation half-life (t 1/2 ) of 4-NP in Reactor A, C and D with inocula of Pc were 2.079, 2.558, 2.424days, while in Reactor B without inocula of Pc it was 3.239days, respectively. Correlation analysis showed that the contents of 4-NP in sediment in Reactor A and D were negatively correlated with the actives of laccase, whereas no obvious correlation was observed in Reactor B and C. All these findings also indicated that Pc enhanced the maturity of compost, and the best composting C/N ratio was 25.46:1. Copyright © 2016 Elsevier Ltd. All rights reserved.

Choline intake influences phosphatidylcholine DHA enrichment in nonpregnant women but not in pregnant women in the third trimester.

PubMed

West, Allyson A; Yan, Jian; Jiang, Xinyin; Perry, Cydne A; Innis, Sheila M; Caudill, Marie A

2013-04-01

Phosphatidylcholine (PC) produced via the S-adenosylmethionine-dependent phosphatidylethanolamine (PE) N-methyltransferase (PEMT) pathway is enriched with docosahexaenoic acid (DHA). DHA plays a critical role in fetal development and is linked to health endpoints in adulthood. It is unknown whether choline, which can serve as a source of S-adenosylmethionine methyl groups, influences PC-DHA or the PC:PE ratio in pregnant and nonpregnant women. This study tested whether choline intake affects indicators of choline-related lipid metabolism, including erythrocyte and plasma PC-DHA and PC:PE ratios, in pregnant women in the third trimester and nonpregnant women. Pregnant (n = 26) and nonpregnant (n = 21) women consumed 480 or 930 mg choline/d and a daily DHA supplement for 12 wk. Blood was collected at baseline and at the midpoint and end of the study. PC-DHA was analyzed as the proportion of total PC fatty acids. Pregnant women had greater (P = 0.002) PC-DHA concentrations than did nonpregnant women at baseline. The proportion of erythrocyte and plasma PC-DHA increased (P ≤ 0.002) in pregnant and nonpregnant women regardless of choline intake. However, in nonpregnant women, consumption of 930 mg choline/d led to greater (P < 0.001) erythrocyte PC-DHA and a more rapid increase (P < 0.001) in plasma PC-DHA. Lower (P = 0.001-0.024) erythrocyte and plasma PC:PE in pregnant women was not modified by choline intake. A higher choline intake may increase PEMT activity, resulting in greater PC-DHA enrichment of the PC molecule in nonpregnant women. Increased production of PC-DHA during pregnancy indicates elevated PEMT activity and a higher demand for methyl donors. This trial was registered at clinicaltrials.gov as NCT01127022.

Using small molecule reagents to help distinguish among prion structural models

USDA-ARS?s Scientific Manuscript database

The only demonstrated difference between infectious prions (PrPSc) and the isosequential normal cellular prion protein (PrPC) is conformation. The structure of PrPC has been determined by a variety of instrumental techniques. The structure of prions remains uncertain. Recent instrumental analysis h...

Insulin is a potential antioxidant for diabetes-associated cognitive decline via regulating Nrf2 dependent antioxidant enzymes.

PubMed

Song, Ying; Ding, Wei; Bei, Yun; Xiao, Yan; Tong, Hai-Da; Wang, Li-Bo; Ai, Li-Yao

2018-05-21

To investigate the neuroprotective effects of insulin on diabetic encephalopathy and its mechanism. The diabetic model was established by injection of streptozotocin. Behavior examinations were conducted by the Morris water maze. Histopathological alterations were detected by HE staining. ROS, CAT levels and SOD activity were measured using a microplate reader. In vitro, the viability of wild type and knock-down PC12 cells was detected by MTT assay, the morphology of cells was monitored under a microscope. The subcellular distribution of Nrf2 was observed by western blotting and immunohistochemistry. Evident oxidative stress injury was observed in diabetic rats and H 2 O 2 -induced PC12 cells. Insulin not only protect diabetic rat from oxidative stress injury but also significantly inhibited H 2 O 2 -induced apoptosis and intracellular ROS in cells. In addition, the level of malondialdehyde was reduced, and the activities of superoxide dismutase, catalase and glutathione peroxidase were augmented in both diabetic rats and PC12 cells. Interestingly, insulin promoted the translocation of Nrf2 into the nucleus and activation of downstream antioxidant protein expression. Further, the Nrf2 knockdown cells suffered more serious H 2 O 2 -induced damage than the wild PC12 cells. Moreover, insulin had no significant protective effect on knockdown cells with H 2 O 2 -damage. Collectively, our results suggested that insulin significantly inhibited neuronal damage through the Nrf2 signaling pathway, which regulates endogenous oxidant-antioxidant balance, therefore, insulin may be a potential protective agent for the treatment of oxidative stress-induced diabetic encephalopathy. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Hydrogen sulfide mediates the anti-survival effect of sulforaphane on human prostate cancer cells.

PubMed

Pei, Yanxi; Wu, Bo; Cao, Qiuhui; Wu, Lingyun; Yang, Guangdong

2011-12-15

Hydrogen sulfide (H(2)S) is a novel gasotransmitter that regulates cell proliferation and other cellular functions. Sulforaphane (SFN) is a sulfur-containing compound that exhibits anticancer properties, and young sprouts of broccoli are particularly rich in SFN. There is consistent epidemiological evidence that the consumption of sulfur-containing vegetables, such as garlic and cruciferous vegetables, may help reduce the occurrence of prostate cancer. Here we found that a large amount of H(2)S is released when SFN is added into cell culture medium or mixed with mouse liver homogenates, respectively. Both SFN and NaHS (a H(2)S donor) decreased the viability of PC-3 cells (a human prostate cancer cell line) in a dose-dependent manner, and supplement of methemoglobin or oxidized glutathione (two H(2)S scavengers) reversed SFN-reduced cell viability. We further found both cystathionine gamma-lyase (CSE) and cystathionine beta-synthase are expressed in PC-3 cells and mouse prostate tissues. H(2)S production in prostate tissues from CSE knockout mice was only 20% of that from wild-type mice, suggesting CSE is a major H(2)S-producing enzyme in prostate. CSE overexpression enhanced H(2)S production and inhibited cell viability in PC-3 cells. In addition, both SFN and NaHS activated p38 mitogen-activated protein kinases (MAPK) and c-Jun N-terminal kinase (JNK). Pre-treatment of PC-3 cells with methemoglobin decreased SFN-stimulated MAPK activities. Suppression of both p38 MAPK and JNK reversed H(2)S- or SFN-reduced viability of PC-3 cells. Our results demonstrated that H(2)S mediates the inhibitory effect of SFN on the proliferation of PC-3 cells, which suggests that H(2)S-releasing diet or drug might be beneficial in the treatment of prostate cancer. Copyright © 2011 Elsevier Inc. All rights reserved.

Epithelial and endothelial expression of the green fluorescent protein reporter gene under the control of bovine prion protein (PrP) gene regulatory sequences in transgenic mice

NASA Astrophysics Data System (ADS)

Lemaire-Vieille, Catherine; Schulze, Tobias; Podevin-Dimster, Valérie; Follet, Jérome; Bailly, Yannick; Blanquet-Grossard, Françoise; Decavel, Jean-Pierre; Heinen, Ernst; Cesbron, Jean-Yves

2000-05-01

The expression of the cellular form of the prion protein (PrPc) gene is required for prion replication and neuroinvasion in transmissible spongiform encephalopathies. The identification of the cell types expressing PrPc is necessary to understanding how the agent replicates and spreads from peripheral sites to the central nervous system. To determine the nature of the cell types expressing PrPc, a green fluorescent protein reporter gene was expressed in transgenic mice under the control of 6.9 kb of the bovine PrP gene regulatory sequences. It was shown that the bovine PrP gene is expressed as two populations of mRNA differing by alternative splicing of one 115-bp 5' untranslated exon in 17 different bovine tissues. The analysis of transgenic mice showed reporter gene expression in some cells that have been identified as expressing PrP, such as cerebellar Purkinje cells, lymphocytes, and keratinocytes. In addition, expression of green fluorescent protein was observed in the plexus of the enteric nervous system and in a restricted subset of cells not yet clearly identified as expressing PrP: the epithelial cells of the thymic medullary and the endothelial cells of both the mucosal capillaries of the intestine and the renal capillaries. These data provide valuable information on the distribution of PrPc at the cellular level and argue for roles of the epithelial and endothelial cells in the spread of infection from the periphery to the brain. Moreover, the transgenic mice described in this paper provide a model that will allow for the study of the transcriptional activity of the PrP gene promoter in response to scrapie infection.

Preconditioning with Gua Lou Gui Zhi decoction enhances H2O2-induced Nrf2/HO-1 activation in PC12 cells

PubMed Central

MAO, JINGJIE; LI, ZUANFANG; LIN, RUHUI; ZHU, XIAOQIN; LIN, JIUMAO; PENG, JUN; CHEN, LIDIAN

2015-01-01

Spasticity is common in various central neurological conditions, including after a stroke. Such spasticity may cause additional problems, and often becomes a primary concern for afflicted individuals. A number of studies have identified nuclear factor (erythroid-derived 2)-like 2 (Nrf2) as a key regulator in the adaptive survival response to oxidative stress. Elevated expression of Nrf2, combined with heme oxygenase 1 (HO-1) resistance, in the central nervous system is known to elicit key internal and external oxidation protection. Gua Lou Gui Zhi decoction (GLGZD) is a popular traditional Chinese formula with a long history of clinical use in China for the treatment of muscular spasticity following a stroke, epilepsy or a spinal cord injury. However, the mechanism underlying the efficacy of the medicine remains unclear. In the present study, the antioxidative effects of GLGZD were evaluated and the underlying molecular mechanisms were investigated, using hydrogen peroxide (H2O2)-induced rat pheochromocytoma cells (PC12 cells) as an in vitro oxidative stress model of neural cells. Upon application of different concentrations of GLGZD, a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay and ATP measurement were conducted to assess the impact on PC12 cell proliferation. In addition, inverted microscopy observations, and the MTT and ATP assessments, revealed that GLGZD attenuated H2O2-induced oxidative damage and signaling repression in PC12 cells. Furthermore, the mRNA and protein expression levels of Nrf2 and HO-1, which are associated with oxidative stress, were analyzed using reverse transcription quantitative polymerase chain reaction (PCR) and confocal microscopy. Confocal microscopy observations, as well as the quantitative PCR assay, revealed that GLGZD exerted a neuroprotective function against H2O2-induced oxidative damage in PC12 cells. Therefore, the results demonstrated that GLGZD protected PC12 cells injured by H2O2, which may be associated with the upregulation of Nrf2 and HO-1 mRNA and protein expression levels in PC12 cells. PMID:26622408

The role of chalcones: helichrysetin, xanthohumol, and flavokawin-C in promoting neurite outgrowth in PC12 Adh cells.

PubMed

Phan, Chia-Wei; Sabaratnam, Vikineswary; Yong, Wai-Kuan; Abd Malek, Sri Nurestri

2018-05-01

Chalcones are a group of compounds widely distributed in plant kingdom. The aim of this study was to assess the neurite outgrowth stimulatory activity of selected chalcones, namely helichrysetin, xanthohumol and flavokawin-C. Using adherent rat pheochromocytoma (PC12 Adh) cells, the chalcones were subjected to neurite outgrowth assay and the extracellular nerve growth factor (NGF) levels were determined. Xanthohumol (10 μg/mL) displayed the highest (p < 0.05) percentage of neurite-bearing PC12 Adh cells and the highest (p < 0.05) NGF level in the culture medium of xanthohumol-treated cells. While, helichrysetin induced a moderately high numbers of neurite-bearing cells, flavokawin-C did not stimulate neurite outgrowth. This work supports the potential use of xanthohumol as a potential neuroactive compound to stimulate neurite outgrowth.

Anti-podocalyxin antibody exerts antitumor effects via antibody-dependent cellular cytotoxicity in mouse xenograft models of oral squamous cell carcinoma.

PubMed

Itai, Shunsuke; Ohishi, Tomokazu; Kaneko, Mika K; Yamada, Shinji; Abe, Shinji; Nakamura, Takuro; Yanaka, Miyuki; Chang, Yao-Wen; Ohba, Shun-Ichi; Nishioka, Yasuhiko; Kawada, Manabu; Harada, Hiroyuki; Kato, Yukinari

2018-04-27

Podocalyxin (PODXL) overexpression is associated with progression, metastasis, and poor outcomes in cancers. We recently produced the novel anti-PODXL monoclonal antibody (mAb) PcMab-47 (IgG 1 , kappa). Herein, we engineered PcMab-47 into 47-mG 2a , a mouse IgG 2a -type mAb, to add antibody-dependent cellular cytotoxicity (ADCC). We further developed 47-mG 2a -f, a core fucose-deficient type of 47-mG 2a to augment its ADCC. Immunohistochemical analysis of oral cancer tissues using PcMab-47 and 47-mG 2a revealed that the latter stained oral squamous cell carcinoma (OSCC) cells in a cytoplasmic pattern at a much lower concentration. PcMab-47 and 47-mG 2a detected PODXL in 163/201 (81.1%) and in 197/201 (98.0%) OSCC samples, respectively. 47-mG 2a -f also detected PODXL in OSCCs at a similar frequency as 47-mG 2a . In vitro analysis revealed that both 47-mG 2a and 47-mG 2a -f exhibited strong complement-dependent cytotoxicity (CDC) against CHO/hPODXL cells. In contrast, 47-mG 2a -f exhibited much stronger ADCC than 47-mG 2a against OSCC cells, indicating that ADCC and CDC of those anti-PODXL mAbs depend on target cells. In vivo analysis revealed that both 47-mG 2a and 47-mG 2a -f exerted antitumor activity in CHO/hPODXL xenograft models at a dose of 100 μg or 500 μg/mouse/week administered twice. 47-mG 2a -f, but not 47-mG 2a , exerted antitumor activity in SAS and HSC-2 xenograft models at a dose of 100 μg/mouse/week administered three times. Although both 47-mG 2a and 47-mG 2a -f exerted antitumor activity in HSC-2 xenograft models at a dose of 500 μg/mouse/week administered twice, 47-mG 2a -f also showed higher antitumor activity than 47-mG 2a . These results suggested that a core fucose-deficient anti-PODXL mAb could be useful for antibody-based therapy against PODXL-expressing OSCCs.

Anti-podocalyxin antibody exerts antitumor effects via antibody-dependent cellular cytotoxicity in mouse xenograft models of oral squamous cell carcinoma

PubMed Central

Itai, Shunsuke; Ohishi, Tomokazu; Kaneko, Mika K.; Yamada, Shinji; Abe, Shinji; Nakamura, Takuro; Yanaka, Miyuki; Chang, Yao-Wen; Ohba, Shun-Ichi; Nishioka, Yasuhiko; Kawada, Manabu; Harada, Hiroyuki; Kato, Yukinari

2018-01-01

Podocalyxin (PODXL) overexpression is associated with progression, metastasis, and poor outcomes in cancers. We recently produced the novel anti-PODXL monoclonal antibody (mAb) PcMab-47 (IgG1, kappa). Herein, we engineered PcMab-47 into 47-mG2a, a mouse IgG2a-type mAb, to add antibody-dependent cellular cytotoxicity (ADCC). We further developed 47-mG2a-f, a core fucose-deficient type of 47-mG2a to augment its ADCC. Immunohistochemical analysis of oral cancer tissues using PcMab-47 and 47-mG2a revealed that the latter stained oral squamous cell carcinoma (OSCC) cells in a cytoplasmic pattern at a much lower concentration. PcMab-47 and 47-mG2a detected PODXL in 163/201 (81.1%) and in 197/201 (98.0%) OSCC samples, respectively. 47-mG2a-f also detected PODXL in OSCCs at a similar frequency as 47-mG2a. In vitro analysis revealed that both 47-mG2a and 47-mG2a-f exhibited strong complement-dependent cytotoxicity (CDC) against CHO/hPODXL cells. In contrast, 47-mG2a-f exhibited much stronger ADCC than 47-mG2a against OSCC cells, indicating that ADCC and CDC of those anti-PODXL mAbs depend on target cells. In vivo analysis revealed that both 47-mG2a and 47-mG2a-f exerted antitumor activity in CHO/hPODXL xenograft models at a dose of 100 μg or 500 μg/mouse/week administered twice. 47-mG2a-f, but not 47-mG2a, exerted antitumor activity in SAS and HSC-2 xenograft models at a dose of 100 μg/mouse/week administered three times. Although both 47-mG2a and 47-mG2a-f exerted antitumor activity in HSC-2 xenograft models at a dose of 500 μg/mouse/week administered twice, 47-mG2a-f also showed higher antitumor activity than 47-mG2a. These results suggested that a core fucose-deficient anti-PODXL mAb could be useful for antibody-based therapy against PODXL-expressing OSCCs. PMID:29854293

Subcellular colocalization of the cellular and scrapie prion proteins in caveolae-like membranous domains

PubMed Central

Vey, Martin; Pilkuhn, Susanne; Wille, Holger; Nixon, Randal; DeArmond, Stephen J.; Smart, Eric J.; Anderson, Richard G. W.; Taraboulos, Albert; Prusiner, Stanley B.

1996-01-01

Results of transgenetic studies argue that the scrapie isoform of the prion protein (PrPSc) interacts with the substrate cellular PrP (PrPC) during conversion into nascent PrPSc. While PrPSc appears to accumulate primarily in lysosomes, caveolae-like domains (CLDs) have been suggested to be the site where PrPC is converted into PrPSc. We report herein that CLDs isolated from scrapie-infected neuroblastoma (ScN2a) cells contain PrPC and PrPSc. After lysis of ScN2a cells in ice-cold Triton X-100, both PrP isoforms and an N-terminally truncated form of PrPC (PrPC-II) were found concentrated in detergent-insoluble complexes resembling CLDs that were isolated by flotation in sucrose gradients. Similar results were obtained when CLDs were purified from plasma membranes by sonication and gradient centrifugation; with this procedure no detergents are used, which minimizes artifacts that might arise from redistribution of proteins among subcellular fractions. The caveolar markers ganglioside GM1 and H-ras were found concentrated in the CLD fractions. When plasma membrane proteins were labeled with the impermeant reagent sulfo-N-hydroxysuccinimide-biotin, both PrPC and PrPSc were found biotinylated in CLD fractions. Similar results on the colocalization of PrPC and PrPSc were obtained when CLDs were isolated from Syrian hamster brains. Our findings demonstrate that both PrPC and PrPSc are present in CLDs and, thus, support the hypothesis that the PrPSc formation occurs within this subcellular compartment. PMID:8962161

Label-free in vitro prostate cancer cell detection via photonic-crystal biosensor

NASA Astrophysics Data System (ADS)

DeLuna, Frank; Ding, XiaoFei; Sagredo, Ismael; Bustamante, Gilbert; Sun, Lu-Zhe; Ye, Jing Yong

2018-02-01

Prostate-specific antigen (PSA) biomarker assays are the current clinical method for mass screening of prostate cancer. However, high false-positive rates are often reported due to PSA's low specificity, leading to an urgent need for the development of a more specific detection system independent of PSA levels. In our previous research, we demonstrated the feasibility of using cellular refractive indices (RI) as a unique contrast parameter to accomplish label-free detection of prostate cancer cells via variance testing, but were unable to determine if a specific cell was cancerous or noncancerous. In this paper, we report the use of our Photonic-Crystal biosensor in a Total-Internal-Reflection (PC-TIR) configuration to construct a label-free imaging system, which allows for the detection of individual prostate cancer cells utilizing cellular RI as the only contrast parameter. Noncancerous prostate (BPH-1) cells and prostate cancer (PC-3) cells were mixed at varied ratios and measured concurrently. Additionally, we isolated and induced PC-3 cells to undergo epithelial-mesenchymal transition (EMT) by exposing these cells to soluble factors such as TGF-β1. The biophysical characteristics of the cellular RI were quantified extensively in comparison to non-induced PC-3 cells as well as BPH-1 cells. EMT is a crucial mechanism for the invasion and metastasis of epithelial tumors characterized by the loss of cell-cell adhesion and increased cell mobility. Our study shows promising clinical potential in utilizing the PC-TIR biosensor imaging system to not only detect prostate cancer cells, but also evaluate prostate cancer progression.

Availability of the key metabolic substrates dictates the respiratory response of cancer cells to the mitochondrial uncoupling.

PubMed

Zhdanov, Alexander V; Waters, Alicia H C; Golubeva, Anna V; Dmitriev, Ruslan I; Papkovsky, Dmitri B

2014-01-01

Active glycolysis and glutaminolysis provide bioenergetic stability of cancer cells in physiological conditions. Under hypoxia, metabolic and mitochondrial disorders, or pharmacological treatment, a deficit of key metabolic substrates may become life-threatening to cancer cells. We analysed the effects of mitochondrial uncoupling by FCCP on the respiration of cells fed by different combinations of Glc, Gal, Gln and Pyr. In cancer PC12 and HCT116 cells, a large increase in O2 consumption rate (OCR) upon uncoupling was only seen when Gln was combined with either Glc or Pyr. Inhibition of glutaminolysis with BPTES abolished this effect. Despite the key role of Gln, addition of FCCP inhibited respiration and induced apoptosis in cells supplied with Gln alone or Gal/Gln. For all substrate combinations, amplitude of respiratory responses to FCCP did not correlate with Akt, Erk and AMPK phosphorylation, cellular ATP, and resting OCR, mitochondrial Ca(2+) or membrane potential. However, we propose that proton motive force could modulate respiratory response to FCCP by regulating mitochondrial transport of Gln and Pyr, which decreases upon mitochondrial depolarisation. As a result, an increase in respiration upon uncoupling is abolished in cells, deprived of Gln or Pyr (Glc). Unlike PC12 or HCT116 cells, mouse embryonic fibroblasts were capable of generating pronounced response to FCCP when deprived of Gln, thus exhibiting lower dependence on glutaminolysis. Overall, the differential regulation of the respiratory response to FCCP by metabolic environment suggests that mitochondrial uncoupling has a potential for substrate-specific inhibition of cell function, and can be explored for selective cancer treatment. © 2013.

Alterations in mitochondrial respiratory functions, redox metabolism and apoptosis by oxidant 4-hydroxynonenal and antioxidants curcumin and melatonin in PC12 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Raza, Haider; John, Annie; Brown, Eric M.

Cellular oxidative stress and alterations in redox metabolisms have been implicated in the etiology and pathology of many diseases including cancer. Antioxidant treatments have been proven beneficial in controlling these diseases. We have recently shown that 4-hydroxynonenal (4-HNE), a by-product of lipid peroxidation, induces oxidative stress in PC12 cells by compromising the mitochondrial redox metabolism. In this study, we have further investigated the deleterious effects of 4-HNE on mitochondrial respiratory functions and apoptosis using the same cell line. In addition, we have also compared the effects of two antioxidants, curcumin and melatonin, used as chemopreventive agents, on mitochondrial redox metabolismmore » and respiratory functions in these cells. 4-HNE treatment has been shown to cause a reduction in glutathione (GSH) pool, an increase in reactive oxygen species (ROS), protein carbonylation and apoptosis. A marked inhibition in the activities of the mitochondrial respiratory enzymes, cytochrome c oxidase and aconitase was observed after 4-HNE treatment. Increased nuclear translocation of NF-kB/p65 protein was also observed after 4-HNE treatment. Curcumin and melatonin treatments, on the other hand, maintained the mitochondrial redox and respiratory functions without a marked effect on ROS production and cell viability. These results suggest that 4-HNE-induced cytotoxicity may be associated, at least in part, with the altered mitochondrial redox and respiratory functions. The alterations in mitochondrial energy metabolism and redox functions may therefore be critical in determining the difference between cell death and survival.« less

Hydrolytically Degradable Poly(Ethylene Glycol) Hydrogel Scaffolds as a Cell Delivery Vehicle: Characterization of PC12 Cell Response

PubMed Central

Zustiak, Silviya P.; Pubill, Stephanie; Ribeiro, Andreia; Leach, Jennie B.

2013-01-01

The central nervous system (CNS) has a low intrinsic potential for regeneration following injury and disease, yet neural stem/progenitor cell (NPC) transplants show promise to provide a dynamic therapeutic in this complex tissue environment. Moreover, biomaterial scaffolds may improve the success of NPC-based therapeutics by promoting cell viability and guiding cell response. We hypothesized that a hydrogel scaffold could provide a temporary neurogenic environment that supports cell survival during encapsulation, and degrades completely in a temporally controlled manner to allow progression of dynamic cellular processes such as neurite extension. We utilized PC12 cells as a model cell line with an inducible neuronal phenotype to define key properties of hydrolytically-degradable poly(ethylene glycol) hydrogel scaffolds that impact cell viability and differentiation following release from the degraded hydrogel. Adhesive peptide ligands (RGDS, IKVAV or YIGSR), were required to maintain cell viability during encapsulation; as compared to YIGSR, the RGDS and IKVAV ligands were associated with a higher percentage of PC12 cells that differentiated to the neuronal phenotype following release from the hydrogel. Moreover, among the hydrogel properties examined (e.g., ligand type, concentration), total polymer density within the hydrogel had the most prominent effect on cell viability, with densities above 15% w/v leading to decreased cell viability likely due to a higher shear modulus. Thus, by identifying key properties of degradable hydrogels that affect cell viability and differentiation following release from the hydrogel, we lay the foundation for application of this system towards future applications of the scaffold as a neural cell delivery vehicle. PMID:24474590

Elevated expression of glutathione peroxidase in PC12 cells results in protection against methamphetamine but not MPTP toxicity.

PubMed

Hom, D G; Jiang, D; Hong, E J; Mo, J Q; Andersen, J K

1997-06-01

In vivo administration of either 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) or methamphetamine (MA) produces damage to the dopaminergic nervous system which may be due in part to the generation of reactive oxygen species (ROS). The resistance of superoxide dismutase (SOD) over-expressing transgenic mice to the effects of both MPTP and MA suggests the involvement of superoxide in the resulting neurotoxicity of both compounds. Superoxide can be converted by SOD to hydrogen peroxide, which itself can cause cellular degeneration by reacting with free iron to produce highly reactive hydroxyl radicals resulting in damage to proteins, nucleic acids and membrane phospholipids. Hydrogen peroxide has also been reported to be produced via inhibition of NADH dehydrogenase by MPP + formed during oxidation of MPTP by MAO-B and by dopamine auto-oxidation following MA-induced dopamine release from synaptic vesicles within nerve terminals. To test whether hydrogen peroxide is an important factor in the toxicity of either of these two neurotoxins, we created clonal PC12 lines expressing elevated levels of the hydrogen peroxide-reducing enzyme glutathione peroxidase (GSHPx). Elevation of GSHPx levels in PC12 was found to diminish the rise in ROS levels and lipid peroxidation resulting from MA but not MPTP treatment. Elevated levels of GSHPx also appeared to prevent decreases in transport-mediated dopamine uptake produced via MA administration as well as to attenuate toxin-induced cell loss as measured by either MTT reduction or LDH release. Our data, therefore, suggest that hydrogen peroxide production likely contributes to MA toxicity in dopaminergic neurons.

Isorhynchophylline Protects PC12 Cells Against Beta-Amyloid-Induced Apoptosis via PI3K/Akt Signaling Pathway

PubMed Central

Xian, Yan-Fang; Lin, Zhi-Xiu; Mao, Qing-Qiu; Chen, Jian-Nan; Su, Zi-Ren; Lai, Xiao-Ping; Ip, Paul Siu-Po

2013-01-01

The neurotoxicity of amyloid-β (Aβ) has been implicated as a critical cause of Alzheimer's disease. Isorhynchophylline (IRN), an oxindole alkaloid isolated from Uncaria rhynchophylla, exerts neuroprotective effect against Aβ 25–35-induced neurotoxicity in vitro. However, the exact mechanism for its neuroprotective effect is not well understood. The present study aimed to investigate the molecular mechanisms underlying the protective action of IRN against Aβ 25–35-induced neurotoxicity in cultured rat pheochromocytoma (PC12) cells. Pretreatment with IRN significantly increased the cell viability, inhibited the release of lactate dehydrogenase and the extent of DNA fragmentation in Aβ 25–35-treated cells. IRN treatment was able to enhance the protein levels of phosphorylated Akt (p-Akt) and glycogen synthase kinase-3β (p-GSK-3β). Lithium chloride blocked Aβ 25–35-induced cellular apoptosis in a similar manner as IRN, suggesting that GSK-3β inhibition was involved in neuroprotective action of IRN. Pretreatment with LY294002 completely abolished the protective effects of IRN. Furthermore, IRN reversed Aβ 25–35-induced attenuation in the level of phosphorylated cyclic AMP response element binding protein (p-CREB) and the effect of IRN could be blocked by the PI3K inhibitor. These experimental findings unambiguously suggested that the protective effect of IRN against Aβ 25–35-induced apoptosis in PC12 cells was associated with the enhancement of p-CREB expression via PI3K/Akt/GSK-3β signaling pathway. PMID:24319473

Enhanced contraception of canine zona pellucida 3 DNA vaccine via targeting DEC-205 in mice.

PubMed

Wang, Ying; Zhang, Beibei; Li, Jinyao; Aipire, Adila; Li, Yijie; Zhang, Fuchun

2018-06-01

Zona pellucida 3 (ZP3) is a potential antigen for the development of contraceptive vaccines to control animal population. In this study, we designed a canine ZP3 (CZP3) DNA vaccine through targeting DEC-205 (named as pcD-scFv-CZP3c) and investigated its contraceptive effect in mice. Female BALB/c mice were intramuscularly immunized 3 times at 2 weeks intervals. After immunization, humoral and cellular immune responses were detected by ELISA and flow cytometry. The results showed that pcD-CZP3 and pcD-scFv-CZP3c induced CZP3-specific antibody (Ab) responses both in serum and vaginal secretions compared to pcDNA3.1. Additionally, compared to pcD-CZP3, pcD-scFv-CZP3c increased the levels of CZP3-specific Abs after a third immunization. Abs induced by these two DNA vaccines could bind with mice and dogs oocytes. Moreover, pcD-scFv-CZP3c enhanced the activation of CD4 + T cells characterized by the increased frequencies of CD4 + CD44 + T cells. Finally, the contraceptive effect was evaluated in the immunized mice. These two DNA vaccines significantly decreased a mean litter size of mice compared to pcDNA3.1, but pcD-scFv-CZP3c group showed the smallest mean litter size. The mean litter size of pcD-scFv-CZP3 were 3.2 ± 0.742 and 4.6 ± 1.118 in two mating tests, which were significantly lower than pcDNA3.1(P < 0.001 and P < 0.05). Our results suggest that the CZP3 DNA vaccine targeted with DEC-205 may be a potential strategy for developing a contraceptive DNA vaccine. Copyright © 2018 Elsevier Inc. All rights reserved.

Activation of GPR30 inhibits growth of prostate cancer cells via sustained activation of Erk1/2, c-jun/c-fos-dependent upregulation of p21, and induction of G2 cell-cycle arrest

PubMed Central

Chan, Queeny K.Y.; Lam, Hung-Ming; Ng, Chi-Fai; Lee, Amy Y.Y.; Chan, Eddie S.Y.; Ng, Ho-Keung; Ho, Shuk-Mei; Lau, Kin-Mang

2010-01-01

G protein–coupled receptor 30 (GPR30) exhibits estrogen-binding affinity and mediates nongenomic signaling of estrogen to regulate cell growth. We here demonstrated for the first time, in contrast to the reported promoting action of GPR30 on the growth of breast and ovarian cancer cells, that activation of GPR30 by the receptor-specific, non-estrogenic ligand G-1 inhibited growth of androgen-dependent and -independent prostate cancer (PCa) cells in vitro and PC-3 xenografts in vivo. However, G-1 elicited no growth or histological changes in the prostates of intact mice and did not inhibit growth in quiescent BPH-1, an immortalized benign prostatic epithelial cell line. Treatment of PC-3 cells with G-1-induced cell-cycle arrest at the G2 phase and reduced the expression of G2-checkpoint regulators (cyclin A2, cyclin B1, cdc25c, and cdc2) and the phosphorylation of their common transcriptional regulator NF-YA in PC-3 cells. With the extensive use of siRNA knockdown experiments and the MEK inhibitor PD98059 in the present study, we dissected the mechanism underlying G-1–induced inhibition of PC-3 cell growth, which was mediated through GPR30, followed by a sustained activation of Erk1/2 and a c-jun/c-fos-dependent upregulation of p21, resulting in the arrest of PC-3 growth at the G2 phase. The discovery of this signaling pathway lays the foundation for future development of GPR30-based therapies for PCa. PMID:20203690

Neuroprotection against 6-OHDA toxicity in PC12 cells and mice through the Nrf2 pathway by a sesquiterpenoid from Tussilago farfara.

PubMed

Lee, Joohee; Song, Kwangho; Huh, Eugene; Oh, Myung Sook; Kim, Yeong Shik

2018-06-01

Oxidative stress plays a key role in neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Therefore, the nuclear factor-E2-related factor 2 (Nrf2), a key regulator of the antioxidative response, is considered to be important as a therapeutic target for neurodegenerative diseases. We investigated the underlying mechanism of Nrf2-mediated neuroprotective effects against oxidative stress in the PC12 cell line by 7β-(3-ethyl-cis-crotonoyloxy)-1α-(2-methylbutyryloxy)-3,14-dehydro-Z-notonipetranone (ECN), one of the sesquiterpenoids in Farfarae Flos. Pretreatment of PC12 cells with ECN had a protective effect against hydrogen peroxide (H 2 O 2 )- or 6-hydroxydopamine (6-OHDA)-induced cytotoxicity. ECN upregulated the ARE-luciferase activity and induced the mRNA expression of Nrf2 and antioxidant enzyme heme oxygenase-1 (HO-1). Knockdown of Nrf2 by small, interfering RNA (siRNA) abrogated the upregulation of HO-1, indicating that ECN had induced HO-1 via the Nrf2 pathway. Pretreatment with the thiol reducing agents, N-acetylcysteine (NAC) or dithiothreitol (DTT), attenuated Nrf2 activation and HO-1 expression. However, the non-thiol reducing antioxidant, Trolox, failed to inhibit HO-1 induction by ECN. These results suggest that ECN may directly interact with Kelch-like ECH-associated protein 1 (Keap1) and modify critical cysteine thiols present in the proteins responsible for Nrf2-mediated upregulation of HO-1. In a 6-OHDA-induced mouse model of PD, administration of ECN ameliorated motor impairments and dopaminergic neuronal damage. Taken together, ECN exerts neuroprotective effects by activating the Nrf2/HO-1 signaling pathway in both PC12 cells and mice. Thus, ECN, as an Nrf2 activator, could be an attractive therapeutic candidate for the neuroprotection or treatment of neurodegenerative diseases. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

The Nutrient and Energy Sensor Sirt1 Regulates the Hypothalamic-Pituitary-Adrenal (HPA) Axis by Altering the Production of the Prohormone Convertase 2 (PC2) Essential in the Maturation of Corticotropin-releasing Hormone (CRH) from Its Prohormone in Male Rats*

PubMed Central

Toorie, Anika M.; Cyr, Nicole E.; Steger, Jennifer S.; Beckman, Ross; Farah, George; Nillni, Eduardo A.

2016-01-01

Understanding the role of hypothalamic neuropeptides and hormones in energy balance is paramount in the search for approaches to mitigate the obese state. Increased hypothalamic-pituitary-adrenal axis activity leads to increased levels of glucocorticoids (GC) that are known to regulate body weight. The axis initiates the production and release of corticotropin-releasing hormone (CRH) from the paraventricular nucleus (PVN) of the hypothalamus. Levels of active CRH peptide are dependent on the processing of its precursor pro-CRH by the action of two members of the family of prohormone convertases 1 and 2 (PC1 and PC2). Here, we propose that the nutrient sensor sirtuin 1 (Sirt1) regulates the production of CRH post-translationally by affecting PC2. Data suggest that Sirt1 may alter the preproPC2 gene directly or via deacetylation of the transcription factor Forkhead box protein O1 (FoxO1). Data also suggest that Sirt1 may alter PC2 via a post-translational mechanism. Our results show that Sirt1 levels in the PVN increase in rats fed a high fat diet for 12 weeks. Furthermore, elevated Sirt1 increased PC2 levels, which in turn increased the production of active CRH and GC. Collectively, this study provides the first evidence supporting the hypothesis that PVN Sirt1 activates the hypothalamic-pituitary-adrenal axis and basal GC levels by enhancing the production of CRH through an increase in the biosynthesis of PC2, which is essential in the maturation of CRH from its prohormone, pro-CRH. PMID:26755731

Water-Soluble Dinitrosyl Iron Complex (DNIC): a Nitric Oxide Vehicle Triggering Cancer Cell Death via Apoptosis.

PubMed

Wu, Shou-Cheng; Lu, Chung-Yen; Chen, Yi-Lin; Lo, Feng-Chun; Wang, Ting-Yin; Chen, Yu-Jen; Yuan, Shyng-Shiou; Liaw, Wen-Feng; Wang, Yun-Ming

2016-09-19

Nitric oxide (NO) is an important cellular signaling molecule that modulates various physiological activities. Angiogenesis-promoting activities of NO-donor drugs have been explored in both experimental and clinical studies. In this study, a structurally well characterized and water-soluble neutral {Fe(NO)2}(9) DNIC [(S(CH2)2OH)(S(CH2)2NH3)Fe(NO)2] (DNIC 2) was synthesized to serve as a NO-donor species. The antitumor activity of DNIC 2 was determined by MTT assay, confocal imaging, and Annexin-V/PI staining. The IC50 values of DNIC 2 were 18.8, 42.9, and 38.6 μM for PC-3, SKBR-3, and CRL5866 tumor cells, respectively. Moreover, DNIC 2 promoted apoptotic cell death via activation of apoptosis-associated proteins and inhibition of survival associated proteins. In particular, DNIC 2 treatment suppressed PC-3 tumor growth by 2.34- and 19.3-fold at 7 and 21 days, in comparison with the control group. These results indicate that water-soluble DNIC 2 may serve as a promising drug for cancer therapy.

Lead Intoxication Synergies of the Ethanol-Induced Toxic Responses in Neuronal Cells--PC12.

PubMed

Kumar, V; Tripathi, V K; Jahan, S; Agrawal, M; Pandey, A; Khanna, V K; Pant, A B

2015-12-01

Lead (Pb)-induced neurodegeneration and its link with widespread neurobehavioral changes are well documented. Experimental evidences suggest that ethanol could enhance the absorption of metals in the body, and alcohol consumption may increase the susceptibility to metal intoxication in the brain. However, the underlying mechanism of ethanol action in affecting metal toxicity in brain cells is poorly understood. Thus, an attempt was made to investigate the modulatory effect of ethanol on Pb intoxication in PC12 cells, a rat pheochromocytoma. Cells were co-exposed to biological safe doses of Pb (10 μM) and ethanol (200 mM), and data were compared to the response of cells which received independent exposure to these chemicals at similar doses. Ethanol (200 mM) exposure significantly aggravated the Pb-induced alterations in the end points associated with oxidative stress and apoptosis. The finding confirms the involvement of reactive oxygen species (ROS)-mediated oxidative stress, and impairment of mitochondrial membrane potential, which subsequently facilitate the translocation of triggering proteins between cytoplasm and mitochondria. We further confirmed the apoptotic changes due to induction of mitochondria-mediated caspase cascade. These cellular changes were found to recover significantly, if the cells are exposed to N-acetyl cysteine (NAC), a known antioxidant. Our data suggest that ethanol may potentiate Pb-induced cellular damage in brain cells, but such damaging effects could be recovered by inhibition of ROS generation. These results open up further possibilities for the design of new therapeutics based on antioxidants to prevent neurodegeneration and associated health problems.

Enhancement of pheochromocytoma nerve cell growth by consecutive fractionization of Angelica gigas Nakai extracts

PubMed Central

Jeong, Myoung Hoon; Kim, Ji Seon; Zou, Yunyun; Yoon, Chang Soon; Lim, Hye Won; Ahn, Juhee

2010-01-01

This work was to investigate the effect of flavonoids from Angelica gigas Nakai on the proliferation and differentiation of PC12 cells. Several solvents including hexane, chloroform, ethyl acetate, butanol and water consecutively partitioned. We determined the ethanol crude extract of Angelica gigas Nakai. The hexane fraction was shown to contain the highest number of flavonoids as follows; 21.48 mg/g and the composition of the flavonoids was as follows: 12.24 mg/g of quercetin, 4.39 mg/g of myricetin and 2.58 mg/g of catechin. In addition, this hexane fraction greatly increased both cell growth and outgrowth of the neurite, and whose effects were three times higher than those of the other fractions. The length of the neurites was measured as ca. 110 μm in adding 50 μg/mL of the hexane fraction, which was about the same as the case of adding 50 ng/mL of NGF as a positive control. This result indicates that the differentiation of PC12 cells by the addition of the hexane fraction was comparable to the case of adding NGF. The hexane fraction was also determined to prevent apoptosis of PC12 cells by suppressing DNA fragmentation. It is interesting that the mixture of three major flavonoids, quercetin, myricetin and catechin showed stronger activity on, both PC12 cell growth and neuritis outgrowth, than when adding each flavonoid alone. We believe this was due to the synergistic effects of the three flavonoids. The activities of these flavonoids from Angelica gigas Nakai are reported for the first time in this study. PMID:20936343

Protective effect of Nigella sativa extract and thymoquinone on serum/glucose deprivation-induced PC12 cells death.

PubMed

Mousavi, S H; Tayarani-Najaran, Z; Asghari, M; Sadeghnia, H R

2010-05-01

The serum/glucose deprivation (SGD)-induced cell death in cultured PC12 cells represents a useful in vitro model for the study of brain ischemia and neurodegenerative disorders. Nigella sativa L. (family Ranunculaceae) and its active component thymoquinone (TQ) has been known as a source of antioxidants. In the present study, the protective effects of N. sativa and TQ on cell viability and reactive oxygen species (ROS) production in cultured PC12 cells were investigated under SGD conditions. PC12 cells were cultured in DMEM medium containing 10% (v/v) fetal bovine serum, 100 units/ml penicillin, and 100 microg/ml streptomycin. Cells were seeded overnight and then deprived of serum/glucose for 6 and 18 h. Cells were pretreated with different concentrations of N. sativa extract (15.62-250 microg/ml) and TQ (1.17-150 microM) for 2 h. Cell viability was quantitated by MTT assay. Intracellular ROS production was measured by flow cytometry using 2',7'-dichlorofluorescin diacetate (DCF-DA) as a probe. SGD induced significant cells toxicity after 6, 18, or 24 h (P < 0.001). Pretreatment with N. sativa (15.62-250 microg/ml) and TQ (1.17-37.5 microM) reduced SGD-induced cytotoxicity in PC12 cells after 6 and 18 h. A significant increase in intracellular ROS production was seen following SGD (P < 0.001). N. sativa (250 microg/ml, P < 0.01) and TQ (2.34, 4.68, 9.37 microM, P < 0.01) pretreatment reversed the increased ROS production following ischemic insult. The experimental results suggest that N. sativa extract and TQ protects the PC12 cells against SGD-induced cytotoxicity via antioxidant mechanisms. Our findings might raise the possibility of potential therapeutic application of N. sativa extract and TQ for managing cerebral ischemic and neurodegenerative disorders.

Acrylamide-induced oxidative stress and inflammatory response are alleviated by N-acetylcysteine in PC12 cells: Involvement of the crosstalk between Nrf2 and NF-κB pathways regulated by MAPKs.

PubMed

Pan, Xiaoqi; Wu, Xu; Yan, Dandan; Peng, Cheng; Rao, Chaolong; Yan, Hong

2018-05-15

Acrylamide (ACR) is a classic neurotoxin in animals and humans. However, the mechanism underlying ACR neurotoxicity remains controversial, and effective prevention and treatment measures against this condition are scarce. This study focused on clarifying the crosstalk between the involved signaling pathways in ACR-induced oxidative stress and inflammatory response and investigating the protective effect of antioxidant N-acetylcysteine (NAC) against ACR in PC12 cells. Results revealed that ACR exposure led to oxidative stress characterized by significant increase in reactive oxygen species (ROS) and malondialdehyde (MDA) levels and glutathione (GSH) consumption. Inflammatory response was observed based on the dose-dependently increased levels of pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin 6 (IL-6). NAC attenuated ACR-induced enhancement of MDA and ROS levels and TNF-α generation. In addition, ACR activated nuclear transcription factor E2-related factor 2 (Nrf2) and nuclear factor-κB (NF-κB) signaling pathways. Knockdown of Nrf2 by siRNA significantly blocked the increased NF-κB p65 protein expression in ACR-treated PC12 cells. Down-regulation of NF-κB by specific inhibitor BAY11-7082 similarly reduced ACR-induced increase in Nrf2 protein expression. NAC treatment increased Nrf2 expression and suppressed NF-κB p65 expression to ameliorate oxidative stress and inflammatory response caused by ACR. Further results showed that mitogen-activated protein kinases (MAPKs) pathway was activated prior to the activation of Nrf2 and NF-κB pathways. Inhibition of MAPKs blocked Nrf2 and NF-κB pathways. Collectively, ACR activated Nrf2 and NF-κB pathways which were regulated by MAPKs. A crosstalk between Nrf2 and NF-κB pathways existed in ACR-induced cell damage. NAC protected against oxidative damage and inflammatory response induced by ACR by activating Nrf2 and inhibiting NF-κB pathways in PC12 cells. Copyright © 2018 Elsevier B.V. All rights reserved.

Effects of huperzine A on secretion of nerve growth factor in cultured rat cortical astrocytes and neurite outgrowth in rat PC12 cells.

PubMed

Tang, Li-li; Wang, Rui; Tang, Xi-can

2005-06-01

To study the effects of huperzine A (HupA) on neuritogenic activity and the expression of nerve growth factor (NGF). After being treated with 10 micromol/L HupA, neurite outgrowth of PC12 cells was observed and counted under phase-contrast microscopy. Mitogenic activity was assayed by [3H]thymidine incorporation. Cell cytotoxicity was evaluated by lactate dehydrogenase (LDH) release. AChE activity, mRNA and protein expression were measured by the Ellman method, RT-PCR, and Western blot, respectively. NGF mRNA and protein levels were determined by RT-PCR and ELISA assays. Treatment of PC12 cells with 10 micromol/L HupA for 48 h markedly increased the number of neurite-bearing cells, but caused no significant alteration in cell viability or other signs of cytotoxicity. In addition to inhibiting AChE activity, 10 micromol/L HupA also increased the mRNA and protein levels of this enzyme. In addition, following 2 h exposure of the astrocytes to 10 micromol/L HupA, there was a significant up-regulation of mRNA for NGF and P75 low-affinity NGF receptor. The protein level of NGF was also increased after 24 h treatment with HupA. Our findings demonstrate for the first time that HupA has a direct or indirect neurotrophic activity, which might be beneficial in treatment of neurodegenerative disorders such as Alzheimer disease.

Gluten-free vegan diet induces decreased LDL and oxidized LDL levels and raised atheroprotective natural antibodies against phosphorylcholine in patients with rheumatoid arthritis: a randomized study

PubMed Central

Elkan, Ann-Charlotte; Sjöberg, Beatrice; Kolsrud, Björn; Ringertz, Bo; Hafström, Ingiäld; Frostegård, Johan

2008-01-01

Introduction The purpose of this study was to investigate the effects of vegan diet in patients with rheumatoid arthritis (RA) on blood lipids oxidized low-density lipoprotein (oxLDL) and natural atheroprotective antibodies against phosphorylcholine (anti-PCs). Methods Sixty-six patients with active RA were randomly assigned to either a vegan diet free of gluten (38 patients) or a well-balanced non-vegan diet (28 patients) for 1 year. Thirty patients in the vegan group completed more than 3 months on the diet regimen. Blood lipids were analyzed by routine methods, and oxLDL and anti-PCs were analyzed by enzyme-linked immunosorbent assay. Data and serum samples were obtained at baseline and after 3 and 12 months. Results Mean ages were 50.0 years for the vegan group and 50.8 years for controls. Gluten-free vegan diet induced lower body mass index (BMI) and low-density lipoprotein (LDL) and higher anti-PC IgM than control diet (p < 0.005). In the vegan group, BMI, LDL, and cholesterol decreased after both 3 and 12 months (p < 0.01) and oxLDL after 3 months (p = 0.021) and trendwise after 12 months (p = 0.090). Triglycerides and high-density lipoprotein did not change. IgA anti-PC levels increased after 3 months (p = 0.027) and IgM anti-PC levels increased trendwise after 12 months (p = 0.057). There was no difference in IgG anti-PC levels. In the control diet group, IgM anti-PC levels decreased both after 3 and 12 months (p < 0.01). When separating vegan patients into clinical responders and non-responders at 12 months, the effects on oxLDL and anti-PC IgA were seen only in responders (p < 0.05). Conclusion A gluten-free vegan diet in RA induces changes that are potentially atheroprotective and anti-inflammatory, including decreased LDL and oxLDL levels and raised anti-PC IgM and IgA levels. PMID:18348715

Gluten-free vegan diet induces decreased LDL and oxidized LDL levels and raised atheroprotective natural antibodies against phosphorylcholine in patients with rheumatoid arthritis: a randomized study.

PubMed

Elkan, Ann-Charlotte; Sjöberg, Beatrice; Kolsrud, Björn; Ringertz, Bo; Hafström, Ingiäld; Frostegård, Johan

2008-01-01

The purpose of this study was to investigate the effects of vegan diet in patients with rheumatoid arthritis (RA) on blood lipids oxidized low-density lipoprotein (oxLDL) and natural atheroprotective antibodies against phosphorylcholine (anti-PCs). Sixty-six patients with active RA were randomly assigned to either a vegan diet free of gluten (38 patients) or a well-balanced non-vegan diet (28 patients) for 1 year. Thirty patients in the vegan group completed more than 3 months on the diet regimen. Blood lipids were analyzed by routine methods, and oxLDL and anti-PCs were analyzed by enzyme-linked immunosorbent assay. Data and serum samples were obtained at baseline and after 3 and 12 months. Mean ages were 50.0 years for the vegan group and 50.8 years for controls. Gluten-free vegan diet induced lower body mass index (BMI) and low-density lipoprotein (LDL) and higher anti-PC IgM than control diet (p < 0.005). In the vegan group, BMI, LDL, and cholesterol decreased after both 3 and 12 months (p < 0.01) and oxLDL after 3 months (p = 0.021) and trendwise after 12 months (p = 0.090). Triglycerides and high-density lipoprotein did not change. IgA anti-PC levels increased after 3 months (p = 0.027) and IgM anti-PC levels increased trendwise after 12 months (p = 0.057). There was no difference in IgG anti-PC levels. In the control diet group, IgM anti-PC levels decreased both after 3 and 12 months (p < 0.01). When separating vegan patients into clinical responders and non-responders at 12 months, the effects on oxLDL and anti-PC IgA were seen only in responders (p < 0.05). A gluten-free vegan diet in RA induces changes that are potentially atheroprotective and anti-inflammatory, including decreased LDL and oxLDL levels and raised anti-PC IgM and IgA levels.

Gas, dust, stars, star formation, and their evolution in M 33 at giant molecular cloud scales

NASA Astrophysics Data System (ADS)

Komugi, Shinya; Miura, Rie E.; Kuno, Nario; Tosaki, Tomoka

2018-06-01

We report on a multi-parameter analysis of giant molecular clouds (GMCs) in the nearby spiral galaxy M 33. A catalog of GMCs identifed in 12CO(J = 3-2) was used to compile associated 12CO(J = 1-0), dust, stellar mass, and star formation rate. Each of the 58 GMCs are categorized by their evolutionary stage. Applying the principal component analysis on these parameters, we construct two principal components, PC1 and PC2, which retain 75% of the information from the original data set. PC1 is interpreted as expressing the total interstellar matter content, and PC2 as the total activity of star formation. Young (< 10 Myr) GMCs occupy a distinct region in the PC1-PC2 plane, with lower interstellar medium (ISM) content and star formation activity compared to intermediate-age and older clouds. Comparison of average cloud properties in different evolutionary stages imply that GMCs may be heated or grow denser and more massive via aggregation of diffuse material in their first ˜ 10 Myr. The PCA also objectively identified a set of tight relations between ISM and star formation. The ratio of the two CO lines is nearly constant, but weakly modulated by massive star formation. Dust is more strongly correlated with the star formation rate than the CO lines, supporting recent findings that dust may trace molecular gas better than CO. Stellar mass contributes weakly to the star formation rate, reminiscent of an extended form of the Schmidt-Kennicutt relation with the molecular gas term substituted by dust.

Gas, dust, stars, star formation, and their evolution in M 33 at giant molecular cloud scales

NASA Astrophysics Data System (ADS)

Komugi, Shinya; Miura, Rie E.; Kuno, Nario; Tosaki, Tomoka

2018-04-01

We report on a multi-parameter analysis of giant molecular clouds (GMCs) in the nearby spiral galaxy M 33. A catalog of GMCs identifed in 12CO(J = 3-2) was used to compile associated 12CO(J = 1-0), dust, stellar mass, and star formation rate. Each of the 58 GMCs are categorized by their evolutionary stage. Applying the principal component analysis on these parameters, we construct two principal components, PC1 and PC2, which retain 75% of the information from the original data set. PC1 is interpreted as expressing the total interstellar matter content, and PC2 as the total activity of star formation. Young (< 10 Myr) GMCs occupy a distinct region in the PC1-PC2 plane, with lower interstellar medium (ISM) content and star formation activity compared to intermediate-age and older clouds. Comparison of average cloud properties in different evolutionary stages imply that GMCs may be heated or grow denser and more massive via aggregation of diffuse material in their first ˜ 10 Myr. The PCA also objectively identified a set of tight relations between ISM and star formation. The ratio of the two CO lines is nearly constant, but weakly modulated by massive star formation. Dust is more strongly correlated with the star formation rate than the CO lines, supporting recent findings that dust may trace molecular gas better than CO. Stellar mass contributes weakly to the star formation rate, reminiscent of an extended form of the Schmidt-Kennicutt relation with the molecular gas term substituted by dust.

The Reverse Transcription Inhibitor Abacavir Shows Anticancer Activity in Prostate Cancer Cell Lines

PubMed Central

Molinari, Agnese; Parisi, Chiara; Bozzuto, Giuseppina; Toccacieli, Laura; Formisano, Giuseppe; De Orsi, Daniela; Paradisi, Silvia; Grober, OlÌ Maria Victoria; Ravo, Maria; Weisz, Alessandro; Arcieri, Romano; Vella, Stefano; Gaudi, Simona

2010-01-01

Background Transposable Elements (TEs) comprise nearly 45% of the entire genome and are part of sophisticated regulatory network systems that control developmental processes in normal and pathological conditions. The retroviral/retrotransposon gene machinery consists mainly of Long Interspersed Nuclear Elements (LINEs-1) and Human Endogenous Retroviruses (HERVs) that code for their own endogenous reverse transcriptase (RT). Interestingly, RT is typically expressed at high levels in cancer cells. Recent studies report that RT inhibition by non-nucleoside reverse transcriptase inhibitors (NNRTIs) induces growth arrest and cell differentiation in vitro and antagonizes growth of human tumors in animal model. In the present study we analyze the anticancer activity of Abacavir (ABC), a nucleoside reverse transcription inhibitor (NRTI), on PC3 and LNCaP prostate cancer cell lines. Principal Findings ABC significantly reduces cell growth, migration and invasion processes, considerably slows S phase progression, induces senescence and cell death in prostate cancer cells. Consistent with these observations, microarray analysis on PC3 cells shows that ABC induces specific and dose-dependent changes in gene expression, involving multiple cellular pathways. Notably, by quantitative Real-Time PCR we found that LINE-1 ORF1 and ORF2 mRNA levels were significantly up-regulated by ABC treatment. Conclusions Our results demonstrate the potential of ABC as anticancer agent able to induce antiproliferative activity and trigger senescence in prostate cancer cells. Noteworthy, we show that ABC elicits up-regulation of LINE-1 expression, suggesting the involvement of these elements in the observed cellular modifications. PMID:21151977

Quantifying the relative amounts of PrP polymorphisms present in prions isolated from heterozygous prion-infected animals

USDA-ARS?s Scientific Manuscript database

Prions cause protein misfolding diseases, such as transmissible spongiform encephalopathy. They propagate infections by converting a normal cellular prion protein into a prion (PrPSc). PrPC and PrPSc are isosequential and differ only in their respective conformations. PrPC is monomeric and sensit...

Evaluation of the immune response in Shitou geese (Anser anser domesticus) following immunization with GPV-VP1 DNA-based and live attenuated vaccines.

PubMed

Deng, Shu-xuan; Cai, Ming-sheng; Cui, Wei; Huang, Jin-lu; Li, Mei-li

2014-01-01

Goose parvovirus (GPV) is a highly contagious and deadly disease for goslings and Muscovy ducklings. To compare the differences in immune response of geese immunized with GPV-VP1 DNA-based and live attenuated vaccines. Shitou geese were immunized once with either 20 μg pcDNA-GPV-VP1 DNA gene vaccine by gene gun bombardment via intramuscular injection, or 300 μg by i.m. injection, or 300 μL live attenuated vaccine by i.m. injection, whereas 300 μg pcDNA3.1 (+) i.m. or 300 μL saline i.m. were used as positive and negative controls, respectively. Each group comprised 28 animals. Peripheral blood samples were collected from 2-210 days after immunization and the proliferation of T lymphocytes, the number of CD4(+) and CD8(+) T cells and the level of IgG assessed. Statistical analysis was performed using a one-way analysis of variance with group multiple comparisons via Tukey's test. The pcDNA-GPV-VP1 DNA and attenuated vaccine induced cellular and humoral responses, and there were no differences between the 20 and 300 μg group in the responses of proliferation of T lymphocyte and the CD8(+) T-cell. However, as to CD4(+) T-cell response and humoral immunity, the 20 μg group performed better than the 300 μg group, which induced better cellular and humoral immunity than live attenuated vaccine. This study showed that it is possible to induce both cellular and humoral response using DNA-based vaccines and that the pcDNA-GPV-VP1 DNA gene vaccine induced better cellular and humoral immunity than live attenuated vaccine.

Molecular Organization of Cholesterol in Docosahexaenoate-Rich Lipid Bilayers

NASA Astrophysics Data System (ADS)

Wassall, Stephen R.; Brzustowicz, Michael R.; Cherezov, Vadim; Caffrey, Martin; Zerouga, Mustapha; Stillwell, William

2002-03-01

We investigated the molecular organization of cholesterol in 1,2-didocosahexaenoylphosphatidylcholine (22:6-22:6PC) and 1-stearoyl-2-docosahexaenoylphosphatidylcholine (18:0-22:6PC) bilayers. The solubility of the sterol measured by wide-angle x-ray diffraction was < 20 mol% in 22:6-22:6PC bilayers which contrasts with > 50 mol% in 18:0-22:6PC. Solid state ^2H NMR of [3α-^2H_1]cholesterol suggests that the reduction in solubility correlates with a larger tilt angle α0 = 24^o for the steroid moiety in 22:6-22:6PC bilayers than in 18:0-22:6PC where α0 < 24^o. We attribute the low solubility of cholesterol in 22:6-22:6PC membranes to steric incompatibility between the rigid steroid moiety and the highly polyunsaturated docosahexaenoic acid (DHA) chain, which has the potential to promote lateral heterogeneity within neural membranes rich in DHA and has implications for receptor activation.

Extracellular matrix specific protein fingerprints measured in serum can separate pancreatic cancer patients from healthy controls

PubMed Central

2013-01-01

Background Pancreatic cancer (PC) is an aggressive disease with an urgent need for biomarkers. Hallmarks of PC include increased collagen deposition (desmoplasia) and increased matrix metalloproteinase (MMP) activity. The aim of this study was to investigate whether protein fingerprints of specific MMP-generated collagen fragments differentiate PC patients from healthy controls when measured in serum. Methods The levels of biomarkers reflecting MMP-mediated degradation of type I (C1M), type III (C3M) and type IV (C4M, C4M12a1) collagen were assessed in serum samples from PC patients (n = 15) and healthy controls (n = 33) using well-characterized and validated competitive ELISAs. Results The MMP-generated collagen fragments were significantly elevated in serum from PC patients as compared to controls. The diagnostic power of C1M, C3M, C4M and C4M12 were ≥83% (p < 0.001) and when combining all biomarkers 99% (p < 0.0001). Conclusions A panel of serum biomarkers reflecting altered MMP-mediated collagen turnover is able to differentiate PC patients from healthy controls. These markers may increase the understanding of mode of action of the disease and, if validated in larger clinical studies, provide an improved and additional tool in the PC setting. PMID:24261855

Icariin Prevents Amyloid Beta-Induced Apoptosis via the PI3K/Akt Pathway in PC-12 Cells

PubMed Central

Zhang, Dongdong; Wang, Zhe; Sheng, Chenxia; Peng, Weijun; Hui, Shan; Gong, Wei; Chen, Shuai

2015-01-01

Icariin is a prenylated flavonol glycoside derived from the Chinese herb Epimedium sagittatum that exerts a variety of pharmacological activities and shows promise in the treatment and prevention of Alzheimer's disease. In this study, we investigated the neuroprotective effects of icariin against amyloid beta protein fragment 25–35 (Aβ 25–35) induced neurotoxicity in cultured rat pheochromocytoma PC12 cells and explored potential underlying mechanisms. Our results showed that icariin dose-dependently increased cell viability and decreased Aβ 25–35-induced apoptosis, as assessed by MTT assay and Annexin V/propidium iodide staining, respectively. Results of western blot analysis revealed that the selective phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 suppressed icariin-induced Akt phosphorylation, suggesting that the protective effects of icariin are associated with activation of the PI3K/Akt signaling pathway. LY294002 also blocked the icariin-induced downregulation of proapoptotic factors Bax and caspase-3 and upregulation of antiapoptotic factor Bcl-2 in Aβ 25–35-treated PC12 cells. These findings provide further evidence for the clinical efficacy of icariin in the treatment of Alzheimer's disease. PMID:25705234

6-demethoxynobiletin, a nobiletin-analog citrus flavonoid, enhances extracellular signal-regulated kinase phosphorylation in PC12D cells.

PubMed

Kimura, Junko; Nemoto, Kiyomitsu; Yokosuka, Akihito; Mimaki, Yoshihiro; Degawa, Masakuni; Ohizumi, Yasushi

2013-01-01

We previously demonstrated that nobiletin, a polymethoxylated flavone isolated from citrus peels, has the potential to improve cognitive dysfunction in patients with Alzheimer's disease (AD). Recent studies suggest that the generation of intraneuronal amyloid-beta (Aβ) oligomers is an early event in the pathogenesis of AD. Aβ oligomers cause deficits in the regulation of the extracellular signal-regulated kinase (ERK) signaling which is critical for consolidation of the memory. Our previous studies revealed that nobiletin activated ERK signaling and subsequent cyclic AMP response element-dependent transcription. In this study, the effects of five nobiletin analogs, 6-demethoxynobiletin, tangeretin, 5-demethylnobiletin, sinensetin, and 6-demethoxytangeretin, isolated from citrus peels were assessed on ERK phosphorylation in PC12D cells, and the structure-activity relationships were examined. PC12D cells were treated with nobiletin or its analogs, and the cell extracts were analyzed by Western blotting using an antibody specific to phosphorylated ERK. 6-Demethoxynobiletin markedly enhanced ERK phosphorylation in a concentration-dependent manner. These results may be useful in developing drugs and functional foods using citrus peels for the treatment of dementia including AD.

The marijuana component cannabidiol inhibits beta-amyloid-induced tau protein hyperphosphorylation through Wnt/beta-catenin pathway rescue in PC12 cells.

PubMed

Esposito, Giuseppe; De Filippis, Daniele; Carnuccio, Rosa; Izzo, Angelo A; Iuvone, Teresa

2006-03-01

Alzheimer's disease (AD) is the most common age-related neurodegenerative disorder. A massive accumulation of beta-amyloid (Abeta) peptide aggregates has been proposed as pivotal event in AD. Abeta-induced toxicity is accompanied by a variegated combination of events including oxidative stress. The Wnt pathway has multiple actions in the cascade of events triggered by Abeta, and drugs that rescue Wnt activity may be considered as novel therapeutics for AD treatment. Cannabidiol, a non-psychoactive marijuana component, has been recently proposed as an antioxidant neuroprotective agent in neurodegenerative diseases. Moreover, it has been shown to rescue PC12 cells from toxicity induced by Abeta peptide. However, the molecular mechanism of cannabidiol-induced neuroprotective effect is still unknown. Here, we report that cannabidiol inhibits hyperphosphorylation of tau protein in Abeta-stimulated PC12 neuronal cells, which is one of the most representative hallmarks in AD. The effect of cannabidiol is mediated through the Wnt/beta-catenin pathway rescue in Abeta-stimulated PC12 cells. These results provide new molecular insight regarding the neuroprotective effect of cannabidiol and suggest its possible role in the pharmacological management of AD, especially in view of its low toxicity in humans.

Asarone from Acori Tatarinowii Rhizoma Potentiates the Nerve Growth Factor-Induced Neuronal Differentiation in Cultured PC12 Cells: A Signaling Mediated by Protein Kinase A

PubMed Central

Lam, Kelly Y. C.; Chen, Jianping; Lam, Candy T. W.; Wu, Qiyun; Yao, Ping; Dong, Tina T. X.; Lin, Huangquan; Tsim, Karl W. K.

2016-01-01

Acori Tatarinowii Rhizoma (ATR), the rhizome of Acorus tatarinowii Schott, is being used clinically to treat neurological disorders. The volatile oil of ATR is being considered as an active ingredient. Here, α-asarone and β-asarone, accounting about 95% of ATR oil, were evaluated for its function in stimulating neurogenesis. In cultured PC12 cells, application of ATR volatile oil, α-asarone or β-asarone, stimulated the expression of neurofilaments, a bio-marker for neurite outgrowth, in a concentration-dependent manner. The co-treatment of ATR volatile oil, α-asarone or β-asarone, with low concentration of nerve growth factor (NGF) potentiated the NGF-induced neuronal differentiation in cultured PC12 cells. In addition, application of protein kinase A inhibitors, H89 and KT5720, in cultures blocked the ATR-induced neurofilament expression, as well as the phosphorylation of cAMP-responsive element binding protein (CREB). In the potentiation of NGF-induced signaling in cultured PC12 cells, α-asarone and β-asarone showed synergistic effects. These results proposed the neurite-promoting asarone, or ATR volatile oil, could be useful in finding potential drugs for treating various neurodegenerative diseases, in which neurotrophin deficiency is normally involved. PMID:27685847

Insights into the Morphological Instability of Bulk Heterojunction PTB7-Th/PCBM Solar Cells upon High-Temperature Aging

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hsieh, Yen-Ju; Huang, Yu-Ching; Liu, Wei-Shin

The impact of the morphological stability of the donor/acceptor mixture under thermal stress on the photovoltaic properties of bulk heterojunction (BHJ) solar cells based on the poly[4,8-bis(5-(2-ethylhexyl)thiophen-2-yl)benzo[1,2-b;4,5-b']-di-thiophene-2,6-diyl-alt-(4-(2 ethylhexyl)-3-fluorothieno[3,4-b]-thiophene)-2-carboxylate-2,6-diyl]/phenyl-C61-butyric acid methylester (PTB7-Th/PC61BM) blend is extensively investigated. Both optical microscopy and transmission electron microscopy micrographs show that long-term high-temperature aging stimulates the formation of microscale clusters, the size of which, however, is about 1 order of magnitude smaller than those observed in thermally annealed poly(3hexylthiophene)/PC61BM composite film. The multilength-scale evolution of the morphology of PTB7-Th/PC61BM film from the scattering profiles of grazing incidence small-angle and wide-angle X-ray scattering indicates the PC61BM moleculesmore » spatially confine the self-organization of polymer chains into large domains during cast drying and upon thermal activation. Moreover, some PC61BM molecules accumulate into ~30-40 nm clusters, the number of which increases with heating time. Therefore, the hole mobility in the active layer decays much more rapidly than the electron mobility, leading to unbalanced charge transport and degraded cell performance. Importantly, the three-component blend that is formed by replacing a small amount of PC61BM in the active layer with the bis-adduct of PC61BM (bis-P61M) exhibits robust morphology against thermal stress. Accordingly, the PTB7-Th/PC61BM:bis-PC61BM (8 wt %) device has an extremely stable power conversion efficiency.« less

Effect of Ultrasonic Extraction on Production and Structural Changes of C-Phycocyanin from Marine Spirulina maxima.

PubMed

Choi, Woon Yong; Lee, Hyeon Yong

2018-01-11

This work first showed that very high amounts of phycocyanins, such as 11.3 mg/mL C-phycocyanin (C-PC), 3.1 mg/mL allophycocyanin (APC), and 0.8 mg/mL phycoerythrin (PE), can be obtained using an ultrasonic extraction process (UE) with a 60 kHz frequency and 3 h of process time at 25 °C, without any other pretreatments. These yields were higher than those from most conventional water extractions at 4 °C for 24 h (Control condition) or at 25 °C for 24 h (WE), namely, 9.8 and 5.7 mg/mL C-PC, 2.3 and 1.2 mg/mL APC, and 0.7 and 0.3 mg/mL PE, respectively. These yields were also shown to be even higher than yields from other reported data. Structural changes in C-PC in the extracts were also found for the first time, according to extraction conditions, showing that the total concentration of C-PC and of the α-subunit of C-PC in the UE were much higher than in the WE, with little difference in the amount of β-subunit of C-PC in the UE or WE. It was also shown that the structural changes in C-PC in the WE decreased both antioxidant and anti-inflammation activities-29.83% vs. 32.09% of α,α-diphenyl-β-picrylhydrazyl (DPPH) scavenging activity and 8.21 vs. 7.25 µM of NO production for the WE and UE, respectively-while the UE, with similar patterns to standard C-PC, showed very high biological effects, which may suggest that the biologically active part is the α-subunit of C-PC, not the β-subunit.

Enhanced Toxic Metal Accumulation in Engineered Bacterial Cells Expressing Arabidopsis thaliana Phytochelatin Synthase

PubMed Central

Sauge-Merle, Sandrine; Cuiné, Stéphan; Carrier, Patrick; Lecomte-Pradines, Catherine; Luu, Doan-Trung; Peltier, Gilles

2003-01-01

Phytochelatins (PCs) are metal-binding cysteine-rich peptides, enzymatically synthesized in plants and yeasts from glutathione in response to heavy metal stress by PC synthase (EC 2.3.2.15). In an attempt to increase the ability of bacterial cells to accumulate heavy metals, the Arabidopsis thaliana gene encoding PC synthase (AtPCS) was expressed in Escherichia coli. A marked accumulation of PCs was observed in vivo together with a decrease in the glutathione cellular content. When bacterial cells expressing AtPCS were placed in the presence of heavy metals such as cadmium or the metalloid arsenic, cellular metal contents were increased 20- and 50-fold, respectively. We discuss the possibility of using genes of the PC biosynthetic pathway to design bacterial strains or higher plants with increased abilities to accumulate toxic metals, and also arsenic, for use in bioremediation and/or phytoremediation processes. PMID:12514032

Electrospun nanofibers: Formation, characterization, and evaluation for nerve tissue engineering applications

NASA Astrophysics Data System (ADS)

Zander, Nicole E.

The effects of fiber alignment and surface chemistry, including the covalent attachment and physical adsorption of the extracellular matrix (ECM) proteins laminin and collagen, on the neurite outgrowth of neuron-like PC12 cells were examined. Neuron-like PC12 cells responded to fiber orientation, and were successfully contact-guided by aligned electrospun nanofibers. In addition, fibers with attached protein, either physically adsorbed or covalently attached, improved neurite outgrowth lengths. Furthermore, aligning the fibers and attaching the ECM protein laminin, in particular, significantly improved neurite outgrowth over randomly oriented fibers with laminin. Since this research suggested that protein concentration on the fibers was the dominant driving force for improved neurite outgrowth, the effect of protein concentration, incorporated onto the surface of the nanofibers, on neurite outgrowth was examined. Two ways to control protein concentration on the fibers were explored—the variation of the fiber-protein reaction time and the variation of the protein soaking solution concentration. In addition, analytical methods to quantify the concentration of protein, as well as the protein coverage, on the surface of the fibers were developed. Although most of the fiber mats had multilayer protein coverage, and hence physically adsorbed proteins which could potentially mean a loss in bioactivity, the neuron-like PC12 cell neurites responded in a dose-dependent manner with increased neurite lengths on scaffolds with higher protein concentrations. The work was extended further by forming protein gradients on the fiber mats in hopes of locally directing neurite outgrowth and orientation. Fiber mats with both linear gradients (continuous change in protein concentration) and step gradients (six regions of uniform protein coverage, with protein concentration increasing from region to region) were fabricated and analyzed. The step gradients formed in the aligned fiber direction showed the most promise for use in cell culture assays. While surface chemistry and topography are important, porosity of the scaffold is also critical to control cellular infiltration and tissue formation. To enhance the porosity of our electrospun nanofiber scaffolds and improve the infiltration of cells, two methods were explored to control porosity. In the first method, the scaffold polymer polycaprolactone was co-electrospun with a sacrificial polymer polyethylene oxide, which was removed after the bi-component fiber mat was formed. In doing so, the void space was increased. In the second method, the spinning solution concentration of polycaprolactone was varied to control fiber diameter and porosity. The second method proved to be more effective at improving the cellular infiltration of PC12 cells. Two orders of magnitude range of fiber diameters were achieved, and nearly full infiltration of PC12 cells was observed for the mats with the highest porosity. The pore sizes of these mats were on the order of the size of the cell bodies (approximately 6-10 µm). Although the majority of this work focuses on using conventional electrospinning to generate solid-core fibers, core-shell fibers, have many applications in tissue engineering, among other fields. We explored an efficient way to generate these fibers from an emulsion solution using a conventional electrospinning apparatus. We characterized the fibers using an atomic force microscope (AFM) elastic modulus mapping technique, along with AFM phase imaging, angle-resolved x-ray photoelectron spectroscopy and thermal gravimetric analysis, to determine the chemical and molar composition of the core and shell layers. This work presents novel analytical techniques for the characterization of core-shell nanofibers in order to better predict and understand their material properties. (Abstract shortened by UMI.).

Expression of peroxisome proliferator-activated receptor (PPAR) in human prostate cancer.

PubMed

Segawa, Yoshihiro; Yoshimura, Rikio; Hase, Taro; Nakatani, Tatsuya; Wada, Seiji; Kawahito, Yutaka; Kishimoto, Taketoshi; Sano, Hajime

2002-05-01

Recent studies have demonstrated that peroxisome proliferator activator-receptors (PPAR)-gamma is expressed in some cancer cells such as breast, lung, and gastric cancer, and its ligand induces growth arrest of these cancer cells through apoptosis. However, the expression and localization of PPARs in prostate have not been examined. In this study, PPARs expression was investigated in human prostate cancer (PC), prostatic intraepithelial neoplasia (PIN), benign prostatic hyperplasia (BPH), and normal prostate (NP) tissues. Tumor specimens were obtained from 156 patients with PC, 15 with PIN, 20 with BPH, and 12 patients with NP tissues. The expressions were investigated by RT-PCR and immunohistochemical methods. Immunoreactive PPAR-alpha and -beta were significantly apparent in PC tissues. Marked expressions of PPAR-alpha and -beta were also detected in PIN, BPH, and NP groups. However, very weak or no expression of immunoreactive PPAR-gamma was found in BPH and NP cases. In contrast, we found significant expression of immunoreactive PPAR-gamma in cancer cells in PC group and in PIN group. Our results demonstrated that PPAR-gamma is induced in PC, and suggest that PPAR-gamma ligands may mediate its own potent antiproliferative effect against PC cells through differentiation. Copyright 2002 Wiley-Liss, Inc.

Elevated hydrostatic pressures induce apoptosis and oxidative stress through mitochondrial membrane depolarization in PC12 neuronal cells: A cell culture model of glaucoma.

PubMed

Tök, Levent; Nazıroğlu, Mustafa; Uğuz, Abdülhadi Cihangir; Tök, Ozlem

2014-10-01

Despite the importance of oxidative stress and apoptosis through mitochondrial depolarization in neurodegenerative diseases, their roles in etiology of glaucoma are poorly understood. We aimed to investigate whether oxidative stress and apoptosis formation are altered in rat pheochromocytoma-derived cell line-12 (PC12) neuronal cell cultures exposed to elevated different hydrostatic pressures as a cell culture model of glaucoma. Cultured PC12 cells were subjected to 0, 15 and 70 mmHg hydrostatic pressure for 1 and 24 h. Then, the following values were analyzed: (a) cell viability; (b) lipid peroxidation and intracellular reactive oxygen species production; (c) mitochondrial membrane depolarization; (d) cell apoptosis; (e) caspase-3 and caspase-9 activities; (f) reduced glutathione (GSH) and glutathione peroxidase (GSH-Px). The hydrostatic pressures (15 and 70 mmHg) increased oxidative cell damage through a decrease of GSH and GSH-Px values, and increasing mitochondrial membrane potential. Additionally, 70 mmHg hydrostatic pressure for 24 h indicated highest apoptotic effects, as demonstrated by plate reader analyses of apoptosis, caspase-3 and -9 values. The present data indicated oxidative stress, apoptosis and mitochondrial changes in PC12 cell line during different hydrostatic pressure as a cell culture model of glaucoma. This findings support the view that mitochondrial oxidative injury contributes early to glaucomatous optic neuropathy.

Differential inhibitory effects of 2-azafluorenones on PI-PLC activation but not on PC-PLC- or PC-PLD-activation induced by histamine, PAF, PMA or A23187 in C6 glioma cells.

PubMed

Wang, Hai-Long; Wang, Li-Chuan; Wei, Jiann-Wu

2013-02-28

In this study, C6 glioma cells were used to test the effects of 2-azafluorenone and its related compounds on membrane phosphatidylinositol (PI) and phosphatidylcholine (PC) turnover. An increase of [³H]-labeled inositol phosphate (IP1) formation by histamine (100 μM) or A23187 (100 nM) via the activation of phosphatidylinositol-specific phospholipase C (PI-PLC) to breakdown labeled substrate was observed, and this effect could be partially blocked by about half at 100 μM of 2-azafluorenones. Histamine induced the increase of IP1 formation, but failed to cause an increase in extracellularly releasing of [3H]choline metabolites, or intracellular accumulation of [³H]phosphscholine. However, platelet activation factor (PAF) from 0.2 to 1 μM, and phorbol 12-myristate-13-acetate (PMA) at 1 μM caused an increase in extracellularly releasing of [³H]choline metabolites, and intracellular accumulation of [³H]phosphocholine via the activation on phosphatidylcholine (PC)-PLC. These responses of PAF and PMA were not affected by 2-azafluorenone or 4-methyl-2-azafluorenone even at high concentration (10⁻⁴ M). A23187 induced an increase of intracellular [³H]choline release via the activation of PCphospholipase D (PLD). This increasing effect of 100 nM A23187 was not affected by 2-azafluorenone or 4-methyl-2-azafluorenone even at a high concentration of 10⁻⁴ M. In summary, the inhibitory effect of 2-azafluorenone and its related compound 4-methyl-2-azafluorenone was observed selectively on PIPLC, but not on PC-PLC or PC-PLD based on changes of products after the activation of these enzymes.

Model of Oxygen and Glucose Deprivation in PC12 Cells and Detection of HSP70 Protein

NASA Astrophysics Data System (ADS)

He, Jinting; Yang, Le; Shao, Yankun

2018-01-01

Objective: PC12 cell was used to set up a ischemia model by OGD and detected HSP70 protein. Methods: Use of PC12 cells induced by NGF stimulation into nerve cells, oxygen and glucose deprivation to build the nerve cells of oxygen and glucose deprivation model; using Western blot analysis of PC12 cells into neuron-like cells and oxygen-glucose deprivation model established. Results: The application of a final concentration of 50 ng / ml of NGF in DMEM complete mediumPC12 cells showed a typical neuronal morphology with the increase in cell culture time. NGF culture time showed a positive correlation, the establishment of oxygen and glucose deprivation (OGD) training environment, the OGD after nerve element appears different degrees of damage, OGD can effectively induce the expression of HSP70. Conclusion: PC12 cell transformed into cells by NGF; the cell model of OGD was established.

Dietary uridine-5'-monophosphate supplementation increases potassium-evoked dopamine release and promotes neurite outgrowth in aged rats.

PubMed

Wang, Lei; Pooler, Amy M; Albrecht, Meredith A; Wurtman, Richard J

2005-01-01

Membrane phospholipids like phosphatidylcholine (PC) are required for cellular growth and repair, and specifically for synaptic function. PC synthesis is controlled by cellular levels of its precursor, cytidine-5'-diphosphate choline (CDP-choline), which is produced from cytidine triphosphate (CTP) and phosphocholine. In rat PC12 cells exogenous uridine was shown to elevate intracellular CDP-choline levels, by promoting the synthesis of uridine triphosphate (UTP), which was partly converted to CTP. In such cells uridine also enhanced the neurite outgrowth produced by nerve growth factor (NGF). The present study assessed the effect of dietary supplementation with uridine-5'-monophosphate disodium (UMP-2Na+, an additive in infant milk formulas) on striatal dopamine (DA) release in aged rats. Male Fischer 344 rats consumed either a control diet or one fortified with 2.5% UMP for 6 wk, ad libitum. In vivo microdialysis was then used to measure spontaneous and potassium (K+)-evoked DA release in the right striatum. Potassium (K+)-evoked DA release was significantly greater among UMP-treated rats, i.e., 341+/-21% of basal levels vs. 283+/-9% of basal levels in control rats (p<0.05); basal DA release was unchanged. In general, each animal's K+-evoked DA release correlated with its striatal DA content, measured postmortem. The levels of neurofilament-70 and neurofilament-M proteins, biomarkers of neurite outgrowth, increased to 182+/-25% (p<0.05) and 221+/-34% (p<0.01) of control values, respectively, with UMP consumption. Hence, UMP treatment not only enhances membrane phosphatide production but also can modulate two membrane-dependent processes, neurotransmitter release and neurite outgrowth, in vivo.

Osteopontin splice variants expression is involved on docetaxel resistance in PC3 prostate cancer cells.

PubMed

Nakamura, K D M; Tilli, T M; Wanderley, J L; Palumbo, A; Mattos, R M; Ferreira, A C; Klumb, C E; Nasciutti, L E; Gimba, E R

2016-02-01

Osteopontin (OPN) is a phosphoprotein that activates several aspects of tumor progression. Alternative splicing of the OPN primary transcript generates three splicing isoforms, OPNa, OPNb and OPNc. In this report, we investigated some cellular mechanisms by which OPN splice variants could mediate PC3 prostate cancer (PCa) cell survival and growth in response to docetaxel (DXT)-induced cell death. Cell survival before and after DXT treatment was analyzed by phase-contrast microscopy and crystal-violet staining assays. Quantitative real-time PCR and immunocytochemical staining assays were used to evaluate the putative involvement of epithelial-mesenchymal transition (EMT) and OPN isoforms on mediating PC3 cell survival. Upon DXT treatment, PC3 cells overexpressing OPNb or OPNc isoforms showed higher cell densities, compared to cells overexpressing OPNa and controls. Notably, cells overexpressing OPNb or OPNc isoforms showed a downregulated pattern of EMT epithelial cell markers, while mesenchymal markers were mostly upregulated in these experimental conditions. We concluded that OPNc or OPNb overexpression in PC3 cells can mediate resistance and cell survival features in response to DXT-induced cell death. Our data also provide evidence the EMT program could be one of the molecular mechanisms mediating survival in OPNb- or OPNc-overexpressing cells in response to DXT treatment. These data could further contribute to a better understanding of the mechanisms by which PCa cells acquire resistance to DXT treatment.

Diospyrin derivative, an anticancer quinonoid, regulates apoptosis at endoplasmic reticulum as well as mitochondria by modulating cytosolic calcium in human breast carcinoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kumar, Binod; Radiation and Cancer Biology Section, Radiation Biology and Health Sciences Division, Bhabha Atomic Research Centre, Mumbai 400085; Kumar, Amit

2012-01-13

Highlights: Black-Right-Pointing-Pointer Diospyrin diethylether (D7) caused oxidative stress-dependent activation of PC-PLC. Black-Right-Pointing-Pointer Activated PC-PLC induced a sustained-release of Ca{sup 2+} from endoplasmic reticulum. Black-Right-Pointing-Pointer The elevated cytosolic Ca{sup +2} led to the calpain-caspase12 dependent apoptosis. Black-Right-Pointing-Pointer D7-Induced Ca{sup +2} also found to accentuate the mitochondrial pathway of apoptosis. -- Abstract: Diospyrin diethylether (D7), a bisnaphthoquinonoid derivative, exhibited an oxidative stress-dependent apoptosis in several human cancer cells and tumor models. The present study was aimed at evaluation of the increase in cytosolic calcium [Ca{sup 2+}]{sub c} leading to the apoptotic cell death triggered by D7 in MCF7 human breast carcinoma cells.more » A phosphotidylcholine-specific phospholipase C (PC-PLC) inhibitor, viz. U73122, and an antioxidant, viz. N-acetylcysteine, could significantly prevent the D7-induced rise in [Ca{sup 2+}]{sub c} and PC-PLC activity. Using an endoplasmic reticulum (ER)-Ca{sup 2+} mobilizer (thapsigargin) and an ER-IP3R antagonist (heparin), results revealed ER as a major source of [Ca{sup 2+}]{sub c} which led to the activation of calpain and caspase12, and cleavage of fodrin. These effects including apoptosis were significantly inhibited by the pretreatment of Bapta-AM (a cell permeable Ca{sup 2+}-specific chelator), or calpeptin (a calpain inhibitor). Furthermore, D7-induced [Ca{sup 2+}]{sub c} was found to alter mitochondrial membrane potential and induce cytochrome c release, which was inhibited by either Bapta-AM or ruthenium red (an inhibitor of mitochondrial Ca{sup 2+} uniporter). Thus, these results provided a deeper insight into the D7-induced redox signaling which eventually integrated the calcium-dependent calpain/caspase12 activation and mitochondrial alterations to accentuate the induction of apoptotic cell death.« less

Novel Membrane-Based Electrochemical Sensor for Real-Time Bio-Applications

PubMed Central

Alatraktchi, Fatima AlZahra'a; Bakmand, Tanya; Dimaki, Maria; Svendsen, Winnie E.

2014-01-01

This article presents a novel membrane-based sensor for real-time electrochemical investigations of cellular- or tissue cultures. The membrane sensor enables recording of electrical signals from a cell culture without any signal dilution, thus avoiding loss of sensitivity. Moreover, the porosity of the membrane provides optimal culturing conditions similar to existing culturing techniques allowing more efficient nutrient uptake and molecule release. The patterned sensor electrodes were fabricated on a porous membrane by electron-beam evaporation. The electrochemical performance of the membrane electrodes was characterized by cyclic voltammetry and chronoamperometry, and the detection of synthetic dopamine was demonstrated down to a concentration of 3.1 pM. Furthermore, to present the membrane-sensor functionality the dopamine release from cultured PC12 cells was successfully measured. The PC12 cells culturing experiments showed that the membrane-sensor was suitable as a cell culturing substrate for bio-applications. Real-time measurements of dopamine exocytosis in cell cultures were performed, where the transmitter release was recorded at the point of release. The developed membrane-sensor provides a new functionality to the standard culturing methods, enabling sensitive continuous in vitro monitoring and closely mimicking the in vivo conditions. PMID:25421738

Inhibition of prohormone convertase 1 (PC1) expression in cholecystokinin (CCK) expressing At-T20 cells decreased cellular content and secretion of CCK and caused a shift in molecular forms of CCK secreted.

PubMed

Beinfeld, Margery C; Vishnuvardhan, Daesety; Blum, Alissa; Reynolds, Nicole; Fannous, Sanya; Kitagawa, Kouki; Marchand, James E

2006-04-01

Two different RNAi methods were used to inhibit the expression of prohormone convertase 1 (PC1) in At-T20 cells. Transient transfection of double stranded RNA and stable expression of a vector expressing hairpin-loop RNA targeting PC1 reduced cholecystokinin (CCK) secretion from At-T20 cells. PC1 mRNA and protein were also decreased in the vector transfected cells. This treatment caused a shift in the forms of cholecystokinin (CCK) secreted, decreasing CCK 22 and increasing CCK 8. Stable expression of RNAi effectively decreased PC1 expression. The observed decrease in CCK seen with these RNAi treatments further supports a role for PC1 in CCK processing in these cells.

Screening of phospholipase A activity and its production by new actinomycete strains cultivated by solid-state fermentation.

PubMed

Sutto-Ortiz, Priscila; Camacho-Ruiz, María de Los Angeles; Kirchmayr, Manuel R; Camacho-Ruiz, Rosa María; Mateos-Díaz, Juan Carlos; Noiriel, Alexandre; Carrière, Frédéric; Abousalham, Abdelkarim; Rodríguez, Jorge A

2017-01-01

Novel microbial phospholipases A (PLAs) can be found in actinomycetes which have been poorly explored as producers of this activity. To investigate microbial PLA production, efficient methods are necessary such as high-throughput screening (HTS) assays for direct search of PLAs in microbial cultures and cultivation conditions to promote this activity. About 200 strains isolated with selected media for actinomycetes and mostly belonging to Streptomyces (73%) and Micromonospora (10%) genus were first screened on agar-plates containing the fluorophore rhodamine 6G and egg yolk phosphatidylcholine (PC) to detect strains producing phospholipase activity. Then, a colorimetric HTS assay for general PLA activity detection (cHTS-PLA) using enriched PC (≈60%) as substrate and cresol red as indicator was developed and applied; this cHTS-PLA assay was validated with known PLAs. For the first time, actinomycete strains were cultivated by solid-state fermentation (SSF) using PC as inductor and sugar-cane bagasse as support to produce high PLA activity (from 207 to 2,591 mU/g of support). Phospholipase activity of the enzymatic extracts from SSF was determined using the implemented cHTS-PLA assay and the PC hydrolysis products obtained, were analyzed by TLC showing the presence of lyso-PC. Three actinomycete strains of the Streptomyces genus that stood out for high accumulation of lyso-PC, were selected and analyzed with the specific substrate 1,2-α-eleostearoyl- sn -glycero-3-phosphocholine (EEPC) in order to confirm the presence of PLA activity in their enzymatic extracts. Overall, the results obtained pave the way toward the HTS of PLA activity in crude microbial enzymatic extracts at a larger scale. The cHTS-PLA assay developed here can be also proposed as a routine assay for PLA activity determination during enzyme purification,directed evolution or mutagenesis approaches. In addition, the production of PLA activity by actinomycetes using SSF allow find and produce novel PLAs with potential applications in biotechnology.

Screening of phospholipase A activity and its production by new actinomycete strains cultivated by solid-state fermentation

PubMed Central

Sutto-Ortiz, Priscila; Camacho-Ruiz, María de los Angeles; Kirchmayr, Manuel R.; Camacho-Ruiz, Rosa María; Mateos-Díaz, Juan Carlos; Noiriel, Alexandre; Carrière, Frédéric; Abousalham, Abdelkarim

2017-01-01

Novel microbial phospholipases A (PLAs) can be found in actinomycetes which have been poorly explored as producers of this activity. To investigate microbial PLA production, efficient methods are necessary such as high-throughput screening (HTS) assays for direct search of PLAs in microbial cultures and cultivation conditions to promote this activity. About 200 strains isolated with selected media for actinomycetes and mostly belonging to Streptomyces (73%) and Micromonospora (10%) genus were first screened on agar-plates containing the fluorophore rhodamine 6G and egg yolk phosphatidylcholine (PC) to detect strains producing phospholipase activity. Then, a colorimetric HTS assay for general PLA activity detection (cHTS-PLA) using enriched PC (≈60%) as substrate and cresol red as indicator was developed and applied; this cHTS-PLA assay was validated with known PLAs. For the first time, actinomycete strains were cultivated by solid-state fermentation (SSF) using PC as inductor and sugar-cane bagasse as support to produce high PLA activity (from 207 to 2,591 mU/g of support). Phospholipase activity of the enzymatic extracts from SSF was determined using the implemented cHTS-PLA assay and the PC hydrolysis products obtained, were analyzed by TLC showing the presence of lyso-PC. Three actinomycete strains of the Streptomyces genus that stood out for high accumulation of lyso-PC, were selected and analyzed with the specific substrate 1,2-α-eleostearoyl-sn-glycero-3-phosphocholine (EEPC) in order to confirm the presence of PLA activity in their enzymatic extracts. Overall, the results obtained pave the way toward the HTS of PLA activity in crude microbial enzymatic extracts at a larger scale. The cHTS-PLA assay developed here can be also proposed as a routine assay for PLA activity determination during enzyme purification,directed evolution or mutagenesis approaches. In addition, the production of PLA activity by actinomycetes using SSF allow find and produce novel PLAs with potential applications in biotechnology. PMID:28695068

GEITLERINEMA SPECIES (OSCILLATORIALES, CYANOBACTERIA) REVEALED BY CELLULAR MORPHOLOGY, ULTRASTRUCTURE, AND DNA SEQUENCING(1).

PubMed

Do Carmo Bittencourt-Oliveira, Maria; Do Nascimento Moura, Ariadne; De Oliveira, Mariana Cabral; Sidnei Massola, Nelson

2009-06-01

Geitlerinema amphibium (C. Agardh ex Gomont) Anagn. and G. unigranulatum (Rama N. Singh) Komárek et M. T. P. Azevedo are morphologically close species with characteristics frequently overlapping. Ten strains of Geitlerinema (six of G. amphibium and four of G. unigranulatum) were analyzed by DNA sequencing and transmission electronic and optical microscopy. Among the investigated strains, the two species were not separated with respect to cellular dimensions, and cellular width was the most varying characteristic. The number and localization of granules, as well as other ultrastructural characteristics, did not provide a means to discriminate between the two species. The two species were not separated either by geography or environment. These results were further corroborated by the analysis of the cpcB-cpcA intergenic spacer (PC-IGS) sequences. Given the fact that morphology is very uniform, plus the coexistence of these populations in the same habitat, it would be nearly impossible to distinguish between them in nature. On the other hand, two of the analyzed strains were distinct from all others based on the PC-IGS sequences, in spite of their morphological similarity. PC-IGS sequences indicate that these two strains could be a different species of Geitlerinema. Using morphology, cell ultrastructure, and PC-IGS sequences, it is not possible to distinguish G. amphibium and G. unigranulatum. Therefore, they should be treated as one species, G. unigranulatum as a synonym of G. amphibium. © 2009 Phycological Society of America.

Comparison of the immune responses in BALB/c mice following immunization with DNA-based and live attenuated vaccines delivered via different routes.

PubMed

Cai, Ming-sheng; Deng, Shu-xuan; Li, Mei-li

2013-02-18

The objective of this study was to compare immune responses induced in BALB/c mice following immunization with pcDNA-GPV-VP2 DNA by gene gun bombardment (6 μg) or by intramuscular (im) injection (100 μg) with the responses to live attenuated vaccine by im injection (100 μl). pcDNA3.1 (+) and physiological saline were used as controls. Peripheral blood samples were collected at 3, 7, 14, 21, 28, 35, 49, 63, 77 and 105 d after immunization. T lymphocyte proliferation was analyzed by MTT assay and enumeration of CD4(+), and CD8(+) T cell populations in peripheral blood was performed by flow cytometric analysis. Indirect ELISA was used to detect IgG levels. Cellular and humoral responses were induced by pcDNA-GPV-VP2 DNA and live virus vaccines. No differences were observed in T cell proliferation and CD8(+) T cell responses induced by the genetic vaccine regardless of the route of delivery. However, CD4(+) T cell responses and humoral immunity were enhanced in following gene gun immunization compared with im injection of the genetic vaccine. Cellular and humoral immunity was enhanced in following gene gun delivery of the genetic vaccine compared with the live attenuated vaccine. In conclusion, the pcDNA-GPV-VP2 DNA vaccine induced enhanced cellular and humoral immunity compared with that induced by the live attenuated vaccine. Copyright © 2012 Elsevier Ltd. All rights reserved.

Correlation and the mechanism of lithium ion diffusion with the crystal structure of Li7La3Zr2O12 revealed by an internal friction technique.

PubMed

Wang, X P; Gao, Y X; Xia, Y P; Zhuang, Z; Zhang, T; Fang, Q F

2014-04-21

The correlation and transport mechanism of lithium ions with the crystal structure of a fast lithium ion conductor Li7La3Zr2O12 are mainly investigated by internal friction (IF) and AC impedance spectroscopy techniques. Compared with the poor conductivity of tetragonal Li7La3Zr2O12, the Al stabilized cubic phase exhibits a good ionic conductivity that can be up to 1.9 × 10(-4) S cm(-1) at room temperature, which can be ascribed to the disordered distribution of lithium ions in the cubic phase. A well-pronounced relaxation IF peak (labeled as peak PC) is observed in the cubic phase while a very weak IF peak (labeled as PT) is observed in the tetragonal phase, further evidencing the difference in lithium ion migration in the two phases. Peak PC can be decomposed into two sub-peaks with the activation energy and the pre-exponential factor of relaxation time being E1 = 0.41 eV and τ01 = 1.2 × 10(-14) s for the lower temperature peak PC1 and E2 = 0.35 eV and τ02 = 1.9 × 10(-15) s for the higher temperature PC2 peak, respectively. Based on the crystalline structure of a cubic garnet-type Li7La3Zr2O12 compound, an atomistic mechanism of lithium ion diffusion via vacancies is suggested, i.e. 48g(96h) ↔ 48g(96h) for peak PC1 and 48g(96h) ↔ 24d for peak PC2, respectively. The weak PT peak in the tetragonal phase is preliminarily interpreted as due to the short jump process among neighboring octahedral sites and vacant tetrahedral sites.

The mouse liver displays daily rhythms in the metabolism of phospholipids and in the activity of lipid synthesizing enzymes.

PubMed

Gorné, Lucas D; Acosta-Rodríguez, Victoria A; Pasquaré, Susana J; Salvador, Gabriela A; Giusto, Norma M; Guido, Mario Eduardo

2015-02-01

The circadian system involves central and peripheral oscillators regulating temporally biochemical processes including lipid metabolism; their disruption leads to severe metabolic diseases (obesity, diabetes, etc). Here, we investigated the temporal regulation of glycerophospholipid (GPL) synthesis in mouse liver, a well-known peripheral oscillator. Mice were synchronized to a 12:12 h light-dark (LD) cycle and then released to constant darkness with food ad libitum. Livers collected at different times exhibited a daily rhythmicity in some individual GPL content with highest levels during the subjective day. The activity of GPL-synthesizing/remodeling enzymes: phosphatidate phosphohydrolase 1 (PAP-1/lipin) and lysophospholipid acyltransferases (LPLATs) also displayed significant variations, with higher levels during the subjective day and at dusk. We evaluated the temporal regulation of expression and activity of phosphatidylcholine (PC) synthesizing enzymes. PC is mainly synthesized through the Kennedy pathway with Choline Kinase (ChoK) as a key regulatory enzyme or through the phosphatidylethanolamine (PE) N-methyltransferase (PEMT) pathway. The PC/PE content ratio exhibited a daily variation with lowest levels at night, while ChoKα and PEMT mRNA expression displayed maximal levels at nocturnal phases. Our results demonstrate that mouse liver GPL metabolism oscillates rhythmically with a precise temporal control in the expression and/or activity of specific enzymes.

The Atypical Antipsychotic Agent, Clozapine, Protects Against Corticosterone-Induced Death of PC12 Cells by Regulating the Akt/FoxO3a Signaling Pathway.

PubMed

Zeng, Zhiwen; Wang, Xue; Bhardwaj, Sanjeev K; Zhou, Xuanhe; Little, Peter J; Quirion, Remi; Srivastava, Lalit K; Zheng, Wenhua

2017-07-01

Schizophrenia is one of the most severe psychiatric disorders. Increasing evidence implicates that neurodegeneration is a component of schizophrenia pathology and some atypical antipsychotics are neuroprotective and successful in slowing the progressive morphological brain changes. As an antipsychotic agent, clozapine has superior and unique effects, but the intracellular signaling pathways that mediate clozapine action remain to be elucidated. The phosphatidylinositol-3-kinase/protein kinase B/Forkhead box O3 (PI3K/Akt/FoxO3a) pathway is crucial for neuronal survival. However, little information is available regarding this pathway with clozapine. In the present study, we investigated the protective effect of clozapine on the PC12 cells against corticosterone toxicity. Our results showed that corticosterone decreases the phosphorylation of Akt and FoxO3a, leading to the nuclear localization of FoxO3a and the apoptosis of PC12 cells, while clozapine concentration dependently protected PC12 cells against corticosterone insult. Pathway inhibitors studies displayed that the protective effect of clozapine was reversed by LY294002 and wortmannin, two PI3K inhibitors, or Akt inhibitor VIII although several other inhibitors had no effect. The shRNA knockdown results displayed that downregulated Akt1 or FoxO3a attenuated the protective effect of clozapine. Western blot analyses revealed that clozapine induced the phosphorylation of Akt and FoxO3a by the PI3K/Akt pathway and reversed the reduction of the phosphorylated Akt and FoxO3a and the nuclear translocation of FoxO3a evoked by corticosterone. Together, our data indicates that clozapine protects PC12 cells against corticosterone-induced cell death by modulating activity of the PI3K/Akt/FoxO3a pathway.

Evidences that beta1 integrin and Rac1 are involved in the overriding effect of laminin on myelin-associated glycoprotein inhibitory activity on neuronal cells.

PubMed

Laforest, Sullivan; Milanini, Julie; Parat, Fabrice; Thimonier, Jean; Lehmann, Maxime

2005-11-01

During neurite elongation, migrating growth cones encounter both permissive and inhibitory substrates, such as laminin and MAG (myelin-associated glycoprotein), respectively. Here, we demonstrated on two neuronal cell lines (PC12 and N1E-115), that laminin and collagen hampered, in a dose-dependent manner, MAG inhibitory activity on several integrin functions, i.e., neurite growth, cell adhesion and cell spreading. Using a function blocking antibody, in PC12 cells, we showed that alpha1beta1 integrin is required in these phenomena. In parallel, we observed that MAG perturbs actin dynamics and lamellipodia formation during early steps of cell spreading. This seemed to be independent of RhoA activation, but dependent of Rac-1 inhibition by MAG. Laminin overrode MAG activity on actin and prevented MAG inhibition NGF-induced Rac1 activation. In conclusion, we evidenced antagonistic signaling between MAG receptors and beta1 integrins, in which Rac-1 may have a central function.

Synthetic Chalcones with Potent Antioxidant Ability on H2O2-Induced Apoptosis in PC12 Cells

PubMed Central

Wu, Jian-Zhang; Cheng, Chan-Chan; Shen, Lai-Lai; Wang, Zhan-Kun; Wu, Shou-Biao; Li, Wu-Lan; Chen, Su-Hua; Zhou, Rong-Ping; Qiu, Pei-Hong

2014-01-01

Chalcone derivatives (E)-3-(4-hydroxy-3-methoxyphenyl)-1-(4-methoxyphenyl) prop-2-en-1-one and (E)-3-(4-hydroxyphenyl)-1-(4-methoxyphenyl) prop-2-en-1-one (Compounds 1 and 2) have been demonstrated to be potent anti-inflammatory agents in our previous study. In light of the relationship of intracellular mechanisms between anti-inflammatories and antioxidants, we further designed and synthesized a series of chalcone derivatives based on 1 and 2, to explore their antioxidant efficacy. The majority of the derivatives exhibited strong protective effects on PC12 (PC12 rat pheochromocytoma) cells exposed to H2O2, and all compounds were nontoxic. A preliminary structure-activity relationship was proposed. Compounds 1 and 1d ((E)-2-methoxy-4-(3-(4-methoxyphenyl)-3-oxoprop-1-en-1-yl) phenyl acrylate) exerted the action in a good dose-dependent manner. Quantitative RT-PCR (qRT-PCR) and western blot analysis showed that 1 and 1d significantly improve the expression of nuclear factor erythroid 2 p45-related factor 2 (Nrf2)-dependent antioxidant genes g-Glutamylcysteine Ligase Catalytic Subunit (GCLC) and heme oxygenase-1 (HO-1) and their corresponding proteins (γ-glutamyl cysteine synthase (γ-GCS) and HO-1) in PC12 cells. Inhibition of GCLC and HO-1 by specific inhibitors, l-buthionine-S-sulfoximine (BSO) and zinc protoporphyrin (ZnPP), respectively, partially reduce the protective effect of 1 and 1d. These data present a series of novel chalcone analogs, especially compounds 1 and 1d, as candidates for treating oxidative stress-related disease by activating the Nrf2-antioxidant responsive element (ARE) pathway. PMID:25318055

Shikonin protects dopaminergic cell line PC12 against 6-hydroxydopamine-mediated neurotoxicity via both glutathione-dependent and independent pathways and by inhibiting apoptosis.

PubMed

Esmaeilzadeh, Emran; Gardaneh, Mossa; Gharib, Ehsan; Sabouni, Farzaneh

2013-08-01

We have investigated the mechanism of shikonin function on protection of dopaminergic neurons against 6-OHDA-induced neurotoxicity. Treatment of rat pheochromocytoma cell line PC12 by serial dilutions of shikonin determined 10 μM of the compound as its optimum concentration for protection saving nearly 70 % of the cells against toxicity. Reverse transcription-PCR analysis of shikonin-treated cells showed threefold increase in mRNA levels of glutathione peroxidase-1 (GPX-1) as a representative component of the intracellular anti-oxidant defense system. To elucidate shikonin-GPX1 relationships and maximize protection, we transduced PC12 cells using recombinant lentivirus vectors that harbored GPX-1 coding sequence. This change upregulated GPX-1 expression, increased peroxidase activity and made neuronal cells resistant to 6-OHDA-mediated toxicity. More importantly, addition of shikonin to GPX1-overexpressing PC12 cells augmented GPX-1 protein content by eightfold leading to fivefold increase of enzymatic activity, 91 % cell survival against neurotoxicity and concomitant increases in intracellular glutathione (GSH) levels. Depletion of intracellular GSH rendered all cell groups highly susceptible to toxicity; however, shikonin was capable of partially saving them. Subsequently, GSH-independent superoxide dismutase mRNA was found upregulated by shikonin. As signs of apoptosis inhibition, the compound upregulated Bcl-2, downregulated Bax, and prevented cell nuclei from undergoing morphological changes typical of apoptosis. Also, a co-staining method demonstrated GPX-1 overexpression significantly increases the percent of live cells that is maximized by shikonin treatment. Our data indicate that shikonin as an antioxidant compound protects dopaminergic neurons against 6-OHDA toxicity and enhances their survival via both glutathione-dependent and direct anti-apoptotic pathways.

Surface topography and chemistry shape cellular behavior on wide band-gap semiconductors.

PubMed

Bain, Lauren E; Collazo, Ramon; Hsu, Shu-Han; Latham, Nicole Pfiester; Manfra, Michael J; Ivanisevic, Albena

2014-06-01

The chemical stability and electrical properties of gallium nitride make it a promising material for the development of biocompatible electronics, a range of devices including biosensors as well as interfaces for probing and controlling cellular growth and signaling. To improve the interface formed between the probe material and the cell or biosystem, surface topography and chemistry can be applied to modify the ways in which the device interacts with its environment. PC12 cells are cultured on as-grown planar, unidirectionally polished, etched nanoporous and nanowire GaN surfaces with and without a physisorbed peptide sequence that promotes cell adhesion. While cells demonstrate preferential adhesion to roughened surfaces over as-grown flat surfaces, the topography of that roughness also influences the morphology of cellular adhesion and differentiation in neurotypic cells. Addition of the peptide sequence generally contributes further to cellular adhesion and promotes development of stereotypic long, thin neurite outgrowths over alternate morphologies. The dependence of cell behavior on both the topographic morphology and surface chemistry is thus demonstrated, providing further evidence for the importance of surface modification for modulating bio-inorganic interfaces. Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Pulsed Electromagnetic Field Exposure Reduces Hypoxia and Inflammation Damage in Neuron-Like and Microglial Cells.

PubMed

Vincenzi, Fabrizio; Ravani, Annalisa; Pasquini, Silvia; Merighi, Stefania; Gessi, Stefania; Setti, Stefania; Cadossi, Ruggero; Borea, Pier Andrea; Varani, Katia

2017-05-01

In the present study, the effect of low-frequency, low-energy pulsed electromagnetic fields (PEMFs) has been investigated by using different cell lines derived from neuron-like cells and microglial cells. In particular, the primary aim was to evaluate the effect of PEMF exposure in inflammation- and hypoxia-induced injury in two different neuronal cell models, the human neuroblastoma-derived SH-SY5Y cells and rat pheochromocytoma PC12 cells and in N9 microglial cells. In neuron-like cells, live/dead and apoptosis assays were performed in hypoxia conditions from 2 to 48 h. Interestingly, PEMF exposure counteracted hypoxia damage significantly reducing cell death and apoptosis. In the same cell lines, PEMFs inhibited the activation of the hypoxia-inducible factor 1α (HIF-1α), the master transcriptional regulator of cellular response to hypoxia. The effect of PEMF exposure on reactive oxygen species (ROS) production in both neuron-like and microglial cells was investigated considering their key role in ischemic injury. PEMFs significantly decreased hypoxia-induced ROS generation in PC12, SH-SY5Y, and N9 cells after 24 or 48 h of incubation. Moreover, PEMFs were able to reduce some of the most well-known pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6, and IL-8 release in N9 microglial cells stimulated with different concentrations of LPS for 24 or 48 h of incubation time. These results show a protective effect of PEMFs on hypoxia damage in neuron-like cells and an anti-inflammatory effect in microglial cells suggesting that PEMFs could represent a potential therapeutic approach in cerebral ischemic conditions. J. Cell. Physiol. 232: 1200-1208, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Amelioration of neurodegenerative changes in cellular and rat models of diabetes-related Alzheimer's disease by exendin-4.

PubMed

Chen, Song; Liu, Ai-ran; An, Feng-mao; Yao, Wen-bing; Gao, Xiang-dong

2012-10-01

Growing evidence suggests that type 2 diabetes mellitus (DM) is associated with age-dependent Alzheimer's disease (AD), the latter of which has even been considered as type 3 diabetes. Several physiopathological features including hyperglycemia, oxidative stress, and dysfunctional insulin signaling relate DM to AD. In this study, high glucose-, oxidative stress-induced neuronal injury and intracerebroventricular-streptozotocin (ICV-STZ) animals as the possible models for diabetes-related AD were employed to investigate the effects of exendin-4 (Ex-4), a long-acting glucagon-like peptide-1 (GLP-1) receptor agonist, on diabetes-associated Alzheimer-like changes as well as the molecular mechanisms involved. Our study demonstrated that GLP-1/Ex-4 could exert a protective effect against reduced viability of PC12 cells caused by high glucose and that this protective effect was mediated via the PI3-kinase pathway. In addition, GLP-1/Ex-4 ameliorated oxidative stress-induced injury in PC12 cells. In rat models, bilateral ICV-STZ administration was used to produce impaired insulin signaling in the brain. Fourteen days following ICV-STZ injection, rats treated with twice-daily Ex-4 had better learning and memory performance in the Morris water maze test compared with rats treated with saline. Additionally, histopathological evaluation confirmed the protective effects of Ex-4 treatment on hippocampal neurons against degeneration. Furthermore, we demonstrated that Ex-4 reversed ICV-STZ-induced tau hyperphosphorylation through downregulation of GSK-3β activity, a key kinase in both DM and AD. Our findings suggests that Ex-4 can protect neurons from diabetes-associated glucose metabolic dysregulation insults in vitro and from ICV-STZ insult in vivo, and that Ex-4 may prove of therapeutic value in the treatment of AD especially DM-related AD.

Lion's Mane Medicinal Mushroom, Hericium erinaceus (Agaricomycetes), Modulates Purinoceptor-Coupled Calcium Signaling and Murine Nociceptive Behavior.

PubMed

Liu, Pei-Shan; Chueh, Sheau-Huei; Chen, Chin-Chu; Lee, Li-Ya; Shiu, Li-Yen

2017-01-01

Hericium erinaceus is well known for the neurotrophic effect it confers by promoting nerve growth factor biosynthesis. We discovered a novel bioactivity of H. erinaceus in its ability to suppress adenosine triphosphate (ATP)-induced calcium signaling in neuronal PC12 cells. ATP, known primarily as a neurotransmitter, also acts on purinoceptors (P2 purinergic receptor [P2R]) to generate the cellular calcium signaling and secretion that mediate P2R physiological manifestations, including pain. Chronic pain reduces quality of life. However, constant analgesic administration can cause liver and kidney injury, as well as loss of the analgesic effect because of desensitization. In this study we investigated the analgesic potential of H. erinaceus through measurements of ATP-induced Ca2+ signaling in cell lines and observation of pain behaviors in mice. In P2R-coupled Ca2+ signaling measurements, extracts of H. erinaceus mycelia (HEEs) blocked ATP-induced Ca2+ signaling in both rat PC12 cells and human HOS cells. HEEs completely blocked ATP-induced Ca2+ signaling in human HOS cells, suggesting that this effect of HEEs is exerted through the P2R subtypes present in HOS cells, which include the P2X4, P2X7, P2Y2, and P2Y4 subtypes. In observations of animal behavior during pain, HEEs significantly reduced heat-induced pain, including postponing both the tail-flick response to heat stimulation and the paw-lifting response to a hot plate. This study demonstrates novel characteristics of H. erinaceus in reducing nociceptive behavior and blocking the functional activity of P2R. Further studies are required to verify this linkage and its molecular mechanisms.

Neutral endopeptidase promotes phorbol ester-induced apoptosis in prostate cancer cells by inhibiting neuropeptide-induced protein kinase C delta degradation.

PubMed

Sumitomo, M; Shen, R; Goldberg, J S; Dai, J; Navarro, D; Nanus, D M

2000-12-01

Phorbol esters induce apoptosis in androgen-sensitive LNCaP cells, which express neutral endopeptidase (NEP), but not in androgen-independent prostate cancer (PC) cells, which lack NEP expression. We investigated the role of NEP in PC cell susceptibility to 12-O-tetradecanoylphorbol-13-acetate (TPA). Western analysis showed that expression of NEP and protein kinase Cdelta (PKCdelta) correlated with PC cell sensitivity to TPA-induced growth arrest and apoptosis in LNCaP cells and in TSU-Prl cells expressing an inducible wild-type NEP protein. Inhibition of NEP enzyme activity using the specific NEP inhibitor CGS24592, or inhibition of PKCdelta using Rottlerin at concentrations that inhibit PKCdelta but not PKCalpha, significantly inhibited TPA-induced growth inhibition and cell death. Furthermore, pulse-chase experiments showed PKCdelta is stabilized in LNCaP cells and in TSU-Pr1 cells overexpressing wild-type NEP compared with PC cells lacking NEP expression. This results from NEP inactivation of its neuropeptide substrates (bombesin and endothelin-1), which in the absence of NEP stimulate cSrc kinase activity and induce rapid degradation of PKCdelta protein. These results indicate that expression of enzymatically active NEP by PC cells is necessary for TPA-induced apoptosis, and that NEP inhibits neuropeptide-induced, cSrc-mediated PKCdelta degradation.

Use of 8-methoxypsoralen and long-wavelength ultraviolet radiation for decontamination of platelet concentrates

NASA Astrophysics Data System (ADS)

Corash, Laurence; Lin, Lily; Wiesehahn, Gary; Cimino, George

1992-06-01

Transmission of viral diseases through blood products remains a problem in transfusion medicine. A number of methods have been developed to inactivate viral pathogens in plasma and plasma fractions, including: dry heating, wet heating, solvent-detergent treatment, and immunoaffinity purification. While some of these methods successfully inactivate pathogenic viruses, inactivation may be incomplete or result in damage to labile plasma proteins and cells. We have developed a photochemical decontamination system (PCD) for platelet concentrates (PC) utilizing treatment with long wavelength ultraviolet radiation (UVA, 320 - 400 nm) and 8-methoxypsoralen (8-MOP). This system is capable of inactivating 25 - 30 logs/hr of bacteria E. coli or S. aureus, 6 logs/hr of bacteriophage fd, 0.9 log/hr of bacteriophage R17 and 1.1 logs/hr of feline leukemia virus (FeLV) in PC. Immediately following 6 hrs of PCD treatment, platelet integrity and function of PCD treated and control PC were equivalent. After overnight storage PCD treated and control PC platelet properties were equal, but there was a slight reduction in TXB-2 production of PCD treated PC compared to controls. Following PCD treatment, PC were stored for 48 to 96 hrs. Platelet counts, morphology scores, extracellular LDH levels, aggregation response, dense body (db) content, and alpha granule ((alpha) g) content of PCD treated and control PC were comparable. We assessed the ability of the PCD technique to inactivate intracellular and extracellular virus, quantified the degree of DNA adduct formation in contaminating lymphocytes, and measured the inhibition of polymerase chain reaction (PCR) mediated amplification of intracellular DNA. High titers of cell-free murine cytomegalovirus added to human platelet concentrates (final concentration 106) were inactivated by PCD within 30 min. Cat renal fibroblasts infected at high levels with feline rhinotracheitis virus (FeRTV) were seeded into PC followed by PCD treatment with inactivation of 4.8 logs of FeRTV within 10 minutes. Purified human lymphocytes were seeded into PC and treated with PCD in the presence of 3H 8-MOP. Six hours of PCD treatment resulted in the formation of 9.3 to 12.8 8-MOP adducts per 1000 base pairs (bp) of DNA. PCR amplification of a 242 bp segment at the HLA-DQ(alpha) locus was examined. Inhibition of PCR DNA amplification was dependent on the numbers of 8-MOP adducts formed, and no amplification was present when greater than 12 adducts per 1000 bp were formed. These studies indicate that PCD can effectively inactivate high titers of cell-associated and cell-free virus seeded into standard human PC. The efficiency of DNA adduct formation can be quantitated, and the level of 8-MOP adduct formation in lymphocytes contaminating PC is comparable to the level of adduct formation in cellular DNA reported in the absence of platelets.

Synergistic inhibition of influenza A virus replication by a plant polyphenol-rich extract and epsilon-aminocaproic acid in vitro and in vivo.

PubMed

Serkedjieva, J; Nikolova, E; Kirilov, N

2010-01-01

A combined antiviral effect of a polyphenol-rich extract of the medicinal plant Geranium sanguineum L. (PC) and a protease inhibitor, epsilon-aminocaproic acid (ACA) was examined in Influenza A virus (IAV)-infected MDCK cell cultures and mice. Synergistic, antagonistic, or indifferent antiviral effects were distinguished on the basis of virus yields, namely fractional yields of individual compounds and yields of both compounds in combination. Combinations of PC and ACA in particular concentrations proved synergistic in the inhibition of virus replication in MDCK cells and in protection of mice against virus infection as determined by virus titers, lung weight, mean survival time (MST), mortality rate, and protection rate (PR). Following the application of a combination of PC and ACA to the virus-infected mice, the levels of the lung protease and protease-inhibitory activity, which were increased due to the virus infection, were brought to normal. These results demonstrate the rationale for a combined application of viral inhibitors with different modes of action to the treatment of IAV infection, in particular PC as a natural inhibitor of early viral transcription and translation and ACA as a synthetic inhibitor of cellular proteases. Influenza A virus; antiviral effect; synergism; plant polyphenol extract; epsilon-aminocaproic acid; protease inhibitors.

PDMS substrate stiffness affects the morphology and growth profiles of cancerous prostate and melanoma cells.

PubMed

Prauzner-Bechcicki, Szymon; Raczkowska, Joanna; Madej, Ewelina; Pabijan, Joanna; Lukes, Jaroslav; Sepitka, Josef; Rysz, Jakub; Awsiuk, Kamil; Bernasik, Andrzej; Budkowski, Andrzej; Lekka, Małgorzata

2015-01-01

A deep understanding of the interaction between cancerous cells and surfaces is particularly important for the design of lab-on-chip devices involving the use of polydimethylsiloxane (PDMS). In our studies, the effect of PDMS substrate stiffness on mechanical properties of cancerous cells was investigated in conditions where the PDMS substrate is not covered with any of extracellular matrix proteins. Two human prostate cancer (Du145 and PC-3) and two melanoma (WM115 and WM266-4) cell lines were cultured on two groups of PDMS substrates that were characterized by distinct stiffness, i.e. 0.75 ± 0.06 MPa and 2.92 ± 0.12 MPa. The results showed the strong effect on cellular behavior and morphology. The detailed analysis of chemical and physical properties of substrates revealed that cellular behavior occurs only due to substrate elasticity. Copyright © 2014 Elsevier Ltd. All rights reserved.

Covalent surface modification of prions: a mass spectrometry-based means of detecting distinctive structural features of prion strains

USDA-ARS?s Scientific Manuscript database

Prions (PrPSc) are molecular pathogens that are able to convert the isosequential normal cellular prion protein (PrPC) into a prion. The only demonstrated differences between PrPC and PrPSc is conformational, they are isoforms. A given host can be infected by more than one kind or strain of prion. F...

Novel epitopes identified by Anti-PrP monoclonal antibodies produced following immunization of Prnp0/0 Balb/cJ mice with purified scrapie prions

USDA-ARS?s Scientific Manuscript database

Prions, or infectious proteins, cause a class of uniformly fatal neurodegenerative diseases. Prions are composed solely of an aberrantly folded isoform(PrPSc)of a normal cellular protein (PrPC). Shared sequence identity of PrPSc with PrPC has limited the detection sensitivity of immunochemical assay...

Subphthalocyanines Axially Substituted with a Tetracyanobuta-1,3-diene-Aniline Moiety: Synthesis, Structure, and Physicochemical Properties.

PubMed

Winterfeld, Kim A; Lavarda, Giulia; Guilleme, Julia; Sekita, Michael; Guldi, Dirk M; Torres, Tomás; Bottari, Giovanni

2017-04-19

A 1,1,4,4-tetracyanobuta-1,3-diene (TCBD)-aniline moiety has been introduced, for the first time, at the axial position of two subphthalocyanines (SubPcs) peripherally substituted with hydrogen (H 12 SubPc) or fluorine atoms (F 12 SubPc). Single-crystal X-ray analysis of both SubPc-TCBD-aniline systems showed that each conjugate is a racemic mixture of two atropisomers resulting from the almost orthogonal geometry adopted by the axial TCBD unit, which were separated by chiral high-performance liquid chromatography. Remarkably, the single-crystal X-ray structure of one atropisomer of each SubPc-TCBD-aniline conjugate has been solved, allowing to unambiguously assign the atropisomers' absolute configuration, something, to the best of our knowledge, unprecedented in TCBD-based conjugates. Moreover, the physicochemical properties of both SubPc-TCBD-aniline racemates have been investigated using a wide range of electrochemical as well as steady-state and time-resolved spectroscopic techniques. Each of the two SubPc-TCBD-aniline conjugates presents a unique photophysical feature never observed before in SubPc chemistry. As a matter of fact, H 12 SubPc-TCBD-aniline showed significant ground-state charge transfer interactions between the H 12 SubPc macrocycle and the electron-withdrawing TCBD unit directly attached at its axial position. In contrast, F 12 SubPc-TCBD-aniline gave rise to an intense, broad emission, which red shifts upon increasing the solvent polarity and stems from an excited complex (i.e., an exciplex). Such an exciplex emission, which has also no precedent in TCBD chemistry, results from intramolecular interactions in the excited state between the electron-rich aniline and the F 12 SubPc π-surface, two molecular fragments kept in spatial proximity by the "unique" three-dimensional geometry adopted by the F 12 SubPc-TCBD-aniline. Complementary transient absorption studies were carried out on both SubPc-TCBD-aniline derivatives, showing the occurrence, in both cases, of photoinduced charge separation and corroborating the formation of the aforementioned intramolecular exciplex in terms of a radical ion pair stabilized through-space.

Tanshinone IIA protects PC12 cells from β-amyloid(25-35)-induced apoptosis via PI3K/Akt signaling pathway.

PubMed

Dong, Huimin; Mao, Shanping; Mao, Shanpin; Wei, Jiajun; Liu, Baohui; Zhang, Zhaohui; Zhang, Qian; Yan, Mingmin

2012-06-01

For the aging populations of any nation, Dementia is becoming a primary problem and Alzheimer’s dementia (AD) is the most common type. However, until now, there is no effective treatment for AD. Tanshinone IIA (Tan IIA) has been reported for neuroprotective potential to against amyloid β peptides (Aβ)-induced cytotoxicity in the rat pheochromocytoma cell line PC-12, which is widely used as AD research model, but the mechanism still remains unclear. To investigate the effect of Tan IIA and the possible molecular mechanism in the apoptosis of PC12 cells, we induced apoptosis in PC12 cells with β-amyloid(25-35), and treated cells with Tan IIA. After 24 h treatment, we found that Tan IIA increased the cell viability and reduced the number of apoptotic cells induced by Aβ(25-35). However, neuroprotection of Tan IIA was abolished by PI3K inhibitor LY294002. Meanwhile, Treatment with lithium chloride, a phosphorylation inhibitor of GSK3β, which is a downstream target of PI3K/Akt, can block Aβ(25-35)-induced cell apoptosis in a Tan IIA-like manner. Our findings suggest that Tan IIA is an effective neuroprotective agent and a viable candidate in AD therapy and PI3K/Akt activation and GSK3β phosphorylation are involved in the neuroprotection of Tan IIA.

Scaffold Hopping Toward Agomelatine: Novel 3, 4-Dihydroisoquinoline Compounds as Potential Antidepressant Agents

NASA Astrophysics Data System (ADS)

Yang, Yang; Ang, Wei; Long, Haiyue; Chang, Ying; Li, Zicheng; Zhou, Liangxue; Yang, Tao; Deng, Yong; Luo, Youfu

2016-10-01

A scaffold-hopping strategy toward Agomelatine based on in silico screening and knowledge analysis was employed to design novel antidepressant agents. A series of 3, 4-dihydroisoquinoline compounds were selected for chemical synthesis and biological assessment. Three compounds (6a-1, 6a-2, 6a-9) demonstrated protective effects on corticosterone-induced lesion of PC12 cells. Compound 6a-1 also displayed low inhibitory effects on the growth of HEK293 and L02 normal cells and it was further evaluated for its potential antidepressant effects in vivo. The forced swim test (FST) results revealed that compound 6a-1 remarkably reduced the immobility time of rats and the open field test (OFT) results indicated a better general locomotor activity of the rats treated with compound 6a-1 than those with Agomelatine or Fluoxetine. Mechanism studies implied that compound 6a-1 can significantly reduce PC12 cell apoptosis by up-regulation of GSH and down-regulation of ROS in corticosterone-induced lesion of PC12 cells. Meanwhile, the down-regulation of calcium ion concentration and up-regulation of BDNF level in PC12 cells may account for the neuroprotective effects. Furthermore, compound 6a-1 can increase cell survival and cell proliferation, promote cell maturation in the rat hippocampus after chronic treatment. The acute toxicity data in vivo indicated compound 6a-1 exhibited less hepatotoxicity than Agomelatine.

Flip-Flop of Phospholipids in Proteoliposomes Reconstituted from Detergent Extract of Chloroplast Membranes: Kinetics and Phospholipid Specificity

PubMed Central

Rajasekharan, Archita; Gummadi, Sathyanarayana N.

2011-01-01

Eukaryotic cells are compartmentalized into distinct sub-cellular organelles by lipid bilayers, which are known to be involved in numerous cellular processes. The wide repertoire of lipids, synthesized in the biogenic membranes like the endoplasmic reticulum and bacterial cytoplasmic membranes are initially localized in the cytosolic leaflet and some of these lipids have to be translocated to the exoplasmic leaflet for membrane biogenesis and uniform growth. It is known that phospholipid (PL) translocation in biogenic membranes is mediated by specific membrane proteins which occur in a rapid, bi-directional fashion without metabolic energy requirement and with no specificity to PL head group. A recent study reported the existence of biogenic membrane flippases in plants and that the mechanism of plant membrane biogenesis was similar to that found in animals. In this study, we demonstrate for the first time ATP independent and ATP dependent flippase activity in chloroplast membranes of plants. For this, we generated proteoliposomes from Triton X-100 extract of intact chloroplast, envelope membrane and thylakoid isolated from spinach leaves and assayed for flippase activity using fluorescent labeled phospholipids. Half-life time of flipping was found to be 6±1 min. We also show that: (a) intact chloroplast and envelope membrane reconstituted proteoliposomes can flip fluorescent labeled analogs of phosphatidylcholine in ATP independent manner, (b) envelope membrane and thylakoid reconstituted proteoliposomes can flip phosphatidylglycerol in ATP dependent manner, (c) Biogenic membrane ATP independent PC flipping activity is protein mediated and (d) the kinetics of PC translocation gets affected differently upon treatment with protease and protein modifying reagents. PMID:22174798

Flip-flop of phospholipids in proteoliposomes reconstituted from detergent extract of chloroplast membranes: kinetics and phospholipid specificity.

PubMed

Rajasekharan, Archita; Gummadi, Sathyanarayana N

2011-01-01

Eukaryotic cells are compartmentalized into distinct sub-cellular organelles by lipid bilayers, which are known to be involved in numerous cellular processes. The wide repertoire of lipids, synthesized in the biogenic membranes like the endoplasmic reticulum and bacterial cytoplasmic membranes are initially localized in the cytosolic leaflet and some of these lipids have to be translocated to the exoplasmic leaflet for membrane biogenesis and uniform growth. It is known that phospholipid (PL) translocation in biogenic membranes is mediated by specific membrane proteins which occur in a rapid, bi-directional fashion without metabolic energy requirement and with no specificity to PL head group. A recent study reported the existence of biogenic membrane flippases in plants and that the mechanism of plant membrane biogenesis was similar to that found in animals. In this study, we demonstrate for the first time ATP independent and ATP dependent flippase activity in chloroplast membranes of plants. For this, we generated proteoliposomes from Triton X-100 extract of intact chloroplast, envelope membrane and thylakoid isolated from spinach leaves and assayed for flippase activity using fluorescent labeled phospholipids. Half-life time of flipping was found to be 6 ± 1 min. We also show that: (a) intact chloroplast and envelope membrane reconstituted proteoliposomes can flip fluorescent labeled analogs of phosphatidylcholine in ATP independent manner, (b) envelope membrane and thylakoid reconstituted proteoliposomes can flip phosphatidylglycerol in ATP dependent manner, (c) Biogenic membrane ATP independent PC flipping activity is protein mediated and (d) the kinetics of PC translocation gets affected differently upon treatment with protease and protein modifying reagents.

78 FR 17865 - Airworthiness Directives; PILATUS AIRCRAFT LTD. Airplanes

Federal Register 2010, 2011, 2012, 2013, 2014

2013-03-25

... TBO were moved from Chapter 5: Time Limits/Maintenance Checks, to Chapter 4: Structural, Component and... Directives; PILATUS AIRCRAFT LTD. Airplanes AGENCY: Federal Aviation Administration (FAA), DOT. ACTION: Final... all PILATUS AIRCRAFT LTD. Models PC-12, PC-12/45, and PC-12/47 airplanes. This AD results from...

A Direct Redox Regulation of Protein Kinase C Isoenzymes Mediates Oxidant-induced Neuritogenesis in PC12 Cells*

PubMed Central

Gopalakrishna, Rayudu; Gundimeda, Usha; Schiffman, Jason Eric; McNeill, Thomas H.

2008-01-01

In this study, we have used the PC12 cell model to elucidate the mechanisms by which sublethal doses of oxidants induce neuritogenesis. The xanthine/xanthine oxidase (X/XO) system was used for the steady state generation of superoxide, and CoCl2 was used as a representative transition metal redox catalyst. Upon treatment of purified protein kinase C (PKC) with these oxidants, there was an increase in its cofactor-independent activation. Redox-active cobalt competed with the redoxinert zinc present in the zinc-thiolates of the PKC regulatory domain and induced the oxidation of these cysteine-rich regions. Both CoCl2 and X/XO induced neurite outgrowth in PC12 cells, as determined by an overexpression of neuronal marker genes. Furthermore, these oxidants induced a translocation of PKC from cytosol to membrane and subsequent conversion of PKC to a cofactor-independent form. Isoenzyme-specific PKC inhibitors demonstrated that PKCε plays a crucial role in neuritogenesis. Moreover, oxidant-induced neurite outgrowth was increased with a conditional overexpression of PKCε and decreased with its knock-out by small interfering RNA. Parallel with PKC activation, an increase in phosphorylation of the growth-associated neuronal protein GAP-43 at Ser41 was observed. Additionally, there was a sustained activation of extracellular signal-regulated kinases 1 and 2, which was correlated with activating phosphorylation (Ser133) of cAMP-responsive element-binding protein. All of these signaling events that are causally linked to neuritogenesis were blocked by antioxidant N-acetylcysteine (both l and d-forms) and by a variety of PKC-specific inhibitors. Taken together, these results strongly suggest that sublethal doses of oxidants induce neuritogenesis via a direct redox activation of PKCε. PMID:18375950

Monitoring Pc 4-mediated photodynamic therapy of U87 tumors with 18F- fluorodeoxy-glucose PET imaging in the Athymic Nude Rat

NASA Astrophysics Data System (ADS)

Varghai, Davood; Cross, Nathan; Spring-Robinson, Chandra; Sharma, Rahul; Feyes, Denise K.; Ahmad, Yusra; Oleinick, Nancy L.; Muzic, Raymond F., Jr.; Dean, David

2007-02-01

Introduction: We have previously demonstrated the use of phthalocyanine Pc 4 for the photodynamic therapy (PDT) of ectopic human glial tumors in the athymic nude rat brain. We wish to determine whether 18F-fluorodeoxy-glucose ( 18F-FDG) Positron Emission Tomography (PET) imaging can detect the reduction in tumor metabolism that must occur after Pc 4-PDT-induced necrosis. Methods: 2.5 x 10 5 U87 cells were injected into the brains of 12 athymic nude rats. After 7 days of tumor growth, all 12 animals were imaged functionally by 18F-FDG micro-PET (μPET) and structurally by micro-CT and/or micro-MR. These animals received 0.5 mg/kg b.w. Pc 4 via tail-vein injection. One day later the scalp was re-incised and the tumor illuminated with 30 J/cm2 of 672-nm light from a diode laser. The next day these animals were again 18F-FDG μPET imaged. Next, the animals were euthanized and their brains were explanted for H&E histology. Results: Histology showed that tumors in the 6 Pc 4-PDT-treated animals demonstrated necrosis ranging from full to frank (severe). Preliminary analysis showed that 18F-FDG μPET activity in 3 of the 6 non-PDT group (i.e., no tumor necrosis observed) animals was seen to increase 2.28 times following tumor photoirradiation, whereas 18F-FDG μPET activity in 5 of the 6 PDT group (i.e., tumor necrosis observed) animals was seen to increase 1.15 times following tumor photoirradiation. Discussion: The increased 18F-FDG μPET activity in the PDT group was unexpected. We had expected this activity to decrease and are presently investigating the cause of this observation.

Winds of change - a molecular outflow in NGC 1377?. The anatomy of an extreme FIR-excess galaxy

NASA Astrophysics Data System (ADS)

Aalto, S.; Muller, S.; Sakamoto, K.; Gallagher, J. S.; Martín, S.; Costagliola, F.

2012-10-01

Aims: Our goal was to investigate the molecular gas distribution and kinematics in the extreme far-infrared (FIR) excess galaxy NGC 1377 and to address the nature and evolutionary status of the buried source. Methods: We used high- (0''65 × 0''52, (65 × 52 pc)) and low- (4''88 × 2''93) resolution SubMillimeter Array (SMA) observations to image the 12CO and 13CO 2-1 line emission. Results: We find bright, complex 12CO 2-1 line emission in the inner 400 pc of NGC 1377. The 12CO 2-1 line has wings that are tracing a kinematical component that appears to be perpendicular to the component traced by the line core. Together with an intriguing X-shape of the integrated intensity and dispersion maps, this suggests that the molecular emission of NGC 1377 consists of a disk-outflow system. Lower limits to the molecular mass and outflow rate are Mout(H2) > 1 × 107 M⊙ and Ṁ > 8 M⊙ yr-1. The age of the proposed outflow is estimated to be 1.4 Myr, the extent to be 200 pc and the outflow speed to be Vout = 140 km s-1. The total molecular mass in the SMA map is estimated to Mtot(H2) = 1.5 × 108 M⊙ (on a scale of 400 pc) while in the inner r = 29 pc the molecular mass is Mcore(H2) = 1.7 × 107 M⊙ with a corresponding H2 column density of N(H2) = 3.4 × 1023 cm-2 and an average 12CO 2-1 brightness temperature of 19 K. 13CO 2-1 emission is found at a factor 10 fainter than 12CO in the low-resolution map while C18O 2-1 remains undetected. We find weak 1 mm continuum emission of 2.4 mJy with spatial extent less than 400 pc. Conclusions: Observing the molecular properties of the FIR-excess galaxy NGC 1377 allows us to probe the early stages of nuclear activity and the onset of feedback in active galaxies. The age of the outflow supports the notion that the current nuclear activity is young - a few Myr. The outflow may be powered by radiation pressure from a compact, dust enshrouded nucleus, but other driving mechanisms are possible. The buried source may be an active galactic nucleus (AGN) or an extremely young (1 Myr) compact starburst. Limitations on size and mass lead us to favor the AGN scenario, but additional studies are required to settle this question. In either case, the wind with its implied mass outflow rate will quench the nuclear power source within the very short time of 5-25 Myr. It is possible, however, that the gas is unable to escape the galaxy and may eventually fall back onto NGC 1377 again.

The dissipation of three fungicides in a biobed organic substrate and their impact on the structure and activity of the microbial community.

PubMed

Marinozzi, Maria; Coppola, Laura; Monaci, Elga; Karpouzas, Dimitrios G; Papadopoulou, Evangelia; Menkissoglu-Spiroudi, Urania; Vischetti, Costantino

2013-04-01

Biopurification systems (BPS) have been introduced to minimise the risk for point source contamination of natural water resources by pesticides. Their depuration efficiency relies mostly on the high biodegradation of their packing substrate (biomixture). Despite that, little is known regarding the interactions between biomixture microflora and pesticides, especially fungicides which are expected to have a higher impact on the microbial community. This study reports the dissipation of the fungicides azoxystrobin (AZX), fludioxonil (FL) and penconazole (PC), commonly used in vineyards, in a biomixture composed of pruning residues and straw used in vineyard BPS. The impact of fungicides on the microbial community was also studied via microbial biomass carbon, basal respiration and phospholipid fatty acid analysis. AZX dissipated faster (t1/2 = 30.1 days) than PC (t1/2 = 99.0 days) and FL (t1/2 = 115.5 days). Fungicides differently affected the microbial community. PC showed the highest adverse effect on both the size and the activity of the biomixture microflora. A significant change in the structure of the microbial community was noted for PC and FL, and it was attributed to a rapid inhibition of the fungal fraction while bacteria showed a delayed response which was attributed to indirect effects by the late proliferation of fungi. All effects observed were transitory and a full recovery of microbial indices was observed 60 days post-application. Overall, no clear link between pesticide persistence and microbial responses was observed stressing the complex nature of interactions between pesticides in microflora in BPS.

A Mutation within the Extended X Loop Abolished Substrate-induced ATPase Activity of the Human Liver ATP-binding Cassette (ABC) Transporter MDR3*

PubMed Central

Kluth, Marianne; Stindt, Jan; Dröge, Carola; Linnemann, Doris; Kubitz, Ralf; Schmitt, Lutz

2015-01-01

The human multidrug resistance protein 3 (MDR3/ABCB4) belongs to the ubiquitous family of ATP-binding cassette (ABC) transporters and is located in the canalicular membrane of hepatocytes. There it flops the phospholipids of the phosphatidylcholine (PC) family from the inner to the outer leaflet. Here, we report the characterization of wild type MDR3 and the Q1174E mutant, which was identified previously in a patient with progressive familial intrahepatic cholestasis type 3 (PFIC-3). We expressed different variants of MDR3 in the yeast Pichia pastoris, purified the proteins via tandem affinity chromatography, and determined MDR3-specific ATPase activity in the presence or absence of phospholipids. The ATPase activity of wild type MDR3 was stimulated 2-fold by liver PC or 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine lipids. Furthermore, the cross-linking of MDR3 with a thiol-reactive fluorophore blocked ATP hydrolysis and exhibited no PC stimulation. Similarly, phosphatidylethanolamine, phosphatidylserine, and sphingomyelin lipids did not induce an increase of wild type MDR3 ATPase activity. The phosphate analogues beryllium fluoride and aluminum fluoride led to complete inhibition of ATPase activity, whereas orthovanadate inhibited exclusively the PC-stimulated ATPase activity of MDR3. The Q1174E mutation is located in the nucleotide-binding domain in direct proximity of the leucine of the ABC signature motif and extended the X loop, which is found in ABC exporters. Our data on the Q1174E mutant demonstrated basal ATPase activity, but PC lipids were incapable of stimulating ATPase activity highlighting the role of the extended X loop in the cross-talk of the nucleotide-binding domain and the transmembrane domain. PMID:25533467

Spectroscopic and Theoretical Study of CuI Binding to His111 in the Human Prion Protein Fragment 106–115

PubMed Central

2016-01-01

The ability of the cellular prion protein (PrPC) to bind copper in vivo points to a physiological role for PrPC in copper transport. Six copper binding sites have been identified in the nonstructured N-terminal region of human PrPC. Among these sites, the His111 site is unique in that it contains a MKHM motif that would confer interesting CuI and CuII binding properties. We have evaluated CuI coordination to the PrP(106–115) fragment of the human PrP protein, using NMR and X-ray absorption spectroscopies and electronic structure calculations. We find that Met109 and Met112 play an important role in anchoring this metal ion. CuI coordination to His111 is pH-dependent: at pH >8, 2N1O1S species are formed with one Met ligand; in the range of pH 5–8, both methionine (Met) residues bind to CuI, forming a 1N1O2S species, where N is from His111 and O is from a backbone carbonyl or a water molecule; at pH <5, only the two Met residues remain coordinated. Thus, even upon drastic changes in the chemical environment, such as those occurring during endocytosis of PrPC (decreased pH and a reducing potential), the two Met residues in the MKHM motif enable PrPC to maintain the bound CuI ions, consistent with a copper transport function for this protein. We also find that the physiologically relevant CuI-1N1O2S species activates dioxygen via an inner-sphere mechanism, likely involving the formation of a copper(II) superoxide complex. In this process, the Met residues are partially oxidized to sulfoxide; this ability to scavenge superoxide may play a role in the proposed antioxidant properties of PrPC. This study provides further insight into the CuI coordination properties of His111 in human PrPC and the molecular mechanism of oxygen activation by this site. PMID:26930130

Stimulation of neuronal neurite outgrowth using functionalized carbon nanotubes

NASA Astrophysics Data System (ADS)

Matsumoto, K.; Sato, C.; Naka, Y.; Whitby, R.; Shimizu, N.

2010-03-01

Low concentrations (0.11-1.7 µg ml - 1) of functionalized carbon nanotubes (CNTs), which are multi-walled CNTs modified by amino groups, when added with nerve growth factor (NGF), promoted outgrowth of neuronal neurites in dorsal root ganglion (DRG) neurons and rat pheochromocytoma cell line PC12h cells in culture media. The quantity of active extracellular signal-regulated kinase (ERK) was higher after the addition of both 0.85 µg ml - 1 CNTs and NGF than that with NGF alone. CNTs increased the number of cells with neurite outgrowth in DRG neurons and PC12h cells after the inhibition of the ERK signaling pathway using a mitogen-activated protein kinase (MAPK)/ERK kinase (MEK) inhibitor. Active ERK proteins were detected in MEK inhibitor-treated neurons after the addition of CNTs to the culture medium. These results demonstrate that CNTs may stimulate neurite outgrowth by activation of the ERK signaling pathway. Thus, CNTs are biocompatible and are promising candidates for biological applications and devices.

Cholesterol Balance in Prion Diseases and Alzheimer’s Disease

PubMed Central

Hannaoui, Samia; Shim, Su Yeon; Cheng, Yo Ching; Corda, Erica; Gilch, Sabine

2014-01-01

Prion diseases are transmissible and fatal neurodegenerative disorders of humans and animals. They are characterized by the accumulation of PrPSc, an aberrantly folded isoform of the cellular prion protein PrPC, in the brains of affected individuals. PrPC is a cell surface glycoprotein attached to the outer leaflet of the plasma membrane by a glycosyl-phosphatidyl-inositol (GPI) anchor. Specifically, it is associated with lipid rafts, membrane microdomains enriched in cholesterol and sphinoglipids. It has been established that inhibition of endogenous cholesterol synthesis disturbs lipid raft association of PrPC and prevents PrPSc accumulation in neuronal cells. Additionally, prion conversion is reduced upon interference with cellular cholesterol uptake, endosomal export, or complexation at the plasma membrane. Altogether, these results demonstrate on the one hand the importance of cholesterol for prion propagation. On the other hand, growing evidence suggests that prion infection modulates neuronal cholesterol metabolism. Similar results were reported in Alzheimer’s disease (AD): whereas amyloid β peptide formation is influenced by cellular cholesterol, levels of cholesterol in the brains of affected individuals increase during the clinical course of the disease. In this review, we summarize commonalities of alterations in cholesterol homeostasis and discuss consequences for neuronal function and therapy of prion diseases and AD. PMID:25419621

Physiological Response of Crocosphaera watsonii to Enhanced and Fluctuating Carbon Dioxide Conditions

PubMed Central

Gradoville, Mary R.; White, Angelicque E.; Letelier, Ricardo M.

2014-01-01

We investigated the effects of elevated pCO2 on cultures of the unicellular N2-fixing cyanobacterium Crocosphaera watsonii WH8501. Using CO2-enriched air, cultures grown in batch mode under high light intensity were exposed to initial conditions approximating current atmospheric CO2 concentrations (∼400 ppm) as well as CO2 levels corresponding to low- and high-end predictions for the year 2100 (∼750 and 1000 ppm). Following acclimation to CO2 levels, the concentrations of particulate carbon (PC), particulate nitrogen (PN), and cells were measured over the diurnal cycle for a six-day period spanning exponential and early stationary growth phases. High rates of photosynthesis and respiration resulted in biologically induced pCO2 fluctuations in all treatments. Despite this observed pCO2 variability, and consistent with previous experiments conducted under stable pCO2 conditions, we observed that elevated mean pCO2 enhanced rates of PC production, PN production, and growth. During exponential growth phase, rates of PC and PN production increased by ∼1.2- and ∼1.5-fold in the mid- and high-CO2 treatments, respectively, when compared to the low-CO2 treatment. Elevated pCO2 also enhanced PC and PN production rates during early stationary growth phase. In all treatments, PC and PN cellular content displayed a strong diurnal rhythm, with particulate C:N molar ratios reaching a high of 22∶1 in the light and a low of 5.5∶1 in the dark. The pCO2 enhancement of metabolic rates persisted despite pCO2 variability, suggesting a consistent positive response of Crocosphaera to elevated and fluctuating pCO2 conditions. PMID:25343645

Atomic Force Microscope Studies of the Fusion of Floating Lipid Bilayers

PubMed Central

Abdulreda, Midhat H.; Moy, Vincent T.

2007-01-01

This study investigated the fusion of apposing floating bilayers of egg L-α-phosphatidylcholine (egg PC) or 1,2-dimyristoyl-sn-glycero-3-phosphocholine. Atomic force microscope measurements of fusion forces under different compression rates were acquired to reveal the energy landscape of the fusion process under varied lipid composition and temperature. Between compression rates of ∼1000 and ∼100,000 pN/s, applied forces in the range from ∼100 to ∼500 pN resulted in fusion of floating bilayers. Our atomic force microscope measurements indicated that one main energy barrier dominated the fusion process. The acquired dynamic force spectra were fit with a simple model based on the transition state theory with the assumption that the fusion activation potential is linear. A significant shift in the energy landscape was observed when bilayer fluidity and composition were modified, respectively, by temperature and different cholesterol concentrations (15% ≤ chol ≤ 25%). Such modifications resulted in a more than twofold increase in the width of the fusion energy barrier for egg PC and 1,2-dimyristoyl-sn-glycero-3-phosphocholine floating bilayers. The addition of 25% cholesterol to egg PC bilayers increased the activation energy by ∼1.0 kBT compared with that of bilayers with egg PC alone. These results reveal that widening of the energy barrier and consequently reduction in its slope facilitated membrane fusion. PMID:17400691

Atomic force microscope studies of the fusion of floating lipid bilayers.

PubMed

Abdulreda, Midhat H; Moy, Vincent T

2007-06-15

This study investigated the fusion of apposing floating bilayers of egg L-alpha-phosphatidylcholine (egg PC) or 1,2-dimyristoyl-sn-glycero-3-phosphocholine. Atomic force microscope measurements of fusion forces under different compression rates were acquired to reveal the energy landscape of the fusion process under varied lipid composition and temperature. Between compression rates of approximately 1000 and approximately 100,000 pN/s, applied forces in the range from approximately 100 to approximately 500 pN resulted in fusion of floating bilayers. Our atomic force microscope measurements indicated that one main energy barrier dominated the fusion process. The acquired dynamic force spectra were fit with a simple model based on the transition state theory with the assumption that the fusion activation potential is linear. A significant shift in the energy landscape was observed when bilayer fluidity and composition were modified, respectively, by temperature and different cholesterol concentrations (15% < or = chol < or = 25%). Such modifications resulted in a more than twofold increase in the width of the fusion energy barrier for egg PC and 1,2-dimyristoyl-sn-glycero-3-phosphocholine floating bilayers. The addition of 25% cholesterol to egg PC bilayers increased the activation energy by approximately 1.0 k(B)T compared with that of bilayers with egg PC alone. These results reveal that widening of the energy barrier and consequently reduction in its slope facilitated membrane fusion.

Naringenin-Induced Apoptotic Cell Death in Prostate Cancer Cells Is Mediated via the PI3K/AKT and MAPK Signaling Pathways.

PubMed

Lim, Whasun; Park, Sunwoo; Bazer, Fuller W; Song, Gwonhwa

2017-05-01

Prostate cancer is the most common cancer in men and the second most common cause of cancer-related deaths in men. Although, various drugs targeting the androgen receptor are normally used, the patients frequently undergo recurrence of the disease. To overcome these limitations, natural compounds have been researched for evidence that they suppress progression and metastasis of various cancer cells. In the present study, we investigated effects of naringenin, a natural anti-oxidant flavonoid derived from citrus, on prostate cancer cells (PC3 and LNCaP). Results of present study with PC3 and LNCaP cells revealed that naringenin inhibited proliferation and migration, while inducing apoptosis and ROS production by those cells. In addition, naringenin-induced loss of mitochondrial membrane potential and increased Bax and decreased Bcl-2 proteins in PC3 cells, but not LNCaP cells. In a dose-dependent manner, naringenin decreased phosphorylation of ERK1/2, P70S6K, S6, and P38 in PC3 cells, and reduced phosphorylation of ERK1/2, P53, P38, and JNK proteins in LNCaP cells. However, naringenin activated phosphorylation of AKT in both PC3 and LNCaP cells. Then, targeted signaling proteins associated with viability of PC3 and LNCaP cells were analyzed using pharmacological inhibitors of AKT and ERK1/2 cell signaling pathways. Moreover, we compared the apoptotic effects of naringenin and paclitaxel alone and in combination to find that naringenin enhanced the efficiency of paclitaxel to suppress progression of prostate cancer cell lines. Collectively, these results indicate that naringenin is a potential chemotherapeutic agent for treatment of prostate cancer. J. Cell. Biochem. 118: 1118-1131, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Anticancer activity of the new photosensitizers: dose and cell type dependence

NASA Astrophysics Data System (ADS)

Gyulkhandanyan, Grigor V.; Ghambaryan, Sona S.; Amelyan, Gayane V.; Ghazaryan, Robert K.; Haroutiunian, Samvel G.; Gyulkhandanyan, Aram G.; Gasparyan, Gennadi H.

2005-04-01

The necessity of researches of antitumor efficiency of new photosensitizers (PS) is explained by the opportunity of their application in photodynamic therapy of tumors. PS, selectively accumulated in cancer cells and activated by the light, generate the active oxygen species that cause apoptosis. Earlier, it was shown that PS chlorin e6 (0.3-0.5 μg/ml) induces rat embryo fibroblast-like cell apoptosis. In present work antitumor activity of the new photosensitizers, water-soluble cationic porphyrins and their metal complexes, is investigated. The dose-dependent destruction of cancer cells was shown on PC-12 (pheochromocytoma, rat adrenal gland) and Jurkat (human lymphoma) cell lines. Meso-tetra-[4-N-(2 `- oxyethyl) pyridyl] porphyrin (TOEPyP) and chlorin e6 possessed the same toxicity at LD50 dose on PC-12 cell line, whereas phototoxicity of TOEPyP was 3 times less compared to chlorin e6(LD50=0.2 and 0.075 μg/ml accordingly). The results have shown weak photosensitizing effect of Zn-and Ag-derivatives of TOEPyP on PC-12 cell line. TOEPyP and Zn-TOEPyP (0.1 - 50 μg/ml) were non-toxic for Jurkat cell line, whereas Ag-TOEPyP was toxic at 10 μg/ml (LD90). TOEPyP and chlorin e6 have shown phototoxic effect in the same dose range (LD50=0.5 and 0.3 μg/ml accordingly). The investigation of toxic and phototoxic effects of the new porphyrins revealed significantly different sensitivity of various cell lines to PSs.

Nanostructured Polyaniline Coating on ITO Glass Promotes the Neurite Outgrowth of PC 12 Cells by Electrical Stimulation.

PubMed

Wang, Liping; Huang, Qianwei; Wang, Jin-Ye

2015-11-10

A conducting polymer polyaniline (PANI) with nanostructure was synthesized on indium tin oxide (ITO) glass. The effect of electrical stimulation on the proliferation and the length of neurites of PC 12 cells was investigated. The dynamic protein adsorption on PANI and ITO surfaces in a cell culture medium was also compared with and without electrical stimulation. The adsorbed proteins were characterized using SDS-PAGE. A PANI coating on ITO surface was shown with 30-50 nm spherical nanostructure. The number of PC 12 cells was significantly greater on the PANI/ITO surface than on ITO and plate surfaces after cell seeding for 24 and 36 h. This result confirmed that the PANI coating is nontoxic to PC 12 cells. The electrical stimulation for 1, 2, and 4 h significantly enhanced the cell numbers for both PANI and ITO conducting surfaces. Moreover, the application of electrical stimulation also improved the neurite outgrowth of PC 12 cells, and the number of PC 12 cells with longer neurite lengths increased obviously under electrical stimulation for the PANI surface. From the mechanism, the adsorption of DMEM proteins was found to be enhanced by electrical stimulation for both PANI/ITO and ITO surfaces. A new band 2 (around 37 kDa) was observed from the collected adsorbed proteins when PC 12 cells were cultured on these surfaces, and culturing PC 12 cells also seemed to increase the amount of band 1 (around 90 kDa). When immersing PANI/ITO and ITO surfaces in a DMEM medium without a cell culture, the number of band 3 (around 70 kDa) and band 4 (around 45 kDa) proteins decreased compared to that of PC 12 cell cultured surfaces. These results are valuable for the design and improvement of the material performance for neural regeneration.

Clozapine protects PC-12 cells from death due to oxidative stress induced by hydrogen peroxide via a cell-type specific mechanism involving inhibition of extracellular signal-regulated kinase phosphorylation.

PubMed

Magliaro, Brian C; Saldanha, Colin J

2009-08-04

Recent evidence suggests that some atypical antipsychotic drugs may protect against oxidative stress and consequent neurodegeneration by mechanisms that remain unclear. Using the neuron-like rat pheochromocytoma (PC-12) cell line, Clozapine and N-desmethylclozapine were tested for their ability to protect against cell death due to oxidative stress induced by hydrogen peroxide (H(2)O(2)). These drugs demonstrated significant protection of PC-12 cells, as measured by both the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide (MTT) and Alamar Blue cell viability assays. However, neither viability assay detected a protective effect of Clozapine on human embryonic kidney (HEK293), rat primary cortical neurons, or human neuroblastoma (SH-SY5Y) exposed to H(2)O(2) treatment. The mechanism of protection involves a PC-12 cell-specific differential response to H(2)O(2) treatment vs. the other cell lines. Pre-treatment with 250 microM or 125 microM diethyldithiocarbamate (DETC), a superoxide dismutase (SOD) inhibitor, unexpectedly showed protection of the PC-12 cells from H(2)O(2) treatment. Western blots revealed that Clozapine, N-desmethylclozapine, and DETC reduce the phosphorylation of extracellular signal-regulated kinase (ERK) that is caused by H(2)O(2) exposure in PC-12 cells. In both HEK293 and SH-SY5Y cells, H(2)O(2) exposure did not increase ERK phosphorylation over control, demonstrating a different response to H(2)O(2) vs. PC-12 cells, and explaining why Clozapine could not protect these cells. Also, U0126, a specific MEK inhibitor, was able to protect PC-12 cells from H(2)O(2) exposure, showing that inhibiting ERK phosphorylation is sufficient to provide protection. Cumulatively, these results indicate that Clozapine, N-desmethylclozapine, DETC, and U0126 protect PC-12 cells by blocking the cell-type specific H(2)O(2) induced increase in ERK phosphorylation.

Normal Cellular Prion Protein Protects against Manganese-induced Oxidative Stress and Apoptotic Cell Death

PubMed Central

Choi, Christopher J.; Anantharam, Vellareddy; Saetveit, Nathan J.; Houk, Robert. S.; Kanthasamy, Arthi; Kanthasamy, Anumantha G.

2012-01-01

The normal prion protein is abundantly expressed in the CNS, but its biological function remains unclear. The prion protein has octapeptide repeat regions that bind to several divalent metals, suggesting that the prion proteins may alter the toxic effect of environmental neurotoxic metals. In the present study, we systematically examined whether prion protein modifies the neurotoxicity of manganese (Mn) by comparing the effect of Mn on mouse neural cells expressing prion protein (PrPC -cells) and prion-knockout (PrPKO -cells). Exposure to Mn (10 μM-1 mM) for 24 hr produced a dose-dependent cytotoxic response in both PrPC -cells and PrPKO -cells. Interestingly, PrPC -cells (EC50 117.6μM) were more resistant to Mn-induced cytotoxicity, as compared to PrPKO -cells (EC50 59.9μM), suggesting a protective role for PrPC against Mn neurotoxicity. Analysis of intracellular Mn levels showed less Mn accumulation in PrPC -cells as compared to PrPKO -cells. Furthermore, Mn-induced mitochondrial depolarization and ROS generation were significantly attenuated in PrPC -cells as compared to PrPKO -cells. Measurement of antioxidant status revealed similar basal levels of glutathione (GSH) in PrPC -cells and PrPKO -cells; however, Mn treatment caused greater depletion of GSH in PrPKO -cells. Mn-induced mitochondrial depolarization and ROS production were followed by time- and dose-dependent activation of the apoptotic cell death cascade involving caspase-9 and -3. Notably, DNA fragmentation induced by both Mn treatment and oxidative stress-inducer hydrogen peroxide (100μM) was significantly suppressed in PrPC -cells as compared to PrPKO -cells. Together, these results demonstrate that prion protein interferes with divalent metal Mn uptake and protects against Mn-induced oxidative stress and apoptotic cell death. PMID:17483122

Differential expression of steroid 5alpha-reductase isozymes and association with disease severity and angiogenic genes predict their biological role in prostate cancer.

PubMed

Das, Kakoli; Lorena, Pia D N; Ng, Lai Kuan; Lim, Diana; Shen, Liang; Siow, Woei Yun; Teh, Ming; Reichardt, Juergen K V; Salto-Tellez, Manuel

2010-09-01

The biological role of steroid 5alpha-reductase isozymes (encoded by the SRD5A1 and SRD5A2 genes) and angiogenic factors that play important roles in the pathogenesis and vascularization of prostate cancer (PC) is poorly understood. The sub-cellular expression of these isozymes and vascular endothelial growth factor (VEGF) in PC tissue microarrays (n=62) was examined using immunohistochemistry. The effect of SRD5A inhibition on the angiogenesis pathway genes in PC was also examined in prostate cell lines, LNCaP, PC3, and RWPE-1, by treating them with the SRD5A inhibitors finasteride and dutasteride, followed by western blot, quantitative PCR, and ELISA chip array techniques. In PC tissues, nuclear SRD5A1 expression was strongly associated with higher cancer Gleason scores (P=0.02), higher cancer stage (P=0.01), and higher serum prostate specific antigen (PSA) levels (P=0.01), whereas nuclear SRD5A2 expression was correlated with VEGF expression (P=0.01). Prostate tumor cell viability was significantly reduced in dutasteride-treated PC3 and RWPE-1 cells compared with finasteride-treated groups. Expression of the angiogenesis pathway genes transforming growth factor beta 1 (TGFB1), endothelin (EDN1), TGFalpha (TGFA), and VEGFR1 was upregulated in LNCaP cells, and at least 7 out of 21 genes were upregulated in PC3 cells treated with finasteride (25 muM). Our findings suggest that SRD5A1 expression predominates in advanced PC, and that inhibition of SRD5A1 and SRD5A2 together was more effective in reducing cell numbers than inhibition of SRD5A2 alone. However, these inhibitors did not show any significant difference in prostate cell angiogenic response. Interestingly, some angiogenic genes remained activated after treatment, possibly due to the duration of treatment and tumor resistance to inhibitors.

The association of GSK3 beta with E2F1 facilitates nerve growth factor-induced neural cell differentiation.

PubMed

Zhou, Fangfang; Zhang, Long; Wang, Aijun; Song, Bo; Gong, Kai; Zhang, Lihai; Hu, Min; Zhang, Xiufang; Zhao, Nanming; Gong, Yandao

2008-05-23

It is widely acknowledged that E2F1 and GSK3beta are both involved in the process of cell differentiation. However, the relationship between E2F1 and GSK3beta in cell differentiation has yet to be discovered. Here, we provide evidence that in the differentiation of PC12 cells induced by nerve growth factor (NGF), GSK3beta was increased at both the mRNA and protein levels, whereas E2F1 at these two levels was decreased. Both wild-type GSK3beta and its kinase-defective mutant GSK3beta KM can inhibit E2F1 by promoting its ubiquitination through physical interaction. In addition, the colocalization of GSK3beta and E2F1 and their subcellular distribution, regulated by NGF, were observed in the process of PC12 differentiation. At the tissue level, GSK3beta colocalized and interacted with E2F1 in mouse hippocampus. Furthermore, GSK3beta facilitated neurite outgrowth by rescuing the promoter activities of Cdk inhibitors p21 and p15 from the inhibition caused by E2F1. To summarize, our findings suggest that GSK3beta can promote the ubiquitination of E2F1 via physical interaction and thus inhibit its transcription activity in a kinase activity independent manner, which plays an important role in the NGF-induced PC12 differentiation.

The Role of 6-Gingerol on Inhibiting Amyloid β Protein-Induced Apoptosis in PC12 Cells.

PubMed

Zeng, Gao-feng; Zong, Shao-hui; Zhang, Zhi-yong; Fu, Song-wen; Li, Ke-ke; Fang, Ye; Lu, Li; Xiao, De-Qiang

2015-10-01

Our previous study suggests that ginger root extract can reverse behavioral dysfunction and prevent Alzheimer's disease (AD)-like symptoms induced by the amyloid-β protein (Aβ) in a rat model. 6-Gingerol is the major gingerol in ginger rhizomes, but its effect on the treatment of AD remains unclear. In this study, we aimed to determine if 6-gingerol had a protective effect on Aβ1-42-induced damage and apoptotic death in rat pheochromocytoma cells (PC12 cells) and to investigate the underlying mechanisms by which 6-gingerol may exert its neuroprotective effects. Our results indicated that pre-treatment with 6-gingerol significantly increased cell viability and reduced cell apoptosis in Aβ1-42-treated cells. Moreover, 6-gingerol pretreatment markedly reduced the level of intracellular reactive oxygen species (ROS) and malondialdehyde (MDA), the production of nitric oxide (NO), and the leakage of lactate dehydrogenase (LDH) and increased superoxide dismutase (SOD) activity compared with the Aβ1-42 treatment group. In addition, 6-gingerol pretreatment also significantly enhanced the protein levels of phosphorylated Akt (p-Akt) and glycogen synthase kinase-3β (p-GSK-3β). Overall, these results indicate that 6-gingerol exhibited protective effects on apoptosis induced by Aβ1-42 in cultured PC12 cells by reducing oxidative stress and inflammatory responses, suppressing the activation of GSK-3β and enhancing the activation of Akt, thereby exerting neuroprotective effects. Therefore, 6-gingerol may be useful in the prevention and/or treatment of AD.

Effects of Extremely Low Frequency Magnetic Field on Neurite Outgrowth of PC12 and PC12D Cells and Evaluation by Image Analysis

NASA Astrophysics Data System (ADS)

Sakanishi, Akio; Takatsuki, Hideyo; Yoshikoshi, Akio; Fujiwara, Yasuyoshi

2004-05-01

A pheochromocytoma cell (PC12), and its derivative (PC12D), differentiate to nervelike cells in culture with the nerve growth factor (NGF) and forskolin respectively. We introduced a morphological factor σ=L/2(π A)1/2 for quantitating neurite outgrowth under a microscope in the presence of extremely low-frequency (ELF) magnetic fields for 22 hours, where L and A are the contour length and the area of the cells in clump determined using an image-analysis system. ELF magnetic fields B1 were generated with a single coil or double coils in Helmholtz configuration together with static fields B0 of -53, -20 and 67 μT. σ increased with increasing NGF or forskolin level at B0=-53 μT (geomagnetism), in agreement with the cytometric observation of micrographs. With the addition of an AC field B1 at 60 Hz (100 μT > B1 > 3 μT rms) to B0, neurite outgrowth represented by σ was depressed for PC12 and stimulated for PC12D. We discuss the cyclotron resonance and the ion parametric resonance models.

Effect of Ultrasonic Extraction on Production and Structural Changes of C-Phycocyanin from Marine Spirulina maxima

PubMed Central

Choi, Woon Yong; Lee, Hyeon Yong

2018-01-01

This work first showed that very high amounts of phycocyanins, such as 11.3 mg/mL C-phycocyanin (C-PC), 3.1 mg/mL allophycocyanin (APC), and 0.8 mg/mL phycoerythrin (PE), can be obtained using an ultrasonic extraction process (UE) with a 60 kHz frequency and 3 h of process time at 25 °C, without any other pretreatments. These yields were higher than those from most conventional water extractions at 4 °C for 24 h (Control condition) or at 25 °C for 24 h (WE), namely, 9.8 and 5.7 mg/mL C-PC, 2.3 and 1.2 mg/mL APC, and 0.7 and 0.3 mg/mL PE, respectively. These yields were also shown to be even higher than yields from other reported data. Structural changes in C-PC in the extracts were also found for the first time, according to extraction conditions, showing that the total concentration of C-PC and of the α-subunit of C-PC in the UE were much higher than in the WE, with little difference in the amount of β-subunit of C-PC in the UE or WE. It was also shown that the structural changes in C-PC in the WE decreased both antioxidant and anti-inflammation activities—29.83% vs. 32.09% of α,α-diphenyl-β-picrylhydrazyl (DPPH) scavenging activity and 8.21 vs. 7.25 µM of NO production for the WE and UE, respectively—while the UE, with similar patterns to standard C-PC, showed very high biological effects, which may suggest that the biologically active part is the α-subunit of C-PC, not the β-subunit. PMID:29324668

New analogs of the CART peptide with anorexigenic potency: the importance of individual disulfide bridges.

PubMed

Blechová, Miroslava; Nagelová, Veronika; Záková, Lenka; Demianová, Zuzana; Zelezná, Blanka; Maletínská, Lenka

2013-01-01

The CART (cocaine- and amphetamine-regulated transcript) peptide is an anorexigenic neuropeptide that acts in the hypothalamus. The receptor and the mechanism of action of this peptide are still unknown. In our previous study, we showed that the CART peptide binds specifically to PC12 rat pheochromocytoma cells in both the native and differentiated into neuronal phenotype. Two biologically active forms, CART(55-102) and CART(61-102), with equal biological activity, contain three disulfide bridges. To clarify the importance of each of these disulfide bridges in maintaining the biological activity of CART(61-102), an Ala scan at particular S-S bridges forming cysteines was performed, and analogs with only one or two disulfide bridges were synthesized. In this study, a stabilized CART(61-102) analog with norleucine instead of methionine at position 67 was also prepared and was found to bind to PC12 cells with an anorexigenic potency similar to that of CART(61-102). The binding study revealed that out of all analogs tested, [Ala(68,86)]CART(61-102), which contains two disulfide bridges (positions 74-94 and 88-101), preserved a high affinity to both native PC12 cells and those that had been differentiated into neurons. In food intake and behavioral tests with mice after intracerebroventricular administration, this analog showed strong and long-lasting anorexigenic potency. Therefore, the disulfide bridge between cysteines 68 and 86 in CART(61-102) can be omitted without a loss of biological activity, but the preservation of two other disulfide bridges and the full-length peptide are essential for biological activity. Copyright © 2012 Elsevier Inc. All rights reserved.

TRPC6 channel-mediated neurite outgrowth in PC12 cells and hippocampal neurons involves activation of RAS/MEK/ERK, PI3K, and CAMKIV signaling.

PubMed

Heiser, Jeanine H; Schuwald, Anita M; Sillani, Giacomo; Ye, Lian; Müller, Walter E; Leuner, Kristina

2013-11-01

The non-selective cationic transient receptor canonical 6 (TRPC6) channels are involved in synaptic plasticity changes ranging from dendritic growth, spine morphology changes and increase in excitatory synapses. We previously showed that the TRPC6 activator hyperforin, the active antidepressant component of St. John's wort, induces neuritic outgrowth and spine morphology changes in PC12 cells and hippocampal CA1 neurons. However, the signaling cascade that transmits the hyperforin-induced transient rise in intracellular calcium into neuritic outgrowth is not yet fully understood. Several signaling pathways are involved in calcium transient-mediated changes in synaptic plasticity, ranging from calmodulin-mediated Ras-induced signaling cascades comprising the mitogen-activated protein kinase, PI3K signal transduction pathways as well as Ca(2+) /calmodulin-dependent protein kinase II (CAMKII) and CAMKIV. We show that several mechanisms are involved in TRPC6-mediated synaptic plasticity changes in PC12 cells and primary hippocampal neurons. Influx of calcium via TRPC6 channels activates different pathways including Ras/mitogen-activated protein kinase/extracellular signal-regulated kinases, phosphatidylinositide 3-kinase/protein kinase B, and CAMKIV in both cell types, leading to cAMP-response element binding protein phosphorylation. These findings are interesting not only in terms of the downstream targets of TRPC6 channels but also because of their potential to facilitate further understanding of St. John's wort extract-mediated antidepressant activity. Alterations in synaptic plasticity are considered to play an important role in the pathogenesis of depression. Beside several other proteins, TRPC6 channels regulate synaptic plasticity. This study demonstrates that different pathways including Ras/MEK/ERK, PI3K/Akt, and CAMKIV are involved in the improvement of synaptic plasticity by the TRPC6 activator hyperforin, the antidepressant active constituent of St. John's wort extract. © 2013 International Society for Neurochemistry.

Derivatives of xanthic acid are novel antioxidants: application to synaptosomes.

PubMed

Lauderback, Christopher M; Drake, Jennifer; Zhou, Daohong; Hackett, Janna M; Castegna, Alessandra; Kanski, Jaroslaw; Tsoras, Maria; Varadarajan, Sridhar; Butterfield, D Allan

2003-04-01

Xanthic acids have long been known to act as reducing agents. Recently, D609, a tricyclodecanol derivative of xanthic acid, has been reported to have anti-apoptotic and anti-inflammatory properties that are attributed to specific inhibition of phosphatidyl choline phospholipase C (PC-PLC). However, because oxidative stress is involved in both of these cellular responses, the possibility that xanthates may act as antioxidants was investigated in the current study. Finding that xanthates efficiently scavenge hydroxyl radicals, the mechanism by which D609 and other xanthate derivatives may protect against oxidative damage was further examined. The xanthates studied, especially D609, mimic glutathione (GSH). Xanthates scavenge hydroxyl radicals and hydrogen peroxide, form disulfide bonds (dixanthogens), and react with electrophilic products of lipid oxidation (acrolein) in a manner similar to GSH. Further, upon disulfide formation, dixanthogens are reduced by glutathione reductase to a redox active xanthate. Supporting its role as an antioxidant, D609 significantly (p < 0.01) reduces free radical-induced changes in synaptosomal lipid peroxidation (TBARs), protein oxidation (protein carbonyls), and protein conformation. Thus, in addition to inhibitory effects on PC-PLC, D609 may prevent cellular apoptotic and inflammatory cascades by acting as antioxidants and novel GSH mimics. These results are discussed with reference to potential therapeutic application of D609 in oxidative stress conditions.

Platelets Cellular and Functional Characteristics in Patients with Atrial Fibrillation: A Comprehensive Meta-Analysis and Systematic Review

PubMed Central

Weymann, Alexander; Ali-Hasan-Al-Saegh, Sadeq; Sabashnikov, Anton; Popov, Aron-Frederik; Mirhosseini, Seyed Jalil; Nombela-Franco, Luis; Testa, Luca; Lotfaliani, Mohammadreza; Zeriouh, Mohamed; Liu, Tong; Dehghan, Hamidreza; Yavuz, Senol; de Oliveira Sá, Michel Pompeu Barros; Baker, William L.; Jang, Jae-Sik; Gong, Mengqi; Benedetto, Umberto; Dohmen, Pascal M.; D’Ascenzo, Fabrizio; Deshmukh, Abhishek J.; Biondi-Zoccai, Giuseppe; Calkins, Hugh; Stone, Gregg W.

2017-01-01

Background This systematic review with meta-analysis aimed to determine the strength of evidence for evaluating the association of platelet cellular and functional characteristics including platelet count (PC), MPV, platelet distribution width (PDW), platelet factor 4, beta thromboglobulin (BTG), and p-selectin with the occurrence of atrial fibrillation (AF) and consequent stroke. Material/Methods We conducted a meta-analysis of observational studies evaluating platelet characteristics in patients with paroxysmal, persistent and permanent atrial fibrillations. A comprehensive subgroup analysis was performed to explore potential sources of heterogeneity. Results Literature search of all major databases retrieved 1,676 studies. After screening, a total of 73 studies were identified. Pooled analysis showed significant differences in PC (weighted mean difference (WMD)=−26.93 and p<0.001), MPV (WMD=0.61 and p<0.001), PDW (WMD=−0.22 and p=0.002), BTG (WMD=24.69 and p<0.001), PF4 (WMD=4.59 and p<0.001), and p-selectin (WMD=4.90 and p<0.001). Conclusions Platelets play a critical and precipitating role in the occurrence of AF. Whereas distribution width of platelets as well as factors of platelet activity was significantly greater in AF patients compared to SR patients, platelet count was significantly lower in AF patients. PMID:28302997

Effect of virgin olive oil and thyme phenolic compounds on blood lipid profile: implications of human gut microbiota.

PubMed

Martín-Peláez, Sandra; Mosele, Juana Ines; Pizarro, Neus; Farràs, Marta; de la Torre, Rafael; Subirana, Isaac; Pérez-Cano, Francisco José; Castañer, Olga; Solà, Rosa; Fernandez-Castillejo, Sara; Heredia, Saray; Farré, Magí; Motilva, María José; Fitó, Montserrat

2017-02-01

To investigate the effect of virgin olive oil phenolic compounds (PC) alone or in combination with thyme PC on blood lipid profile from hypercholesterolemic humans, and whether the changes generated are related with changes in gut microbiota populations and activities. A randomized, controlled, double-blind, crossover human trial (n = 12) was carried out. Participants ingested 25 mL/day for 3 weeks, preceded by 2-week washout periods, three raw virgin olive oils differing in the concentration and origin of PC: (1) a virgin olive oil (OO) naturally containing 80 mg PC/kg, (VOO), (2) a PC-enriched virgin olive oil containing 500 mg PC/kg, from OO (FVOO), and (3) a PC-enriched virgin olive oil containing a mixture of 500 mg PC/kg from OO and thyme, 1:1 (FVOOT). Blood lipid values and faecal quantitative changes in microbial populations, short chain fatty acids, cholesterol microbial metabolites, bile acids, and phenolic metabolites were analysed. FVOOT decreased seric ox-LDL concentrations compared with pre-FVOOT, and increased numbers of bifidobacteria and the levels of the phenolic metabolite protocatechuic acid compared to VOO (P < 0.05). FVOO did not lead to changes in blood lipid profile nor quantitative changes in the microbial populations analysed, but increased the coprostanone compared to FVOOT (P < 0.05), and the levels of the faecal hydroxytyrosol and dihydroxyphenylacetic acids, compared with pre-intervention values and to VOO, respectively (P < 0.05). The ingestion of a PC-enriched virgin olive oil, containing a mixture of olive oil and thyme PC for 3 weeks, decreases blood ox-LDL in hypercholesterolemic humans. This cardio-protective effect could be mediated by the increases in populations of bifidobacteria together with increases in PC microbial metabolites with antioxidant activities.

[Study on construction and immune protective effect of recombinant nucleic acid vaccine of Toxoplasma gondii].

PubMed

Wei, Qing-Kuan

2012-04-01

To construct the polyvalent recombinant nucleic acid vaccine of Toxoplasma gondii and measure its protective immune effect. The gene of heat shock protein (HSP70) was amplified by PCR and inserted into the recombinant plasmid of pcDNA3-ROP2-p30 to construct recombinant polyvalent nucleic vaccine (pcDNA3-ROP2-p30-Hsp70). BALB/c mice were immunized with the constructed recombinant nucleic vaccine. CD4+ and CD8+ in the splenic lymphocytes and the lymphocytes in anticoagulant whole blood, the immune indices such as antibodies (IgG, IgM and IgA) and IFN-gamma, TNF, IL-2, IL-4, IL-12 in serum and splenic lymphocytes culture medium were detected, along with the challenge experiment. The protective immune responses that caused by the vaccine was measured by detecting the changes of immune indices of mice and the challenge experiment. 916 bp fragment of HSP70 gene was amplified by PCR. The recombinant polyvalent nucleic vaccine pcDNA3-ROP2-p30-HSP70 that included the whole open reading frame sequence of HSP gene was successfully constructed. The immunization results also showed this polyvalent nucleic vaccine could induce strong cellular and humoral responses by the detection of higher antibody titer in the experimental mice group, the increasing proliferation of CD4+ and CD8+ cells with significant deviations among the groups (F(CD4+) = 45.00, F(CD8+) = 15.01, all P < 0.01) and the apparent up-regulated levels of several cytokines IFN-gamma, IL-2 and IL-12 in serum and cultural supernatant of spleen cells, with more striking effect in serum. As a result of the challenge experiment, the immunized mice showed a longer survival time. The recombinant nucleic acid vaccine pcDNA3-ROP2-p30-HSP70 possesses a strong immunogenicity and is able to induce an immune protection.

Optical imaging of TNF-α induced apoptosis pathway in living PC12 cells

NASA Astrophysics Data System (ADS)

Zhang, Lan; Xing, Da; Chen, Miaojuan

2007-05-01

Tumor necrosis factor-α (TNF-α) elicits a wide range of biological responses, including neuronal apoptosis and neuroprotection, and this functional pleiotropy is essentially determined by the individual molecular orchestration. Two main pathways lead to apoptosis - the 'extrinsic' or death receptor-initiated pathway, and the 'intrinsic' or mitochondrial pathway. In this study we firstly examine the signaling pathways involved in TNF-α induced apoptosis in living PC12 cells by optical imaging. Our results show that the cleavage of BID has been monitored in real time using fluorescence resonance energy transfer (FRET) technique after PC12 cells treated with TNF-α. Then we observe BAX can't translocation to mitochondria during PC12 cells apoptosis induced by TNF-α, and that there is no any evidence of cytochrome C release into cytosol during cell apoptosis. Our data support that TNF-α mediated PC12 cells apoptosis is extrinsic apoptotic pathway which independent of mitochondria.

New Thymoquinol Glycosides and Neuroprotective Dibenzocyclooctane Lignans from the Rattan Stems of Schisandra chinensis.

PubMed

Yang, Bing-You; Guo, Jiang-Tao; Li, Zu-Yi; Wang, Chang-Fu; Wang, Zhi-Bin; Wang, Qiu-Hong; Kuang, Hai-Xue

2016-09-01

Three new lignans (1 - 3), together with four new thymoquinol glycosides (4 - 7), were isolated from 70%-EtOH extract of the rattan stems of Schisandra chinensis. The structures of 1 - 7 were elucidated by detailed spectroscopic analyses, and these new compounds were identified as pinobatol-9-O-β-d-glucopyranoside (1), 1,2,13,14-tetramethoxydibenzocyclooctadiene 3,12-O-β-d-diglucopyranoside (2), 3,7-dihydroxy-1,2,13,14-tetramethoxydibenzocyclooctadiene 12-O-β-d-glucopyranoside (3), thymoquinol 2-O-β-d-apiofuranosyl-(1→6)-β-d-glucopyranoside (4), thymoquinol 2-O-α-d-arabinofuranosyl-(1→6)-β-d-glucopyranoside (5), thymoquinol 5-O-β-d-apiofuranosyl-(1→6)-β-d-glucopyranoside (6), and thymoquinol 5-O-α-d-arabinofuranosyl-(1→6)-β-d-glucopyranoside (7). The neuroprotective activity of 1 - 7 was evaluated on PC12 cells with neurotoxicity induced by amyloid-beta 1 - 42 (Aβ 1 - 42 ). Compounds 2 and 3 showed protecting activity against Aβ-induced toxicity in PC12 cells. © 2016 Wiley-VHCA AG, Zürich.

Lipopolysaccharides-stimulated macrophage products enhance Withaferin A-induced apoptosis via activation of caspases and inhibition of NF-κB pathway in human cancer cells.

PubMed

Piao, Liang; Canguo, Zhao; Wenjie, Lu; Xiaoli, Cheng; Wenli, Shi; Li, Lu

2017-01-01

Macrophages, as a major cellular component in tumor microenvironment, play an important role in tumor progression. However, their roles in modulation of cytotoxic chemotherapy are still not fully understood. Here, we investigated the influence of Lipoplysaccharides (LPS)-stimulated macrophage products (LSMP) on Withaferin A (WA), a natural compound that derived from the medicinal plant Withania somnifera, as an antitumor agent in human breast cancer cells MDA-MB-231 and prostate cancer cells PC-3. Our results revealed that LSMP may enhance WA-induced apoptosis in both cell lines, the underlying mechanisms of which are closely associated with activation of caspase-8, -9 and -3, cleavage of poly ADP-ribose polymerase (PARP), as well as specifically inhibiting the translocation of nuclear factor-κB (NF-κB) and down-regulation of anti-apoptotic proteins X-linked inhibitor of apoptosis protein (XIAP) and inhibitor of apoptosis protein (cIAP1/2). These findings demonstrate that macrophages in tumor microenvironment can modulate tumor responses to chemotoxic agents, providing an effective strategy that targets macrophages to enhance the antitumor efficacy of cytotoxic chemotherapy. Copyright © 2016. Published by Elsevier Ltd.

Potentiometric Dye Imaging for Pheochromocytoma and Cortical Neurons with a Novel Measurement System Using an Integrated Complementary Metal-Oxide-Semiconductor Imaging Device

NASA Astrophysics Data System (ADS)

Kobayashi, Takuma; Tagawa, Ayato; Noda, Toshihiko; Sasagawa, Kiyotaka; Tokuda, Takashi; Hatanaka, Yumiko; Tamura, Hideki; Ishikawa, Yasuyuki; Shiosaka, Sadao; Ohta, Jun

2010-11-01

The combination of optical imaging with voltage-sensitive dyes is a powerful tool for studying the spatiotemporal patterns of neural activity and understanding the neural networks of the brain. To visualize the potential status of multiple neurons simultaneously using a compact instrument with high density and a wide range, we present a novel measurement system using an implantable biomedical photonic LSI device with a red absorptive light filter for voltage-sensitive dye imaging (BpLSI-red). The BpLSI-red was developed for sensing fluorescence by the on-chip LSI, which was designed by using complementary metal-oxide-semiconductor (CMOS) technology. A micro-electro-mechanical system (MEMS) microfabrication technique was used to postprocess the CMOS sensor chip; light-emitting diodes (LEDs) were integrated for illumination and to enable long-term cell culture. Using the device, we succeeded in visualizing the membrane potential of 2000-3000 cells and the process of depolarization of pheochromocytoma cells (PC12 cells) and mouse cerebral cortical neurons in a primary culture with cellular resolution. Therefore, our measurement application enables the detection of multiple neural activities simultaneously.

Hydrogen gas alleviates oxygen toxicity by reducing hydroxyl radical levels in PC12 cells.

PubMed

Yu, Junchao; Yu, Qiuhong; Liu, Yaling; Zhang, Ruiyun; Xue, Lianbi

2017-01-01

Hyperbaric oxygen (HBO) therapy through breathing oxygen at the pressure of above 1 atmosphere absolute (ATA) is useful for varieties of clinical conditions, especially hypoxic-ischemic diseases. Because of generation of reactive oxygen species (ROS), breathing oxygen gas at high pressures can cause oxygen toxicity in the central nervous system, leading to multiple neurological dysfunction, which limits the use of HBO therapy. Studies have shown that Hydrogen gas (H2) can diminish oxidative stress and effectively reduce active ROS associated with diseases. However, the effect of H2 on ROS generated from HBO therapy remains unclear. In this study, we investigated the effect of H2 on ROS during HBO therapy using PC12 cells. PC12 cells cultured in medium were exposed to oxygen gas or mixed oxygen gas and H2 at 1 ATA or 5 ATA. Cells viability and oxidation products and ROS were determined. The data showed that H2 promoted the cell viability and inhibited the damage in the cell and mitochondria membrane, reduced the levels of lipid peroxidation and DNA oxidation, and selectively decreased the levels of •OH but not disturbing the levels of O2•-, H2O2, or NO• in PC12 cells during HBO therapy. These results indicated that H2 effectively reduced •OH, protected cells against oxygen toxicity resulting from HBO therapy, and had no effect on other ROS. Our data supported that H2 could be potentially used as an antioxidant during HBO therapy.

Surface functionalization of a polymeric lipid bilayer for coupling a model biological membrane with molecules, cells, and microstructures.

PubMed

Morigaki, Kenichi; Mizutani, Kazuyuki; Saito, Makoto; Okazaki, Takashi; Nakajima, Yoshihiro; Tatsu, Yoshiro; Imaishi, Hiromasa

2013-02-26

We describe a stable and functional model biological membrane based on a polymerized lipid bilayer with a chemically modified surface. A polymerized lipid bilayer was formed from a mixture of two diacetylene-containing phospholipids, 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine (DiynePC) and 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphoethanolamine (DiynePE). DiynePC formed a stable bilayer structure, whereas the ethanolamine headgroup of DiynePE enabled functional molecules to be grafted onto the membrane surface. Copolymerization of DiynePC and DiynePE resulted in a robust bilayer. Functionalization of the polymeric bilayer provided a route to a robust and biomimetic surface that can be linked with biomolecules, cells, and three-dimensional (3D) microstructures. Biotin and peptides were grafted onto the polymeric bilayer for attaching streptavidin and cultured mammalian cells by molecular recognition, respectively. Nonspecific adsorption of proteins and cells on polymeric bilayers was minimum. DiynePE was also used to attach a microstructure made of an elastomer (polydimethylsiloxan: PDMS) onto the membrane, forming a confined aqueous solution between the two surfaces. The microcompartment enabled us to assay the activity of a membrane-bound enzyme (cyochrome P450). Natural (fluid) lipid bilayers were incorporated together with membrane-bound proteins by lithographically polymerizing DiynePC/DiynePE bilayers. The hybrid membrane of functionalized polymeric bilayers and fluid bilayers offers a novel platform for a wide range of biomedical applications including biosensor, bioassay, cell culture, and cell-based assay.

Protective effect of arctigenin on ethanol-induced neurotoxicity in PC12 cells.

PubMed

Huang, Jia; Xiao, Lan; Wei, Jing-Xiang; Shu, Ya-Hai; Fang, Shi-Qi; Wang, Yong-Tang; Lu, Xiu-Min

2017-04-01

As a neurotropic substance, ethanol can damage nerve cells through an increase in the production of free radicals, interference of neurotrophic factor signaling pathways, activation of endogenous apoptotic signals and other molecular mechanisms. Previous studies have revealed that a number of natural drugs extracted from plants offer protection of nerve cells from damage. Among these, arctigenin (ATG) is a lignine extracted from Arctium lappa (L.), which has been found to exert a neuroprotective effect on scopolamine‑induced memory deficits in mice with Alzheimer's disease and glutamate-induced neurotoxicity in primary neurons. As a result, it may offer beneficial effects on ethanol-induced neurotoxicity. However, the effects of ATG on ethanol‑induced nerve damage remain to be elucidated. To address this issue, the present study used rat pheochromocytoma PC12 cells to investigate the neuroprotective effects of ATG on ethanol-induced cell damage by performing an MTT reduction assay, cell cycle analysis, Hoechst33342/propidium iodide fluorescence staining and flow cytometry to examine apoptosis. The results showed that 10 µM ATG effectively promoted the proliferation of damaged cells, and increased the distribution ratio of the cells at the G2/M and S phases (P<0.05). In addition, the apoptosis and necrosis of the PC12 cells were significantly decreased following treatment with ATG. Therefore, it was concluded that 10 µM ATG had a protective effect on ethanol‑induced injury in PC12 cells.

COPII-coated membranes function as transport carriers of intracellular procollagen I

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gorur, Amita; Yuan, Lin; Kenny, Samuel J.

The coat protein complex II (COPII) is essential for the transport of large cargo, such as 300-nm procollagen I (PC1) molecules, from the endoplasmic reticulum (ER) to the Golgi. Previous work has shown that the CUL3-KLHL12 complex increases the size of COPII vesicles at ER exit sites to more than 300 nm in diameter and accelerates the secretion of PC1. However, the role of large COPII vesicles as PC1 transport carriers was not unambiguously demonstrated. In this study, using stochastic optical reconstruction microscopy, correlated light electron microscopy, and live-cell imaging, we demonstrate the existence of mobile COPII-coated vesicles that completelymore » encapsulate the cargo PC1 and are physically separated from ER. We also developed a cell-free COPII vesicle budding reaction that reconstitutes the capture of PC1 into large COPII vesicles. This process requires COPII proteins and the GTPase activity of the COPII subunit SAR1. We conclude that large COPII vesicles are bona fide carriers of PC1.« less

COPII-coated membranes function as transport carriers of intracellular procollagen I

DOE PAGES

Gorur, Amita; Yuan, Lin; Kenny, Samuel J.; ...

2017-04-20

The coat protein complex II (COPII) is essential for the transport of large cargo, such as 300-nm procollagen I (PC1) molecules, from the endoplasmic reticulum (ER) to the Golgi. Previous work has shown that the CUL3-KLHL12 complex increases the size of COPII vesicles at ER exit sites to more than 300 nm in diameter and accelerates the secretion of PC1. However, the role of large COPII vesicles as PC1 transport carriers was not unambiguously demonstrated. In this study, using stochastic optical reconstruction microscopy, correlated light electron microscopy, and live-cell imaging, we demonstrate the existence of mobile COPII-coated vesicles that completelymore » encapsulate the cargo PC1 and are physically separated from ER. We also developed a cell-free COPII vesicle budding reaction that reconstitutes the capture of PC1 into large COPII vesicles. This process requires COPII proteins and the GTPase activity of the COPII subunit SAR1. We conclude that large COPII vesicles are bona fide carriers of PC1.« less

Gemcitabine diphosphate choline is a major metabolite linked to the Kennedy pathway in pancreatic cancer models in vivo.

PubMed

Bapiro, T E; Frese, K K; Courtin, A; Bramhall, J L; Madhu, B; Cook, N; Neesse, A; Griffiths, J R; Tuveson, D A; Jodrell, D I; Richards, F M

2014-07-15

The modest benefits of gemcitabine (dFdC) therapy in patients with pancreatic ductal adenocarcinoma (PDAC) are well documented, with drug delivery and metabolic lability cited as important contributing factors. We have used a mouse model of PDAC: KRAS(G12D); p53(R172H); pdx-Cre (KPC) that recapitulates the human disease to study dFdC intra-tumoural metabolism. LC-MS/MS and NMR were used to measure drug and physiological analytes. Cytotoxicity was assessed by the Sulphorhodamine B assay. In KPC tumour tissue, we identified a new, Kennedy pathway-linked dFdC metabolite (gemcitabine diphosphate choline (GdPC)) present at equimolar amounts to its precursor, the accepted active metabolite gemcitabine triphosphate (dFdCTP). Utilising additional subcutaneous PDAC tumour models, we demonstrated an inverse correlation between GdPC/dFdCTP ratios and cytidine triphosphate (CTP). In tumour homogenates in vitro, CTP inhibited GdPC formation from dFdCTP, indicating competition between CTP and dFdCTP for CTP:phosphocholine cytidylyltransferase (CCT). As the structure of GdPC precludes entry into cells, potential cytotoxicity was assessed by stimulating CCT activity using linoleate in KPC cells in vitro, leading to increased GdPC concentration and synergistic growth inhibition after dFdC addition. GdPC is an important element of the intra-tumoural dFdC metabolic pathway in vivo.

Onjisaponin B derived from Radix Polygalae enhances autophagy and accelerates the degradation of mutant α-synuclein and huntingtin in PC-12 cells.

PubMed

Wu, An-Guo; Wong, Vincent Kam-Wai; Xu, Su-Wei; Chan, Wai-Kit; Ng, Choi-In; Liu, Liang; Law, Betty Yuen-Kwan

2013-11-15

Emerging evidence indicates important protective roles being played by autophagy in neurodegenerative disorders through clearance of aggregate-prone or mutant proteins. In the current study, we aimed to identify autophagy inducers from Chinese medicinal herbs as a potential neuroprotective agent that enhances the clearance of mutant huntingtin and α-synuclein in PC-12 cells. Through intensive screening using the green fluorescent protein-light chain 3 (GFP-LC3) autophagy detection platform, we found that the ethanol extracts of Radix Polygalae (Yuan Zhi) were capable of inducing autophagy. Further investigation showed that among three single components derived from Radix Polygalae--i.e., polygalacic acid, senegenin and onjisaponin B--onjisaponin B was able to induce autophagy and accelerate both the removal of mutant huntingtin and A53T α-synuclein, which are highly associated with Huntington disease and Parkinson disease, respectively. Our study further demonstrated that onjisaponin B induces autophagy via the AMPK-mTOR signaling pathway. Therefore, findings in the current study provide detailed insights into the protective mechanism of a novel autophagy inducer, which is valuable for further investigation as a new candidate agent for modulating neurodegenerative disorders through the reduction of toxicity and clearance of mutant proteins in the cellular level.

Onjisaponin B Derived from Radix Polygalae Enhances Autophagy and Accelerates the Degradation of Mutant α-Synuclein and Huntingtin in PC-12 Cells

PubMed Central

Wu, An-Guo; Wong, Vincent Kam-Wai; Xu, Su-Wei; Chan, Wai-Kit; Ng, Choi-In; Liu, Liang; Law, Betty Yuen-Kwan

2013-01-01

Emerging evidence indicates important protective roles being played by autophagy in neurodegenerative disorders through clearance of aggregate-prone or mutant proteins. In the current study, we aimed to identify autophagy inducers from Chinese medicinal herbs as a potential neuroprotective agent that enhances the clearance of mutant huntingtin and α-synuclein in PC-12 cells. Through intensive screening using the green fluorescent protein-light chain 3 (GFP-LC3) autophagy detection platform, we found that the ethanol extracts of Radix Polygalae (Yuan Zhi) were capable of inducing autophagy. Further investigation showed that among three single components derived from Radix Polygalae—i.e., polygalacic acid, senegenin and onjisaponin B—onjisaponin B was able to induce autophagy and accelerate both the removal of mutant huntingtin and A53T α-synuclein, which are highly associated with Huntington disease and Parkinson disease, respectively. Our study further demonstrated that onjisaponin B induces autophagy via the AMPK-mTOR signaling pathway. Therefore, findings in the current study provide detailed insights into the protective mechanism of a novel autophagy inducer, which is valuable for further investigation as a new candidate agent for modulating neurodegenerative disorders through the reduction of toxicity and clearance of mutant proteins in the cellular level. PMID:24248062

The critical chemical and mechanical regulation of folic acid on neural engineering.

PubMed

Kim, Gloria B; Chen, Yongjie; Kang, Weibo; Guo, Jinshan; Payne, Russell; Li, Hui; Wei, Qiong; Baker, Julianne; Dong, Cheng; Zhang, Sulin; Wong, Pak Kin; Rizk, Elias B; Yan, Jiazhi; Yang, Jian

2018-04-03

The mandate of folic acid supplementation in grained products has reduced the occurrence of neural tube defects by one third in the U.S since its introduction by the Food and Drug Administration in 1998. However, the advantages and possible mechanisms of action of using folic acid for peripheral nerve engineering and neurological diseases still remain largely elusive. Herein, folic acid is described as an inexpensive and multifunctional niche component that modulates behaviors in different cells in the nervous system. The multiple benefits of modulation include: 1) generating chemotactic responses on glial cells, 2) inducing neurotrophin release, and 3) stimulating neuronal differentiation of a PC-12 cell system. For the first time, folic acid is also shown to enhance cellular force generation and global methylation in the PC-12 cells, thereby enabling both biomechanical and biochemical pathways to regulate neuron differentiation. These findings are evaluated in vivo for clinical translation. Our results suggest that folic acid-nerve guidance conduits may offer significant benefits as a low-cost, off-the-shelf product for reaching the functional recovery seen with autografts in large sciatic nerve defects. Consequently, folic acid holds great potential as a critical and convenient therapeutic intervention for neural engineering, regenerative medicine, medical prosthetics, and drug delivery. Copyright © 2018 Elsevier Ltd. All rights reserved.

Protein C deficiency in insulin-dependent diabetes: a hyperglycemia-related phenomenon.

PubMed

Ceriello, A; Quatraro, A; Dello Russo, P; Marchi, E; Barbanti, M; Milani, M R; Giugliano, D

1990-08-13

In 30 insulin-dependent diabetic patients protein C (PC) antigen and PC activity were significantly lower than those of matched control healthy subjects. An inverse correlation between fasting plasma glucose and both PC concentration and activity was present in diabetics, while a direct correlation between PC concentration and PC activity was observed. Induced hyperglycemia in diabetic and normal subjects was able to decrease both PC antigen levels and PC activity, and heparin reversed in part this effect. In diabetic patients euglycemia obtained by insulin infusion restored to normal the depressed PC levels. Heparin did not alter both the basal PC concentration and activity in healthy controls. These data stress the major role of hyperglycemia in determining PC decrease in diabetics, and suggest that PC reduction is probably associated to hyperglycemia-enhanced thrombin formation.

Synthesis of novel ring-contracted artemisinin dimers with potent anticancer activities.

PubMed

Zhang, Ning; Yu, Zhimei; Yang, Xiaohong; Hu, Ping; He, Yun

2018-04-25

Artemisinin is a potential anticancer agent with an interesting trioxane sesquiterpene structure. In order to improve the biological activity and metabolic stability of artemisinin, a series of novel ring-contracted artemisinin dimers were synthesized. These dimers were evaluated by MTT assay against six cancer cell lines. Most of the dimmers exhibited improved antiproliferative activities over artemisinin. Especially, compound 8b showed the most pronounced anti-cancer activity for PC12 cancer cells with an IC 50 value of 1.56 μM. Thus, PC12 cancer cells were used to further investigate the mechanism of antiproliferation for this series of compounds. Compound 8b arrested cell cycle at G1 phase and induced cell apoptosis via up-regulation of Bad, Bax, caspase-3 and caspase-9 protein expressions while inhibiting the expression of Bcl-xL. The present studies are the first to synthesize the ring-contracted artemisinin as dimers and show that these dimers have potent anti-tumor activities against several cancer cell lines. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

ULF geomagnetic activity effects on tropospheric temperature, specific humidity, and cloud cover in Antarctica, during 2003-2010

NASA Astrophysics Data System (ADS)

Regi, Mauro; Redaelli, Gianluca; Francia, Patrizia; De Lauretis, Marcello

2017-06-01

In the present study we investigated the possible relationship between the ULF geomagnetic activity and the variations of several atmospheric parameters. In particular, we compared the ULF activity in the Pc1-2 frequency band (100 mHz-5 Hz), computed from geomagnetic field measurements at Terra Nova Bay in Antarctica, with the tropospheric temperature T, specific humidity Q, and cloud cover (high cloud cover, medium cloud cover, and low cloud cover) obtained from reanalysis data set. The statistical analysis was conducted during the years 2003-2010, using correlation and Superposed Epoch Analysis approaches. The results show that the atmospheric parameters significantly change following the increase of geomagnetic activity within 2 days. These changes are evident in particular when the interplanetary magnetic field Bz component is oriented southward (Bz<0) and the By component duskward (By>0). We suggest that both the precipitation of electrons induced by Pc1-2 activity and the intensification of the polar cap potential difference, modulating the microphysical processes in the clouds, can affect the atmosphere conditions.

CELLULAR AND MOLECULAR MECHANISMS OF ACTION OF LINURON: AN ANTIANDROGENIC HERBICIDE THAT PRODUCES REPRODUCTIVE MALFORMATIONS IN MALE RATS

EPA Science Inventory

Title: CELLULAR AND MOLECULAR MECHANISMS OF ACTION OF LINURON: AN ANTIANDROGENIC HERBICIDE THAT PRODUCES REPRODUCTIVE MALFORMATIONS IN MALE RATS. C Lambright1, J Ostby, K Bobseine, V Wilson, AK Hotchkiss2, PC Mann3 and LE Gray Jr1.

Antiandrogenic chemicals alter sex d...

Polyamines and Their Metabolites as Diagnostic Markers of Human Diseases

PubMed Central

Park, Myung Hee; Igarashi, Kazuei

2013-01-01

Polyamines, putrescine, spermidine and spermine, are ubiquitous in living cells and are essential for eukaryotic cell growth. These polycations interact with negatively charged molecules such as DNA, RNA, acidic proteins and phospholipids and modulate various cellular functions including macromolecular synthesis. Dysregulation of the polyamine pathway leads to pathological conditions including cancer, inflammation, stroke, renal failure and diabetes. Increase in polyamines and polyamine synthesis enzymes is often associated with tumor growth, and urinary and plasma contents of polyamines and their metabolites have been investigated as diagnostic markers for cancers. Of these, diacetylated derivatives of spermidine and spermine are elevated in the urine of cancer patients and present potential markers for early detection. Enhanced catabolism of cellular polyamines by polyamine oxidases (PAO), spermine oxidase (SMO) or acetylpolyamine oxidase (AcPAO), increases cellular oxidative stress and generates hydrogen peroxide and a reactive toxic metabolite, acrolein, which covalently incorporates into lysine residues of cellular proteins. Levels of protein-conjuagated acrolein (PC-Acro) and polyamine oxidizing enzymes were increased in the locus of brain infarction and in plasma in a mouse model of stroke and also in the plasma of stroke patients. When the combined measurements of PC-Acro, interleukin 6 (IL-6), and C-reactive protein (CRP) were evaluated, even silent brain infarction (SBI) was detected with high sensitivity and specificity. Considering that there are no reliable biochemical markers for early stage of stroke, PC-Acro and PAOs present promising markers. Thus the polyamine metabolites in plasma or urine provide useful tools in early diagnosis of cancer and stroke. PMID:24009852

The statistical mechanics of complex signaling networks: nerve growth factor signaling

NASA Astrophysics Data System (ADS)

Brown, K. S.; Hill, C. C.; Calero, G. A.; Myers, C. R.; Lee, K. H.; Sethna, J. P.; Cerione, R. A.

2004-10-01

The inherent complexity of cellular signaling networks and their importance to a wide range of cellular functions necessitates the development of modeling methods that can be applied toward making predictions and highlighting the appropriate experiments to test our understanding of how these systems are designed and function. We use methods of statistical mechanics to extract useful predictions for complex cellular signaling networks. A key difficulty with signaling models is that, while significant effort is being made to experimentally measure the rate constants for individual steps in these networks, many of the parameters required to describe their behavior remain unknown or at best represent estimates. To establish the usefulness of our approach, we have applied our methods toward modeling the nerve growth factor (NGF)-induced differentiation of neuronal cells. In particular, we study the actions of NGF and mitogenic epidermal growth factor (EGF) in rat pheochromocytoma (PC12) cells. Through a network of intermediate signaling proteins, each of these growth factors stimulates extracellular regulated kinase (Erk) phosphorylation with distinct dynamical profiles. Using our modeling approach, we are able to predict the influence of specific signaling modules in determining the integrated cellular response to the two growth factors. Our methods also raise some interesting insights into the design and possible evolution of cellular systems, highlighting an inherent property of these systems that we call 'sloppiness.'

Antiproliferative mechanisms of the flavonoids 2,2'-dihydroxychalcone and fisetin in human prostate cancer cells.

PubMed

Haddad, Ahmed Q; Fleshner, Neil; Nelson, Colleen; Saour, Basil; Musquera, Mireia; Venkateswaran, Vasundara; Klotz, Laurence

2010-01-01

We have previously demonstrated the antiproliferative effect of two flavonoids-2,2'-dihydroxychalcone (DHC), a novel synthetic flavonoid, and fisetin, a naturally occurring flavonol-in prostate cancer cells. In this study, we further examine the mechanisms of these compounds on survival and proliferation pathways. DHC and fisetin (1-50 microM) caused a dose-dependent reduction in viability, a concomitant increase in apoptosis in PC3 cells at 72 h, and a decrease in clonogenic survival at 24 h treatment. DHC was considerably more potent than fisetin in these cytotoxicity assays. The mechanism of accelerated cellular senescence was not activated by either compound in PC3 or lymph node carcinoma of the prostate (LNCaP) cells. Gene expression alterations in PC3 and LNCaP cells treated with 15 muM DHC and 25 microM fisetin for 6 to 24 h were determined by oligonucleotide array. Amongst the most highly represented functional categories of genes altered by both compounds was the cell cycle category. In total, 100 cell cycle genes were altered by DHC and fisetin including 27 genes with key functions in G2/M phase that were downregulated by both compounds. Other functional categories altered included chromosome organization, apoptosis, and stress response. These results demonstrate the multiple mechanisms of antitumor activity of DHC and fisetin in prostate cancer cells in vitro.

Assessment of the potential activity of major dietary compounds as selective estrogen receptor modulators in two distinct cell models for proliferation and differentiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lecomte, Sylvain; Lelong, Marie; Bourgine, Gaëlle

Estrogen receptors (ERs) α and β are distributed in most tissues of women and men. ERs are bound by estradiol (E2), a natural hormone, and mediate the pleiotropic and tissue-specific effects of E2, such as proliferation of breast epithelial cells or protection and differentiation of neuronal cells. Numerous environmental molecules, called endocrine disrupting compounds, also interact with ERs. Phytoestrogens belong to this large family and are considered potent therapeutic molecules that act through their selective estrogen receptor modulator (SERM) activity. Using breast cancer cell lines as a model of estrogen-dependent proliferation and a stably ER-expressing PC12 cell line as amore » model of neuronal differentiating cells, we studied the SERM activity of major dietary compounds, such as apigenin, liquiritigenin, daidzein, genistein, coumestrol, resveratrol and zearalenone. The ability of these compounds to induce ER-transactivation and breast cancer cell proliferation and enhance Nerve Growth Factor (NGF) -induced neuritogenesis was assessed. Surprisingly, although all compounds were able to activate the ER through an estrogen responsive element reporter gene, they showed differential activity toward proliferation or differentiation. Apigenin and resveratrol showed a partial or no proliferative effect on breast cancer cells but fully contributed to the neuritogenesis effect of NGF. However, daidzein and zearalenone showed full effects on cellular proliferation but did not induce cellular differentiation. In summary, our results suggest that the therapeutic potential of phytoestrogens can diverge depending on the molecule and the phenotype considered. Hence, apigenin and resveratrol might be used in the development of therapeutics for breast cancer and brain diseases. - Highlights: • SERM activity of dietary compounds on proliferation and differentiation is studied. • All the dietary compounds tested transactivate estrogen receptors. • Apigenin and resveratrol could be good candidates for future therapeutics. • Daidzein and zearalenone are to be avoided to maintain human health.« less

Glycoform-independent prion conversion by highly efficient, cell-based, protein misfolding cyclic amplification

PubMed Central

Moudjou, Mohammed; Chapuis, Jérôme; Mekrouti, Mériem; Reine, Fabienne; Herzog, Laetitia; Sibille, Pierre; Laude, Hubert; Vilette, Didier; Andréoletti, Olivier; Rezaei, Human; Dron, Michel; Béringue, Vincent

2016-01-01

Prions are formed of misfolded assemblies (PrPSc) of the variably N-glycosylated cellular prion protein (PrPC). In infected species, prions replicate by seeding the conversion and polymerization of host PrPC. Distinct prion strains can be recognized, exhibiting defined PrPSc biochemical properties such as the glycotype and specific biological traits. While strain information is encoded within the conformation of PrPSc assemblies, the storage of the structural information and the molecular requirements for self-perpetuation remain uncertain. Here, we investigated the specific role of PrPC glycosylation status. First, we developed an efficient protein misfolding cyclic amplification method using cells expressing the PrPC species of interest as substrate. Applying the technique to PrPC glycosylation mutants expressing cells revealed that neither PrPC nor PrPSc glycoform stoichiometry was instrumental to PrPSc formation and strainness perpetuation. Our study supports the view that strain properties, including PrPSc glycotype are enciphered within PrPSc structural backbone, not in the attached glycans. PMID:27384922

PrPc Does Not Mediate Internalization of PrPSc but Is Required at an Early Stage for De Novo Prion Infection of Rov Cells▿

PubMed Central

Paquet, Sophie; Daude, Nathalie; Courageot, Marie-Pierre; Chapuis, Jérôme; Laude, Hubert; Vilette, Didier

2007-01-01

We have studied the interactions of exogenous prions with an epithelial cell line inducibly expressing PrPc protein and permissive to infection by a sheep scrapie agent. We demonstrate that abnormal PrP (PrPSc) and prion infectivity are efficiently internalized in Rov cells, whether or not PrPc is expressed. At odds with earlier studies implicating cellular heparan sulfates in PrPSc internalization, we failed to find any involvement of such molecules in Rov cells, indicating that prions can enter target cells by several routes. We further show that PrPSc taken up in the absence of PrPc was unable to promote efficient prion multiplication once PrPc expression was restored in the cells. This observation argues that interaction of PrPSc with PrPc has to occur early, in a specific subcellular compartment(s), and is consistent with the view that the first prion multiplication events may occur at the cell surface. PMID:17626095

Rapid in situ assessment of physiological activities in bacterial biofilms using fluorescent probes

NASA Technical Reports Server (NTRS)

Yu, F. P.; McFeters, G. A.

1994-01-01

Two rapid in situ enumeration methods using fluorescent probes were used to assess the physiological activities of Klebsiella pneumoniae biofilms on stainless steel. Fluorescent dyes, 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) and rhodamine 123 (Rh 123), were chosen to perform this study. CTC is a soluble redox indicator which can be reduced by respiring bacteria to fluorescent CTC-formazan crystals. Rh 123 is incorporated into bacteria with respect to cellular proton motive force. The intracellular accumulation of these fluorescent dyes can be determined using epifluorescence microscopy. The results obtained with these two fluorescent probes in situ were compared to the plate count (PC) and in situ direct viable count (DVC) methods. Viable cell densities within biofilms determined by the three in situ methods were comparable and always showed approximately 2-fold higher values than those obtained with the PC method. As an additional advantage, the results were observed after 2 h, which was shorter than the 4 h incubation time required for the DVC method and 24 h for colony formation. The results indicate that staining with CTC and Rh 123 provides rapid information regarding cell numbers and physiological activities of bacteria within biofilms.

Differential response of DU145 and PC3 prostate cancer cells to ionizing radiation: role of reactive oxygen species, GSH and Nrf2 in radiosensitivity.

PubMed

Jayakumar, Sundarraj; Kunwar, Amit; Sandur, Santosh K; Pandey, Badri N; Chaubey, Ramesh C

2014-01-01

Radioresistance is the major impediment in radiotherapy of many cancers including prostate cancer, necessitating the need to understand the factors contributing to radioresistance in tumor cells. In the present study, the role of cellular redox and redox sensitive transcription factor, Nrf2 in the radiosensitivity of prostate cancer cell lines PC3 and DU145, has been investigated. Differential radiosensitivity of PC3 and DU145 cells was assessed using clonogenic assay, flow cytometry, and comet assay. Their redox status was measured using DCFDA and DHR probes. Expression of Nrf2 and its dependent genes was measured by EMSA and real time PCR. Knockdown studies were done using shRNA transfection. PC3 and DU145 cells differed significantly in their radiosensitivity as observed by clonogenic survival, apoptosis and neutral comet assays. Both basal and inducible levels of ROS were higher in PC3 cells than that of DU145 cells. DU145 cells showed higher level of basal GSH content and GSH/GSSG ratio than that of PC3 cells. Further, significant increase in both basal and induced levels of Nrf2 and its dependent genes was observed in DU145 cells. Knock-down experiments and pharmacological intervention studies revealed the involvement of Nrf2 in differential radio-resistance of these cells. Cellular redox status and Nrf2 levels play a causal role in radio-resistance of prostate cancer cells. The pivotal role Nrf2 has been shown in the radioresistance of tumor cells and this study will further help in exploiting this factor in radiosensitization of other tumor cell types. © 2013.

The Aqueous Extract of Rhizome of Gastrodia elata Protected Drosophila and PC12 Cells against Beta-Amyloid-Induced Neurotoxicity

PubMed Central

Ng, Chun-Fai; Ko, Chun-Hay; Koon, Chi-Man; Xian, Jia-Wen; Leung, Ping-Chung; Fung, Kwok-Pui; Chan, Ho Yin Edwin; Lau, Clara Bik-San

2013-01-01

This study aims to investigate the neuroprotective effect of the rhizome of Gastrodia elata (GE) aqueous extract on beta-amyloid(Aβ)-induced toxicity in vivo and in vitro. Transgenic Drosophila mutants with Aβ-induced neurodegeneration in pan-neuron and ommatidia were used to determine the efficacy of GE. The antiapoptotic and antioxidative mechanisms of GE were also studied in Aβ-treated pheochromocytoma (PC12) cells. In vivo studies demonstrated that GE (5 mg/g Drosophila media)-treated Drosophila possessed a longer lifespan, better locomotor function, and less-degenerated ommatidia when compared with the Aβ-expressing control (all P < 0.05). In vitro studies illustrated that GE increased the cell viability of Aβ-treated PC12 cells in dose-dependent manner, probably through attenuation of Aβ-induced oxidative and apoptotic stress. GE also significantly upregulated the enzymatic activities of catalase, superoxide dismutase, and glutathione peroxidase, leading to the decrease of reactive oxidation species production and apoptotic marker caspase-3 activity. In conclusion, our current data presented the first evidence that the aqueous extract of GE was capable of reducing the Aβ-induced neurodegeneration in Drosophila, possibly through inhibition of apoptosis and reduction of oxidative stress. GE aqueous extract could be developed as a promising herbal agent for neuroprotection and novel adjuvant therapies for Alzheimer's disease. PMID:24174977

Production of cattle lacking prion protein

PubMed Central

Richt, Jürgen A; Kasinathan, Poothappillai; Hamir, Amir N; Castilla, Joaquin; Sathiyaseelan, Thillai; Vargas, Francisco; Sathiyaseelan, Janaki; Wu, Hua; Matsushita, Hiroaki; Koster, Julie; Kato, Shinichiro; Ishida, Isao; Soto, Claudio; Robl, James M; Kuroiwa, Yoshimi

2010-01-01

Prion diseases are caused by propagation of misfolded forms of the normal cellular prion protein PrPC, such as PrPBSE in bovine spongiform encephalopathy (BSE) in cattle and PrPCJD in Creutzfeldt-Jakob disease (CJD) in humans1. Disruption of PrPC expression in mice, a species that does not naturally contract prion diseases, results in no apparent developmental abnormalities2–5. However, the impact of ablating PrPC function in natural host species of prion diseases is unknown. Here we report the generation and characterization of PrPC-deficient cattle produced by a sequential gene-targeting system6. At over 20 months of age, the cattle are clinically, physiologically, histopathologically, immunologically and reproductively normal. Brain tissue homogenates are resistant to prion propagation in vitro as assessed by protein misfolding cyclic amplification7. PrPC-deficient cattle may be a useful model for prion research and could provide industrial bovine products free of prion proteins. PMID:17195841

Spontaneous Generation of Infectious Prion Disease in Transgenic Mice

PubMed Central

Castilla, Joaquín; Pintado, Belén; Gutiérrez-Adan, Alfonso; Andréoletti, Olivier; Aguilar-Calvo, Patricia; Arroba, Ana-Isabel; Parra-Arrondo, Beatriz; Ferrer, Isidro; Manzanares, Jorge; Espinosa, Juan-Carlos

2013-01-01

We generated transgenic mice expressing bovine cellular prion protein (PrPC) with a leucine substitution at codon 113 (113L). This protein is homologous to human protein with mutation 102L, and its genetic link with Gerstmann–Sträussler–Scheinker syndrome has been established. This mutation in bovine PrPC causes a fully penetrant, lethal, spongiform encephalopathy. This genetic disease was transmitted by intracerebral inoculation of brain homogenate from ill mice expressing mutant bovine PrP to mice expressing wild-type bovine PrP, which indicated de novo generation of infectious prions. Our findings demonstrate that a single amino acid change in the PrPC sequence can induce spontaneous generation of an infectious prion disease that differs from all others identified in hosts expressing the same PrPC sequence. These observations support the view that a variety of infectious prion strains might spontaneously emerge in hosts displaying random genetic PrPC mutations. PMID:24274622

Inhibition of Phosphatidylcholine-Specific Phospholipase C Interferes with Proliferation and Survival of Tumor Initiating Cells in Squamous Cell Carcinoma

PubMed Central

Ferri, Renata; Mercurio, Laura; Canevari, Silvana; Podo, Franca; Miotti, Silvia; Iorio, Egidio

2015-01-01

Purpose The role of phosphatidylcholine-specific phospholipase C (PC-PLC), the enzyme involved in cell differentiation and proliferation, has not yet been explored in tumor initiating cells (TICs). We investigated PC-PLC expression and effects of PC-PLC inhibition in two adherent (AD) squamous carcinoma cell lines (A431 and CaSki), with different proliferative and stemness potential, and in TIC-enriched floating spheres (SPH) originated from them. Results Compared with immortalized non-tumoral keratinocytes (HaCaT) A431-AD cells showed 2.5-fold higher PC-PLC activity, nuclear localization of a 66-kDa PC-PLC isoform, but a similar distribution of the enzyme on plasma membrane and in cytoplasmic compartments. Compared with A431-AD, A431-SPH cells showed about 2.8-fold lower PC-PLC protein and activity levels, but similar nuclear content. Exposure of adherent cells to the PC-PLC inhibitor D609 (48h) induced a 50% reduction of cell proliferation at doses comprised between 33 and 50 μg/ml, without inducing any relevant cytotoxic effect (cell viability 95±5%). In A431-SPH and CaSki-SPH D609 induced both cytostatic and cytotoxic effects at about 20 to 30-fold lower doses (IC50 ranging between 1.2 and 1.6 μg/ml). Furthermore, D609 treatment of A431-AD and CaSki-AD cells affected the sphere-forming efficiency, which dropped in both cells, and induced down-modulation of stem-related markers mRNA levels (Oct4, Nestin, Nanog and ALDH1 in A431; Nestin and ALDH1 in CaSki cells). Conclusions These data suggest that the inhibition of PC-PLC activity may represent a new therapeutic approach to selectively target the most aggressive and tumor promoting sub-population of floating spheres originated from squamous cancer cells possessing different proliferative and stemness potential. PMID:26402860

Differences in levels of platelet-derived microparticles in platelet components prepared using the platelet rich plasma, buffy coat, and apheresis procedures.

PubMed

Noulsri, Egarit; Udomwinijsilp, Prapaporn; Lerdwana, Surada; Chongkolwatana, Viroje; Permpikul, Parichart

2017-04-01

There has been an increased interest in platelet-derived microparticles (PMPs) in transfusion medicine. Little is known about PMP status during the preparation of platelet concentrates for transfusion. The aim of this study is to compare the PMP levels in platelet components prepared using the buffy coat (BC), platelet-rich plasma platelet concentrate (PRP-PC), and apheresis (AP) processes. Platelet components were prepared using the PRP-PC and BC processes. Apheresis platelets were prepared using the Trima Accel and Amicus instruments. The samples were incubated with annexin A5-FITC, CD41-PE, and CD62P-APC. At day 1 after processing, the PMPs and activated platelets were determined using flow cytometry. Both the percentage and number of PMPs were higher in platelet components prepared using the Amicus instrument (2.6±1.8, 32802±19036 particles/μL) than in platelet components prepared using the Trima Accel instrument (0.5±0.4, 7568±5298 particles/μL), BC (1.2±0.6, 12,920±6426 particles/μL), and PRP-PC (0.9±0.6, 10731±5514 particles/μL). Both the percentage and number of activated platelets were higher in platelet components prepared using the Amicus instrument (33.2±13.9, 427553±196965 cells/μL) than in platelet components prepared using the Trima Accel instrument (16.2±6.1, 211209±87706 cells/μL), BC (12.9±3.2, 140624±41003 cells/μL), and PRP-PC (21.1±6.3, 265210±86257 cells/μL). The study suggests high variability of PMPs and activated platelets in platelet components prepared using different processes. This result may be important in validating the instruments involved in platelet blood collection and processing. Copyright © 2016 Elsevier Ltd. All rights reserved.

Material Properties of Matrix Lipids Determine Conformation and Intermolecular Reactivity of a Diacetylenic Phosphatidylcholine in the Lipid Bilayer

PubMed Central

Puri, Anu; Jang, Hyunbum; Yavlovich, Amichai; Masood, M. Athar; Veenstra, Timothy D.; Luna, Carlos; Aranda-Espinoza, Helim; Nussinov, Ruth; Blumenthal, Robert

2011-01-01

Photopolymerizable phospholipid DC8,9PC (1,2-bis-(tricosa-10,12-diynoyl)-sn-glycero-3-phosphocholine) exhibits unique assembly characteristics in the lipid bilayer. Due to the presence of the diacetylene groups, DC8,9PC undergoes polymerization upon UV (254 nm) exposure and assumes chromogenic properties. DC8,9PC photopolymerization in a gel phase matrix lipid 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) monitored by UV-VIS absorption spectroscopy occurred within 2 minutes after UV treatment, whereas no spectral shifts were observed when DC8,9PC was incorporated in a liquid phase matrix 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). Liquid chromatography-tandem mass spectrometry analysis showed a decrease in DC8,9PC monomer in both DPPC and POPC environments without any change in matrix lipids in UV-treated samples. Molecular Dynamics (MD) simulations of DPPC/DC8,9PC and POPC/DC8,9PC bilayers indicate that the DC8,9PC molecules adjust to the thickness of the matrix lipid bilayer. Furthermore, motions of DC8,9PC in the gel phase bilayer are more restricted than in the fluid bilayer. The restricted motional flexibility of DC8,9PC (in the gel phase) enables the reactive diacetylenes in individual molecules to align and undergo polymerization, whereas the unrestricted motions in the fluid bilayer restrict polymerization due to the lack of appropriate alignment of the DC8,9PC fatty acyl chains. Fluorescence microscopy data indicates homogenous distribution of the lipid probe 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-lissamine rhodamine B sulfonyl ammonium salt (N-Rh-PE) in POPC/DC8,9PC monolayers, but domain formation in DPPC/DC8,9PC monolayers. These results show that the DC8,9PC molecules cluster and assume the preferred conformation in the gel phase matrix for UV-triggered polymerization reaction. PMID:22053903

Increased storage and secretion of phosphatidylcholines by senescent human peritoneal mesothelial cells.

PubMed

Bartosova, Maria; Rudolf, Andras; Pichl, Sebastian; Schmidt, Kathrin; Okun, Jürgen G; Straub, Beate K; Rutkowski, Rafael; Witowski, Janusz; Schmitt, Claus P

2016-08-01

Human peritoneal mesothelial cells (HPMC) secrete phosphatidylcholines (PC) which form a lipid bilayer lining the peritoneum. They prevent frictions and adhesions and act as a barrier to the transport of water-soluble solutes while permitting water flux. PC may play an essential role in peritoneal integrity and function, the role of PD induced HPMC senescence on PC homeostasis, however, is unknown. HPMC cell lines were isolated from four non-uremic patients. Expression of the three PC synthesis genes (rt-PCR), and cellular storage and secretion of PC (ESI-mass-spectrometry) were analyzed in young and senescent HPMC (>Hayflick-limit). Senescent cells displayed significantly altered morphology; flow cytometry demonstrated extensive staining for senescence-associated beta galactosidase. Nine different PC were detected in HPMC with palmitoyl-myristoyl phosphatidylcholine (PMPC) being most abundant. In senescent HPMC mRNA expression of the three key PC synthesis genes was 1.5-, 2.4- and 6-fold increased as compared to young HPMC, with the latter, phosphatidylcholine cytidylyltransferase, being rate limiting. Intracellular storage of the nine PC was 75-450 % higher in senescent vs. young HPMC, PC secretion rates were 100-300 % higher. Intracellular PC concentrations were not correlated with the PC secretion rates. Electron microscopy demonstrated lamellar bodies, the primary storage site of PC, in senescent but not in young cells. Senescent HPMC store and secrete substantially more PC than young cells. Our findings indicate a novel protective mechanism, which should counteract peritoneal damage induced by chronic exposure to PD fluids.

PcToll3 was involved in anti-Vibrio response by regulating the expression of antimicrobial peptides in red swamp crayfish, Procambarus clarkii.

PubMed

Lan, Jiang-Feng; Wei, Shun; Wang, Yu-Qing; Dai, Yun-Jia; Tu, Jia-Gang; Zhao, Li-Juan; Li, Xin-Cang; Qin, Qi-Wei; Chen, Nan; Lin, Li

2016-10-01

Tolls and Toll-like receptors (TLRs) play an important role in host immune defenses by regulating the expression of antimicrobial peptides (AMPs) and cytokines, but the functional differences of crustacean Tolls from Drosophila Tolls or Mammal TLRs are largely unknown. A novel Toll receptor, named PcToll3, was identified from red swamp crayfish, Procambarus clarkii. It was widely expressed in all detected tissues, and its transcript in hemocytes was up-regulated at 12 h after Vibrio parahemolyticus (Vibrio) injection or at 24 h post white spot syndrome virus (WSSV) challenge. After knockdown of PcToll3, the activity of bacterial clearance was inhibited, and the expression levels of AMPs including Crustin1 (Cru1), Anti-lippopolysaccharide factor 1 (ALF1), and Lysozymes1 (Lys1), which could be up-regulated by Vibrio, were all affected. Meanwhile, PcToll3 silencing influenced the expression of myeloid differentiation factor 88 (PcMyd88), tumor necrosis factor-associated factor 6 (PcTRAF6), and PcDorsal, which were the counterparts of Drosophila Toll signaling pathway. Interestingly, PcToll3 silencing inhibited translocation of PcDorsal from cytoplasm to nucleus. Furthermore, the knockdown of PcDorsal also impaired the expression of AMPs after Vibrio challenge. Hence, we concluded that, besides participating in antiviral immunity, PcToll3 might also regulate the expression of Cru1 and Lys1 to participate in anti-Vibrio immune responses by promoting PcDorsal translocation into nucleus. Copyright © 2016 Elsevier Ltd. All rights reserved.

Protective effects of NMDA receptor antagonist, memantine, against senescence of PC12 cells: A possible role of nNOS and combined effects with donepezil.

PubMed

Ota, Hidetaka; Ogawa, Sumito; Ouchi, Yasuyoshi; Akishita, Masahiro

2015-12-01

Alzheimer disease (AD) is a neurodegenerative disorder characterized by cognitive dysfunction. The pathology of AD is mainly related to amyloid ß (Aß)-peptides, but glutamate-mediated toxicity is also one of the main processes of memory impairment in AD. Glutamate is the main excitatory neurotransmitter in the central nervous system (CNS) and is particularly involved in synaptic plasticity, memory, and learning. Memantine is a low-affinity voltage-dependent noncompetitive antagonist at glutamatergic NMDA receptors. Here,we investigated whether memantine protects against glutamate-induced senescence. In PC12 cells, treatment with glutamate induced senescent phenotypes as judged by the cell appearance and senescence-associated ß-galactosidase (SA-ßgal) in parallel with decreased SIRT1 and increased p53 expression. However, treatment with memantine decreased glutamate-induced senescent PC12 cells and reversed the changes in SIRT1 and p53 expression. Glutamate is known to stimulate the production of NO and O2(-) and has the capacity to generate ONOO(-) in the CNS. Therefore, we investigated whether glutamate activates nNOS and memantine reverses it. Treatment with glutamate increased nNOS expression, activity, and production of NO,whereas memantine blocked them. Next, the in vivo effects of memantine on cognitive function in senescence-accelerated mouse prone 8 (SAMP8), as a model of AD, were investigated. In the Morris water maze test, SAMP8 showed a marked decline in performance, but memantine administration improved it. Moreover, neuronal senescence and the level of oxidative stress in the hippocampus were decreased by memantine. Finally, the effects of combination treatment with memantine and donepezil, a cholinesterase inhibitor, were investigated. We observed additive effects of memantine and donepezil on the senescent phenotype of PC12 cells and the hippocampus of SAMP8. These results indicate that inhibition of the NMDA receptor by memantine leads to a decrease innNOS activity and results in a reduction of glutamate-induced senescence. Thus, our present study suggests a critical role of memantine in the prevention of neuronal aging, and supports that donepezil has a combined effect with memantine.

Hyperforin activates nonselective cation channels (NSCCs).

PubMed

Treiber, Kristina; Singer, Andrea; Henke, Bettina; Müller, Walter E

2005-05-01

A large body of evidence supports the preclinical antidepressant profile of hyperforin including inhibition of the synaptosomal uptake of several neurotransmitters by hyperforin and studies in behavioural models. In contrast to other antidepressants, hyperforin does not directly inhibit neurotransmitter transporters, but instead uptake inhibition seems to be the consequence of an elevated intracellular sodium concentration ([Na+]i). The mechanism of hyperforin-induced elevation of [Na+]i was investigated using two different cell types: human platelets and rat pheochromocytoma cells (PC12 cells). In both cell systems, hyperforin increased both [Na+]i and free intracellular Ca2+ concentration ([Ca2+]i). One pathway for Na+ and Ca2+ entry is mediated by nonselective cation channels (NSCCs), which can be blocked by SK&F 96365 and LOE 908. LOE 908 is a blocker of both NSCC1 and NSCC2 subclasses, while SK&F 96365 blocks NSCC2 only. Both SK&F 96365 and LOE 908 completely inhibited the hyperforin-induced influx of Na+ and Ca2+ into platelets and PC12 cells. This indicates that hyperforin is mainly active upon NSCC2. The effect of hyperforin is inhibited by La3+ and Gd3+, indicating that there is a potential homology with canonical transient receptor potential protein channels (TRPC channels). Moreover, La3+ and Gd3+ attenuate the effect of hyperforin on serotonin uptake in human platelets. Additionally, hyperforin induces barium influx in PC12 cells and this influx can be inhibited by SK&F 96365, LOE 908, Gd3+ and La3+. In summary, these findings suggest that hyperforin represents a new principle for preclinical antidepressant activity, modulating brain neurotransmission by inhibition of neurotransmitter uptake via activation of NSCCs.British Journal of Pharmacology (2005) 145, 75-83. doi:10.1038/sj.bjp.0706155.

Hyperforin activates nonselective cation channels (NSCCs)

PubMed Central

Treiber, Kristina; Singer, Andrea; Henke, Bettina; Müller, Walter E

2005-01-01

A large body of evidence supports the preclinical antidepressant profile of hyperforin including inhibition of the synaptosomal uptake of several neurotransmitters by hyperforin and studies in behavioural models. In contrast to other antidepressants, hyperforin does not directly inhibit neurotransmitter transporters, but instead uptake inhibition seems to be the consequence of an elevated intracellular sodium concentration ([Na+]i). The mechanism of hyperforin-induced elevation of [Na+]i was investigated using two different cell types: human platelets and rat pheochromocytoma cells (PC12 cells). In both cell systems, hyperforin increased both [Na+]i and free intracellular Ca2+ concentration ([Ca2+]i). One pathway for Na+ and Ca2+ entry is mediated by nonselective cation channels (NSCCs), which can be blocked by SK&F 96365 and LOE 908. LOE 908 is a blocker of both NSCC1 and NSCC2 subclasses, while SK&F 96365 blocks NSCC2 only. Both SK&F 96365 and LOE 908 completely inhibited the hyperforin-induced influx of Na+ and Ca2+ into platelets and PC12 cells. This indicates that hyperforin is mainly active upon NSCC2. The effect of hyperforin is inhibited by La3+ and Gd3+, indicating that there is a potential homology with canonical transient receptor potential protein channels (TRPC channels). Moreover, La3+ and Gd3+ attenuate the effect of hyperforin on serotonin uptake in human platelets. Additionally, hyperforin induces barium influx in PC12 cells and this influx can be inhibited by SK&F 96365, LOE 908, Gd3+ and La3+. In summary, these findings suggest that hyperforin represents a new principle for preclinical antidepressant activity, modulating brain neurotransmission by inhibition of neurotransmitter uptake via activation of NSCCs. PMID:15723093

Human Albumin Prevents 6-Hydroxydopamine-Induced Loss of Tyrosine Hydroxylase in In Vitro and In Vivo

PubMed Central

Zhang, Li-Juan; Xue, Yue-Qiang; Yang, Chun; Yang, Wei-Hua; Chen, Long; Zhang, Qian-Jin; Qu, Ting-Yu; Huang, Shile; Zhao, Li-Ru; Wang, Xiao-Min; Duan, Wei-Ming

2012-01-01

Human albumin has recently been demonstrated to protect brain neurons from injury in rat ischemic brain. However, there is no information available about whether human albumin can prevent loss of tyrosine hydroxylase (TH) expression of dopaminergic (DA) neurons induced by 6-hydroxydopamine (6-OHDA) toxicity that is most commonly used to create a rat model of Parkinson's disease (PD). In the present study, two microliters of 1.25% human albumin were stereotaxically injected into the right striatum of rats one day before or 7 days after the 6-OHDA lesion in the same side. D-Amphetamine-induced rotational asymmetry was measured 7 days, 3 and 10 weeks after 6-OHDA lesion. We observed that intrastriatal administration of human albumin significantly reduced the degree of rotational asymmetry. The number of TH-immunoreactive neurons present in the substantia nigra was greater in 6-OHDA lesioned rats following human albumin-treatment than non-human albumin treatment. TH-immunoreactivity in the 6-OHDA-lesioned striatum was also significantly increased in the human albumin-treated rats. To examine the mechanisms underlying the effects of human albumin, we challenged PC12 cells with 6-OHDA as an in vitro model of PD. Incubation with human albumin prevented 6-OHDA-induced reduction of cell viability in PC12 cell cultures, as measured by MTT assay. Furthermore, human albumin reduced 6-OHDA-induced formation of reactive oxygen species (ROS) and apoptosis in cultured PC12 cells, as assessed by flow cytometry. Western blot analysis showed that human albumin inhibited 6-OHDA-induced activation of JNK, c-Jun, ERK, and p38 mitogen-activated protein kinases (MAPK) signaling in PC12 cultures challenged with 6-OHDA. Human albumin may protect against 6-OHDA toxicity by influencing MAPK pathway followed by anti-ROS formation and anti-apoptosis. PMID:22815976

Visualizing dopamine released from living cells using a nanoplasmonic probe

NASA Astrophysics Data System (ADS)

Qin, W. W.; Wang, S. P.; Li, J.; Peng, T. H.; Xu, Y.; Wang, K.; Shi, J. Y.; Fan, C. H.; Li, D.

2015-09-01

We report the development of an ultrasensitive nanoplasmonic probe for discriminative detection and imaging of dopamine released from living cells. The sensing mechanism is based on the dopamine-induced seeded-growth of Au nanoparticles (Au NPs) that leads to the shift of the plasmon band. This platform allows for the detection of dopamine with a detection limit down to 0.25 pM within 1 min. This nanoplasmonic assay is further applied to visualize the release of dopamine from living rat pheochromocytoma (PC12) cells under ATP-stimulation with dark-field microscopy (DFM). The DFM results together with real time fluorescence imaging of PC12 cells stained with the Fluo calcium indicator, suggested that ATP stimulated-release of dopamine is concomitant with the Ca2+ influx, and the influx of Ca2+ is through ATP-activated channels instead of the voltage-gated Ca2+ channel (VGC).We report the development of an ultrasensitive nanoplasmonic probe for discriminative detection and imaging of dopamine released from living cells. The sensing mechanism is based on the dopamine-induced seeded-growth of Au nanoparticles (Au NPs) that leads to the shift of the plasmon band. This platform allows for the detection of dopamine with a detection limit down to 0.25 pM within 1 min. This nanoplasmonic assay is further applied to visualize the release of dopamine from living rat pheochromocytoma (PC12) cells under ATP-stimulation with dark-field microscopy (DFM). The DFM results together with real time fluorescence imaging of PC12 cells stained with the Fluo calcium indicator, suggested that ATP stimulated-release of dopamine is concomitant with the Ca2+ influx, and the influx of Ca2+ is through ATP-activated channels instead of the voltage-gated Ca2+ channel (VGC). Electronic supplementary information (ESI) available: Fig. S1-S4 and Table S1. See DOI: 10.1039/c5nr04433b

Protective effect of naringin against the LPS-induced apoptosis of PC12 cells: Implications for the treatment of neurodegenerative disorders

PubMed Central

Wang, Hui; Xu, You Song; Wang, Miao Lin; Cheng, Chao; Bian, Rui; Yuan, Hao; Wang, Yi; Guo, Ting; Zhu, Lin Lin; Zhou, Hang

2017-01-01

Several studies have demonstrated that increased apoptosis plays an essential role in neurodegenerative disorders. It has been demonstrated that lipopolysaccharide (LPS) induces apoptosis largely through the production of intracellular reactive oxygen species (ROS) and inflammatory mediators. In this study, we investigated the potential protective mechanisms of naringin (Nar), a pummelo peel extract, on LPS-induced PC12 cell apoptosis. Nar pre-conditioning prior to stimulation with LPS for 18 h was a prerequisite for evaluating PC12 cell viability and the protective mechanisms of Nar. Nar significantly improved cell survival in a time- and concentration-dependent manner. On the one hand, Nar downregulated cytochrome P450 2E1 (CYP2E1), inhibited the release of ROS, mitigated the stimulation of oxidative stress, and rectified the antioxidant protein contents of nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), superoxide dismutase (SOD)2 and glutathione synthetase (GSS). On the other hand, Nar down-regulated inflammatory gene and protein expression, including interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, HMGB1, high mobility group box 1 protein (HMGB1), cyclo-oxygenase-2 (COX-2), the Toll-like receptor 4 (TLR4)-myeloid differentiation factor 88 (MyD88)-TNF receptor-associated factor 6 (TRAF6) path way and downstream mitogen activated protein kinase (MAPK) phosphorylation, activator protein transcription factor-1 (AP-1) and nuclear factor (NF)-κB. Moroever, Nar markedly attenuated the cytochrome c shift from the mitochondria to the cytosol and regulated caspase-3-related protein expression. To the best of our knowledge, this is the first study to report the antioxidant, anti-inflammatory and anti-apoptotic effects of Nar in neuronal-like PC12 cells. These results suggest that Nar can be utilized as a potential drug for the treatment of neurodegenerative disorders. PMID:28260042

Immunological dysregulation in multiple myeloma microenvironment.

PubMed

Romano, Alessandra; Conticello, Concetta; Cavalli, Maide; Vetro, Calogero; La Fauci, Alessia; Parrinello, Nunziatina Laura; Di Raimondo, Francesco

2014-01-01

Multiple Myeloma (MM) is a systemic hematologic disease due to uncontrolled proliferation of monoclonal plasma cells (PC) in bone marrow (BM). Emerging in other solid and liquid cancers, the host immune system and the microenvironment have a pivotal role for PC growth, proliferation, survival, migration, and resistance to drugs and are responsible for some clinical manifestations of MM. In MM, microenvironment is represented by the cellular component of a normal bone marrow together with extracellular matrix proteins, adhesion molecules, cytokines, and growth factors produced by both stromal cells and PC themselves. All these components are able to protect PC from cytotoxic effect of chemo- and radiotherapy. This review is focused on the role of immunome to sustain MM progression, the emerging role of myeloid derived suppressor cells, and their potential clinical implications as novel therapeutic target.

DIFFERENTIAL MODULATION OF CATECHOLAMINES BY CHLOROTRIAZINE HERBICIDES IN PHEOCHROMOCYTOMA (PC12) CELLS IN VITRO

EPA Science Inventory

Differential modulation of catecholamines by chlorotriazine herbicides in pheochromocytoma (PC12) cells in vitro.

Das PC, McElroy WK, Cooper RL.

Curriculum in Toxicology, University of North Carolina, Chapel Hill 27599, USA.

Epidemiological, wildlife, and lab...

Biodegradable polymeric micelles encapsulated JK184 suppress tumor growth through inhibiting Hedgehog signaling pathway

NASA Astrophysics Data System (ADS)

Zhang, Nannan; Liu, Shichang; Wang, Ning; Deng, Senyi; Song, Linjiang; Wu, Qinjie; Liu, Lei; Su, Weijun; Wei, Yuquan; Xie, Yongmei; Gong, Changyang

2015-01-01

JK184 can specially inhibit Gli in the Hedgehog (Hh) pathway, which showed great promise for cancer therapeutics. For developing aqueous formulation and improving anti-tumor activity of JK184, we prepared JK184 encapsulated MPEG-PCL micelles by the solid dispersion method without using surfactants or toxic organic solvents. The cytotoxicity and cellular uptake of JK184 micelles were both increased compared with the free drug. JK184 micelles induced more apoptosis and blocked proliferation of Panc-1 and BxPC-3 tumor cells. In addition, JK184 micelles exerted a sustained in vitro release behavior and had a stronger inhibitory effect on proliferation, migration and invasion of HUVECs than free JK184. Furthermore, JK184 micelles had stronger tumor growth inhibiting effects in subcutaneous Panc-1 and BxPC-3 tumor models. Histological analysis showed that JK184 micelles improved anti-tumor activity by inducing more apoptosis, decreasing microvessel density and reducing expression of CD31, Ki67, and VEGF in tumor tissues. JK184 micelles showed a stronger inhibition of Gli expression in Hh signaling, which played an important role in pancreatic carcinoma. Furthermore, circulation time of JK184 in blood was prolonged after entrapment in polymeric micelles. Our results suggested that JK184 micelles are a promising drug candidate for treating pancreatic tumors with a highly inhibitory effect on Hh activity.JK184 can specially inhibit Gli in the Hedgehog (Hh) pathway, which showed great promise for cancer therapeutics. For developing aqueous formulation and improving anti-tumor activity of JK184, we prepared JK184 encapsulated MPEG-PCL micelles by the solid dispersion method without using surfactants or toxic organic solvents. The cytotoxicity and cellular uptake of JK184 micelles were both increased compared with the free drug. JK184 micelles induced more apoptosis and blocked proliferation of Panc-1 and BxPC-3 tumor cells. In addition, JK184 micelles exerted a sustained in vitro release behavior and had a stronger inhibitory effect on proliferation, migration and invasion of HUVECs than free JK184. Furthermore, JK184 micelles had stronger tumor growth inhibiting effects in subcutaneous Panc-1 and BxPC-3 tumor models. Histological analysis showed that JK184 micelles improved anti-tumor activity by inducing more apoptosis, decreasing microvessel density and reducing expression of CD31, Ki67, and VEGF in tumor tissues. JK184 micelles showed a stronger inhibition of Gli expression in Hh signaling, which played an important role in pancreatic carcinoma. Furthermore, circulation time of JK184 in blood was prolonged after entrapment in polymeric micelles. Our results suggested that JK184 micelles are a promising drug candidate for treating pancreatic tumors with a highly inhibitory effect on Hh activity. Electronic supplementary information (ESI) available. See DOI: 10.1039/c4nr06300g

The Role of 6-Gingerol on Inhibiting Amyloid β Protein-Induced Apoptosis in PC12 Cells

PubMed Central

Zong, Shao-hui; Zhang, Zhi-yong; Fu, Song-wen; Li, Ke-ke; Fang, Ye; Lu, Li; Xiao, De-qiang

2015-01-01

Abstract Our previous study suggests that ginger root extract can reverse behavioral dysfunction and prevent Alzheimer's disease (AD)-like symptoms induced by the amyloid-β protein (Aβ) in a rat model. 6-Gingerol is the major gingerol in ginger rhizomes, but its effect on the treatment of AD remains unclear. In this study, we aimed to determine if 6-gingerol had a protective effect on Aβ1–42-induced damage and apoptotic death in rat pheochromocytoma cells (PC12 cells) and to investigate the underlying mechanisms by which 6-gingerol may exert its neuroprotective effects. Our results indicated that pre-treatment with 6-gingerol significantly increased cell viability and reduced cell apoptosis in Aβ1–42-treated cells. Moreover, 6-gingerol pretreatment markedly reduced the level of intracellular reactive oxygen species (ROS) and malondialdehyde (MDA), the production of nitric oxide (NO), and the leakage of lactate dehydrogenase (LDH) and increased superoxide dismutase (SOD) activity compared with the Aβ1–42 treatment group. In addition, 6-gingerol pretreatment also significantly enhanced the protein levels of phosphorylated Akt (p-Akt) and glycogen synthase kinase-3β (p-GSK-3β). Overall, these results indicate that 6-gingerol exhibited protective effects on apoptosis induced by Aβ1–42 in cultured PC12 cells by reducing oxidative stress and inflammatory responses, suppressing the activation of GSK-3β and enhancing the activation of Akt, thereby exerting neuroprotective effects. Therefore, 6-gingerol may be useful in the prevention and/or treatment of AD. PMID:25811848

Fibulin-3 negatively regulates ALDH1 via c-MET suppression and increases γ-radiation-induced sensitivity in some pancreatic cancer cell lines

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, In-Gyu, E-mail: igkim@kaeri.re.kr; Department of Radiation Biotechnology and Applied Radioisotope, Korea University of Science and Technology; Lee, Jae-Ha

2014-11-21

Highlights: • FBLN-3 gene was poorly expressed in some pancreatic cancer lines. • FBLN-3 promoter region was highly methylated in some pancreatic cancer cell lines. • FBLN-3 inhibited c-MET activation and expression and reduced cellular level of ALDH1. • FBLN-3/c-Met/ALDH1 axis modulates stemness and EMT in pancreatic cancer cells. - Abstract: Fibulin-3 (FBLN-3) has been postulated to be either a tumor suppressor or promoter depending on the cell type, and hypermethylation of the FBLN-3 promoter is often associated with human disease, especially cancer. We report that the promoter region of the FBLN-3 was significantly methylated (>95%) in some pancreatic cancermore » cell lines and thus FBLN-3 was poorly expressed in pancreatic cancer cell lines such as AsPC-1 and MiaPaCa-2. FBLN-3 overexpression significantly down-regulated the cellular level of c-MET and inhibited hepatocyte growth factor-induced c-MET activation, which were closely associated with γ-radiation resistance of cancer cells. Moreover, we also showed that c-MET suppression or inactivation decreased the cellular level of ALDH1 isozymes (ALDH1A1 or ALDH1A3), which serve as cancer stem cell markers, and subsequently induced inhibition of cell growth in pancreatic cancer cells. Therefore, forced overexpression of FBLN-3 sensitized cells to cytotoxic agents such as γ-radiation and strongly inhibited the stemness and epithelial to mesenchymal transition (EMT) property of pancreatic cancer cells. On the other hand, if FBLN3 was suppressed in FBLN-3-expressing BxPC3 cells, the results were opposite. This study provides the first demonstration that the FBLN-3/c-MET/ALDH1 axis in pancreatic cancer cells partially modulates stemness and EMT as well as sensitization of cells to the detrimental effects of γ-radiation.« less

Experimental study on the neurotoxic effect of β-amyloid on the cytoskeleton of PC12 cells

PubMed Central

Shi, Zhenyu; Fan, Wenjuan; Liu, Hongliang; Deng, Jinbo; Deng, Jiexin

2018-01-01

The aim of the present study was to establish a cell model of Alzheimer's disease (AD) and investigate the neurotoxic effects of β-amyloid (Aβ) on the cytoskeleton. PC12 cells were cultured and treated with Aβ25-35, and cell survival was analyzed with the MTT assay. Cell apoptosis was visualized using 4′,6-diamidino-2-phenylindole staining and the terminal deoxynucleotidyl transferase dUTP nick-end labeling assay. Immunocytochemistry and phalloidin staining were used to label the cytoskeleton of PC12 cells. Aβ25-35 was found to induce PC12 cell apoptosis in a dose-dependent manner (P<0.05). Moreover, Aβ25-35 also caused dose-dependent disintegration of the cytoskeleton (P<0.05). Therefore, the PC12 cell cytoskeleton was found to be sensitive to Aβ25-35 neurotoxicity. The disintegration of the cytoskeleton is likely an important pathological alteration in AD, and Aβ is a key molecule involved in AD pathogenesis. PMID:29436599

The effect of bovine IFN-alpha on the immune response in guinea pigs vaccinated with DNA vaccine of foot-and-mouth disease virus.

PubMed

Guo, Hui-Chen; Liu, Zai-Xin; Sun, Shi-Qi; Leng, Qing-Wen; Li, Dong; Liu, Xiang-Tao; Xie, Qing-Ge

2004-10-01

In this study, we constructed recombinant plasmid pcDNA3.1/P12X3C3D including P1, 2A, 3C, 3D and part of 2B gene of FMDV and pcDNA3.1/IFN containing the gene encoding bovine IFN-alpha. We inoculated the DNA vaccine pcDNA3.1/P12X3C3D with or without pcDNA3.1/IFN to evaluate the efficiency of this DNA vaccine and the immunogenicity of DNA vaccine enhanced by the co-delivery with pcDNA3.1/IFN. After two times of vaccination with DNA vaccine, all of guinea pigs were challenged with 103 ID50 FMDV type O. Anti-FMDV antibody levels were detected by ELISA and T lymphocyte proliferation response was tested by MTT assay. The result shows that guinea pigs inoculated by pcDNA3.1/P12X3C3D alone or with pcDNA3.1/IFN generated specific antibodies and induced an FMDV-specific T lymphocyte proliferation response. FMDV challenge tests showed that one in four guinea pigs immunized by pcDNA3.1/P12X3C3D with pcDNA3.1/IFN was protected from the FMDV serotype O infection. This result indicated that the efficiency of the DNA vaccine was enhanced by co-delivery with pcDNA3.1/IFN. However, the protection rate was considerably lower than that immunized with conventional FMD vaccine.

Decursin attenuates the amyloid-β-induced inflammatory response in PC12 cells via MAPK and nuclear factor-κB pathway.

PubMed

Li, Li; Yang, Yiqiu; Zheng, Jingbin; Cai, Guodi; Lee, Yongwoo; Du, Jikun

2018-02-01

Decursin, the major bioactive component of Angelica gigas Nakai, exhibited neuroprotective properties. Our previous studies showed that decursin conferred neuroprotective effects in PC12 cells induced by Amyloid-β (Aβ) 25-35 via antiapoptosis and antioxidant. In this study, the antiinflammatory effects of decursin against PC12 cells injury stimulated by Aβ 25-35 were assessed. Our results demonstrated that decursin suppressed the expression of cyclooxygenase-2 protein and prostaglandin E2 content which was stimulated by Aβ 25-35 in PC12 cells. Meanwhile, the nuclear translocation of nuclear factor-κB in Aβ 25-35 -treated PC12 cells was also inhibited by decursin. In addition, decursin suppressed phosphorylation of the two upstream pathway kinases, p38 and c-Jun N-terminal kinase. Overall, our findings indicate that decursin exerts protective effects against neuroinflammation stimulated by Aβ 25-35 in PC12 cells by abolishing cyclooxygenase-2 protein expression through inactivation of nuclear factor-κB via the upstream kinases including p38 and c-Jun N-terminal kinase. This work provides a new insight into the pharmacological mode of decursin and should facilitate its therapeutic application in treatment of inflammatory disorders. Copyright © 2017 John Wiley & Sons, Ltd.

Anti-austeric activity of phenolic constituents of seeds of Arctium lappa.

PubMed

Tezuka, Yasuhiro; Yamamoto, Keiichi; Awale, Suresh; Lia, Feng; Yomoda, Satoshi; Kadota, Shigetoshi

2013-04-01

From seeds of Arctium lappa L. (Asteraceae) we obtained arctigenin (1), arctiin (2), chlorogenic acid (3), 4,5-dicaffeoylquinic acid (4), 3,5-dicaffeoylquinic acid (5), 3,4-dicaffeoylquinic acid (6), matairesinol (11), isolappaol A (12), lappaol F (14), and lappaol B (15), together with 1:1 mixtures of isolappaol C (7) and lappaol C (8), arctignan E (9) and arctignan D (10), and 12 and lappaol A (13), while 3,3',4'-tri-O-demethylarctigenin (16), 3,3'-di-O-demethyl-4'-dehydroxyarctigenin (17), and 3-O-demethylarctigenin (18) were obtained by anaerobic microbiological metabolism of 1. Then, we evaluated the in vitro preferential cytotoxic activity of these pure compounds and 1:1 mixtures, together with enterodiol (19) and enterolactone (20), against human pancreatic cancer PANC-1 cells in nutrient-deprived medium (NDM). Among them, 1 and 18 showed potent activity, with PC50 values of 1.75 and 4.38 microM, respectively, while 11, 15, and 17 showed mild activity with PC50 values of 31.1, 30.9, and 38.7 microM, respectively. By comparing their structures and PC50 values, the following structural moieties could be concluded to be important for the preferential cytotoxicity of 1: 1) the 3-hydroxy-4-methoxyphenyl group at the 2-position on the gamma-butyrolactone ring, 2) the less polar substituent at the 3-position on the gamma-butyrolactone ring, and 3) the gamma-butyrolactone ring.

Genetic and Functional Assessment of the Role of the rs13431652-A and rs573225-A Alleles in the G6PC2 Promoter That Are Strongly Associated With Elevated Fasting Glucose Levels

PubMed Central

Bouatia-Naji, Nabila; Bonnefond, Amélie; Baerenwald, Devin A.; Marchand, Marion; Bugliani, Marco; Marchetti, Piero; Pattou, François; Printz, Richard L.; Flemming, Brian P.; Umunakwe, Obi C.; Conley, Nicholas L.; Vaxillaire, Martine; Lantieri, Olivier; Balkau, Beverley; Marre, Michel; Lévy-Marchal, Claire; Elliott, Paul; Jarvelin, Marjo-Riitta; Meyre, David; Dina, Christian; Oeser, James K.; Froguel, Philippe; O'Brien, Richard M.

2010-01-01

OBJECTIVE Genome-wide association studies have identified a single nucleotide polymorphism (SNP), rs560887, located in a G6PC2 intron that is highly correlated with variations in fasting plasma glucose (FPG). G6PC2 encodes an islet-specific glucose-6-phosphatase catalytic subunit. This study examines the contribution of two G6PC2 promoter SNPs, rs13431652 and rs573225, to the association signal. RESEARCH DESIGN AND METHODS We genotyped 9,532 normal FPG participants (FPG <6.1 mmol/l) for three G6PC2 SNPs, rs13431652 (distal promoter), rs573225 (proximal promoter), rs560887 (3rd intron). We used regression analyses adjusted for age, sex, and BMI to assess the association with FPG and haplotype analyses to assess comparative SNP contributions. Fusion gene and gel retardation analyses characterized the effect of rs13431652 and rs573225 on G6PC2 promoter activity and transcription factor binding. RESULTS Genetic analyses provide evidence for a strong contribution of the promoter SNPs to FPG variability at the G6PC2 locus (rs13431652: β = 0.075, P = 3.6 × 10−35; rs573225 β = 0.073 P = 3.6 × 10−34), in addition to rs560887 (β = 0.071, P = 1.2 × 10−31). The rs13431652-A and rs573225-A alleles promote increased NF-Y and Foxa2 binding, respectively. The rs13431652-A allele is associated with increased FPG and elevated promoter activity, consistent with the function of G6PC2 in pancreatic islets. In contrast, the rs573225-A allele is associated with elevated FPG but reduced promoter activity. CONCLUSIONS Genetic and in situ functional data support a potential role for rs13431652, but not rs573225, as a causative SNP linking G6PC2 to variations in FPG, though a causative role for rs573225 in vivo cannot be ruled out. PMID:20622168

Enhancement of neurite outgrowth in PC12 cells stimulated with cyclic AMP and NGF by 6-acylated ascorbic acid 2-O-alpha-glucosides (6-Acyl-AA-2G), novel lipophilic ascorbate derivatives.

PubMed

Zhou, Xiaohua; Tai, Akihiro; Yamamoto, Itaru

2003-03-01

It has been shown that ascorbate (AsA) and its stable derivative, ascorbic acid 2-O-alpha-glucoside (AA-2G), do not elicit neurite outgrowth in PC12 cells. However, these ascorbates are synergistically enhanced by both dibutyryl cyclic AMP (Bt(2)cAMP)- and nerve growth factor (NGF)-induced neurite outgrowth in this model. In the present study, the effects of a series of novel lipophilic ascorbate derivatives, 6-acylated ascorbic acid 2-O-alpha-glucosides (6-Acyl-AA-2G), on neurite outgrowth induced by Bt(2)cAMP and NGF were examined in PC12 cells. We found that all the tested acylated ascorbate derivatives enhanced neurite formation induced by both agents in a dose-dependent manner. Of the 6-Acyl-AA-2G derivatives, 6-octanoyl ascorbic acid 2-O-alpha-glucoside (6-Octa-AA-2G) enhanced the Bt(2)cAMP-induced phosphorylated MAPK p44 and p42 expression. A alpha-glucosidase inhibitor, castanospermine, completely abrogated the promotion of neurite outgrowth and MAPK expression by 6-Octa-AA-2G. Addition of 6-Octa-AA-2G (0.5 mM) to PC12 cells caused a rapid and significant increase in intracellular AsA content, which reached a maximum and was maintained from 12 to 24 h after the culture. These findings suggest that 6-Acyl-AA-2G is rapidly hydrolyzed to AsA within the cell and enhances neurite differentiation through the interaction with the inducer-activated MAPK pathway.

Perfluorinated chemicals: differential toxicity, inhibition of aromatase activity and alteration of cellular lipids in human placental cells.

PubMed

Gorrochategui, Eva; Pérez-Albaladejo, Elisabet; Casas, Josefina; Lacorte, Sílvia; Porte, Cinta

2014-06-01

The cytotoxicity of eight perfluorinated chemicals (PFCs), namely, perfluorobutanoic acid (PFBA), perfluorohexanoic acid (PFHxA), perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), perfluorododecanoic acid (PFDoA), perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHxS) and perfluorooctanesulfonate (PFOS) was assessed in the human placental choriocarcinoma cell line JEG-3. Only the long chain PFCs--PFOS, PFDoA, PFNA, PFOA--showed significant cytotoxicity in JEG-3 cells with EC50 values in the range of 107 to 647 μM. The observed cytotoxicity was to some extent related to a higher uptake of the longer chain PFCs by cells (PFDoA>PFOS≫PFNA>PFOA>PFHxA). Moreover, this work evidences a high potential of PFOS, PFOA and PFBS to act as aromatase inhibitors in placental cells with IC50s in the range of 57-80 μM, the inhibitory effect of PFBS being particularly important despite the rather low uptake of the compound by cells. Finally, exposure of JEG-3 cells to a mixture of the eight PFCs (0.6 μM each) led to a relative increase (up to 3.4-fold) of several lipid classes, including phosphatidylcholines (PCs), plasmalogen PC and lyso plasmalogen PC, which suggests an interference of PFCs with membrane lipids. Overall, this work highlights the ability of the PFC mixture to alter cellular lipid pattern at concentrations well below those that generate toxicity, and the potential of the short chain PFBS, often considered a safe substitute of PFOS, to significantly inhibit aromatase activity in placental cells. Copyright © 2014 Elsevier Inc. All rights reserved.

Astrocyte truncated-TrkB mediates BDNF antiapoptotic effect leading to neuroprotection.

PubMed

Saba, Julieta; Turati, Juan; Ramírez, Delia; Carniglia, Lila; Durand, Daniela; Lasaga, Mercedes; Caruso, Carla

2018-05-31

Astrocytes are glial cells that help maintain brain homeostasis and become reactive in neurodegenerative processes releasing both harmful and beneficial factors. We have demonstrated that brain-derived neurotrophic factor (BDNF) expression is induced by melanocortins in astrocytes but BDNF actions in astrocytes are largely unknown. We hypothesize that BDNF may prevent astrocyte death resulting in neuroprotection. We found that BDNF increased astrocyte viability, preventing apoptosis induced by serum deprivation by decreasing active caspase-3 and p53 expression. The antiapoptotic action of BDNF was abolished by ANA-12 (a specific TrkB antagonist) and by K252a (a general Trk antagonist). Astrocytes only express the BDNF receptor TrkB truncated isoform 1, TrkB-T1. BDNF induced ERK, Akt and Src (a non-receptor tyrosine kinase) activation in astrocytes. Blocking ERK and Akt pathways abolished BDNF protection in serum deprivation-induced cell death. Moreover, BDNF protected astrocytes from death by 3-nitropropionic acid (3-NP), an effect also blocked by ANA-12, K252a, and inhibitors of ERK, calcium and Src. BDNF reduced reactive oxygen species (ROS) levels induced in astrocytes by 3-NP and increased xCT expression and glutathione levels. Astrocyte conditioned media (ACM) from untreated astrocytes partially protected PC12 neurons whereas ACM from BDNF-treated astrocytes completely protected PC12 neurons from 3-NP-induced apoptosis. Both ACM from control and BDNF-treated astrocytes markedly reduced ROS levels induced by 3-NP in PC12 cells. Our results demonstrate that BDNF protects astrocytes from cell death through TrkB-T1 signaling, exerts an antioxidant action, and induces release of neuroprotective factors from astrocytes. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

A mutation within the extended X loop abolished substrate-induced ATPase activity of the human liver ATP-binding cassette (ABC) transporter MDR3.

PubMed

Kluth, Marianne; Stindt, Jan; Dröge, Carola; Linnemann, Doris; Kubitz, Ralf; Schmitt, Lutz

2015-02-20

The human multidrug resistance protein 3 (MDR3/ABCB4) belongs to the ubiquitous family of ATP-binding cassette (ABC) transporters and is located in the canalicular membrane of hepatocytes. There it flops the phospholipids of the phosphatidylcholine (PC) family from the inner to the outer leaflet. Here, we report the characterization of wild type MDR3 and the Q1174E mutant, which was identified previously in a patient with progressive familial intrahepatic cholestasis type 3 (PFIC-3). We expressed different variants of MDR3 in the yeast Pichia pastoris, purified the proteins via tandem affinity chromatography, and determined MDR3-specific ATPase activity in the presence or absence of phospholipids. The ATPase activity of wild type MDR3 was stimulated 2-fold by liver PC or 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine lipids. Furthermore, the cross-linking of MDR3 with a thiol-reactive fluorophore blocked ATP hydrolysis and exhibited no PC stimulation. Similarly, phosphatidylethanolamine, phosphatidylserine, and sphingomyelin lipids did not induce an increase of wild type MDR3 ATPase activity. The phosphate analogues beryllium fluoride and aluminum fluoride led to complete inhibition of ATPase activity, whereas orthovanadate inhibited exclusively the PC-stimulated ATPase activity of MDR3. The Q1174E mutation is located in the nucleotide-binding domain in direct proximity of the leucine of the ABC signature motif and extended the X loop, which is found in ABC exporters. Our data on the Q1174E mutant demonstrated basal ATPase activity, but PC lipids were incapable of stimulating ATPase activity highlighting the role of the extended X loop in the cross-talk of the nucleotide-binding domain and the transmembrane domain. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Reperfusion injury protection during Basic Life Support improves circulation and survival outcomes in a porcine model of prolonged cardiac arrest.

PubMed

Debaty, Guillaume; Lurie, Keith; Metzger, Anja; Lick, Michael; Bartos, Jason A; Rees, Jennifer N; McKnite, Scott; Puertas, Laura; Pepe, Paul; Fowler, Raymond; Yannopoulos, Demetris

2016-08-01

Ischemic postconditioning (PC) using three intentional pauses at the start of cardiopulmonary resuscitation (CPR) improves outcomes after cardiac arrest in pigs when epinephrine (epi) is used before defibrillation. We hypothesized PC, performed during basic life support (BLS) in the absence of epinephrine, would reduce reperfusion injury and enhance 24h functional recovery. Prospective animal investigation. Animal laboratory Female farm pigs (n=46, 39±1kg). Protocol A: After 12min of ventricular fibrillation (VF), 28 pigs were randomized to four groups: (A) Standard CPR (SCPR), (B) active compression-decompression CPR with an impedance threshold device (ACD-ITD), (C) SCPR+PC (SCPR+PC) and (D) ACD-ITD CPR+PC. Protocol B: After 15min of VF, 18 pigs were randomized to ACD-ITD CPR or ACD-ITD+PC. The BLS duration was 2.75min in Protocol A and 5min in Protocol B. Following BLS, up to three shocks were delivered. Without return of spontaneous circulation (ROSC), CPR was resumed and epi (0.5mg) and defibrillation delivered. The primary end point was survival without major adverse events. Hemodynamic parameters and left ventricular ejection fraction (LVEF) were also measured. Data are presented as mean±SEM. Protocol A: ACD-ITD+PC (group D) improved coronary perfusion pressure after 3min of BLS versus the three other groups (28±6, 35±7, 23±5 and 47±7 for groups A, B, C, D respectively, p=0.05). There were no significant differences in 24h survival between groups. LVEF 4h post ROSC was significantly higher with ACD-ITD+PC vs ACD-ITD alone (52.5±3% vs. 37.5±6.6%, p=0.045). Survival rates were significantly higher with ACD-ITD+PC vs. ACD-ITD alone (p=0.027). BLS using ACD-ITD+PC reduced post resuscitation cardiac dysfunction and improved functional recovery after prolonged untreated VF in pigs. 12-11. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Hydrogen gas alleviates oxygen toxicity by reducing hydroxyl radical levels in PC12 cells

PubMed Central

Yu, Junchao; Yu, Qiuhong; Liu, Yaling; Zhang, Ruiyun; Xue, Lianbi

2017-01-01

Hyperbaric oxygen (HBO) therapy through breathing oxygen at the pressure of above 1 atmosphere absolute (ATA) is useful for varieties of clinical conditions, especially hypoxic-ischemic diseases. Because of generation of reactive oxygen species (ROS), breathing oxygen gas at high pressures can cause oxygen toxicity in the central nervous system, leading to multiple neurological dysfunction, which limits the use of HBO therapy. Studies have shown that Hydrogen gas (H2) can diminish oxidative stress and effectively reduce active ROS associated with diseases. However, the effect of H2 on ROS generated from HBO therapy remains unclear. In this study, we investigated the effect of H2 on ROS during HBO therapy using PC12 cells. PC12 cells cultured in medium were exposed to oxygen gas or mixed oxygen gas and H2 at 1 ATA or 5 ATA. Cells viability and oxidation products and ROS were determined. The data showed that H2 promoted the cell viability and inhibited the damage in the cell and mitochondria membrane, reduced the levels of lipid peroxidation and DNA oxidation, and selectively decreased the levels of •OH but not disturbing the levels of O2•-, H2O2, or NO• in PC12 cells during HBO therapy. These results indicated that H2 effectively reduced •OH, protected cells against oxygen toxicity resulting from HBO therapy, and had no effect on other ROS. Our data supported that H2 could be potentially used as an antioxidant during HBO therapy. PMID:28362819

Contrasting protective effects of cannabinoids against oxidative stress and amyloid-β evoked neurotoxicity in vitro.

PubMed

Harvey, Benjamin S; Ohlsson, Katharina S; Mååg, Jesper L V; Musgrave, Ian F; Smid, Scott D

2012-01-01

Cannabinoids have been widely reported to have neuroprotective properties in vitro and in vivo. In this study we compared the effects of CB1 and CB2 receptor-selective ligands, the endocannabinoid anandamide and the phytocannabinoid cannabidiol, against oxidative stress and the toxic hallmark Alzheimer's protein, β-amyloid (Aβ) in neuronal cell lines. PC12 or SH-SY5Y cells were selectively exposed to either hydrogen peroxide, tert-butyl hydroperoxide or Aβ, alone or in the presence of the CB1 specific agonist arachidonyl-2'-chloroethylamide (ACEA), CB2 specific agonist JWH-015, anandamide or cannabidiol. Cannabidiol improved cell viability in response to tert-butyl hydroperoxide in PC12 and SH-SY5Y cells, while hydrogen peroxide-mediated toxicity was unaffected by cannabidiol pretreatment. Aβ exposure evoked a loss of cell viability in PC12 cells. Of the cannabinoids tested, only anandamide was able to inhibit Aβ-evoked neurotoxicity. ACEA had no effect on Aβ-evoked neurotoxicity, suggesting a CB1 receptor-independent effect of anandamide. JWH-015 pretreatment was also without protective influence on PC12 cells from either pro-oxidant or Aβ exposure. None of the cannabinoids directly inhibited or disrupted preformed Aβ fibrils and aggregates. In conclusion, the endocannabinoid anandamide protects neuronal cells from Aβ exposure via a pathway unrelated to CB1 or CB2 receptor activation. The protective effect of cannabidiol against oxidative stress does not confer protection against Aβ exposure, suggesting divergent pathways for neuroprotection of these two cannabinoids. Copyright © 2011 Elsevier Inc. All rights reserved.

Gemcitabine diphosphate choline is a major metabolite linked to the Kennedy pathway in pancreatic cancer models in vivo

PubMed Central

Bapiro, T E; Frese, K K; Courtin, A; Bramhall, J L; Madhu, B; Cook, N; Neesse, A; Griffiths, J R; Tuveson, D A; Jodrell, D I; Richards, F M

2014-01-01

Background: The modest benefits of gemcitabine (dFdC) therapy in patients with pancreatic ductal adenocarcinoma (PDAC) are well documented, with drug delivery and metabolic lability cited as important contributing factors. We have used a mouse model of PDAC: KRASG12D; p53R172H; pdx-Cre (KPC) that recapitulates the human disease to study dFdC intra-tumoural metabolism. Methods: LC-MS/MS and NMR were used to measure drug and physiological analytes. Cytotoxicity was assessed by the Sulphorhodamine B assay. Results: In KPC tumour tissue, we identified a new, Kennedy pathway-linked dFdC metabolite (gemcitabine diphosphate choline (GdPC)) present at equimolar amounts to its precursor, the accepted active metabolite gemcitabine triphosphate (dFdCTP). Utilising additional subcutaneous PDAC tumour models, we demonstrated an inverse correlation between GdPC/dFdCTP ratios and cytidine triphosphate (CTP). In tumour homogenates in vitro, CTP inhibited GdPC formation from dFdCTP, indicating competition between CTP and dFdCTP for CTP:phosphocholine cytidylyltransferase (CCT). As the structure of GdPC precludes entry into cells, potential cytotoxicity was assessed by stimulating CCT activity using linoleate in KPC cells in vitro, leading to increased GdPC concentration and synergistic growth inhibition after dFdC addition. Conclusions: GdPC is an important element of the intra-tumoural dFdC metabolic pathway in vivo. PMID:24874484

Viability evaluation of culture cells patterned by femtosecond laser-induced impulsive force

NASA Astrophysics Data System (ADS)

Takizawa, Noriko; Okano, Kazunori; Uwada, Takayuki; Hosokawa, Yoichiroh; Masuhara, Hiroshi

2008-02-01

PC12 cells, which are derived from a rat pheochromocytoma, were independently patterned utilizing an impulsive force resulting in impulsive shockwave and cavitation bubble generation by focused femtosecond laser irradiation. Since the PC12 cells respond reversibly to nerve growth factor by induction of the neuronal phenotype, we can assess an influence that the impulsive force gives to the bioactivity in term of the cell differentiation. The patterned cells were accumulated on an intact dish and cultured for 3 days. The behavior of appearance and cell differentiation was observed by multipoint time-lapse system. On bases of these results, it was proved that the biological activity of the cell is unaffected by the femtosecond laser patterning.

Novel arylalkenylpropargylamines as neuroprotective, potent, and selective monoamine oxidase B inhibitors for the treatment of Parkinson's disease.

PubMed

Huleatt, Paul B; Khoo, Mui Ling; Chua, Yi Yuan; Tan, Tiong Wei; Liew, Rou Shen; Balogh, Balázs; Deme, Ruth; Gölöncsér, Flóra; Magyar, Kalman; Sheela, David P; Ho, Han Kiat; Sperlágh, Beáta; Mátyus, Péter; Chai, Christina L L

2015-02-12

To develop novel neuroprotective agents, a library of novel arylalkenylpropargylamines was synthesized and tested for inhibitory activities against monoamine oxidases. From this, a number of highly potent and selective monoamine oxidase B inhibitors were identified. Selected compounds were also tested for neuroprotection in in vitro studies with PC-12 cells treated with 6-OHDA and rotenone, respectively. It was observed that some of the compounds tested yielded a marked increase in survival in PC-12 cells treated with the neurotoxins. This indicates that these propargylamines are able to confer protection against the effects of the toxins and may also be considered as novel disease-modifying anti-Parkinsonian agents, which are much needed for the therapy of Parkinson's disease.

Identification of a novel phospholipase D with high transphosphatidylation activity and its application in synthesis of phosphatidylserine and DHA-phosphatidylserine.

PubMed

Mao, Xiangzhao; Liu, Qianqian; Qiu, Yongqian; Fan, Xiaoqin; Han, Qingqing; Liu, Yanjun; Zhang, Lujia; Xue, Changhu

2017-05-10

Phosphatidylserine (PS) and docosahexaenoic acid-phosphatidylserine (DHA-PS) have significant nutritional and biological functions, which are extensively used in functional food industries. Phospholipase D (PLD)-mediated transphosphatidylation of phosphatidylcholine (PC) or DHA-PC with l-serine, is an effective method for PS and DHA-PS preparation. However, because of the hydrolysis activity of PLD, PC and DHA-PC would be converted to the undesirable byproduct, phosphatidic acid (PA) and DHA-PA. In this study, a novel phospholipase D (PLD a2 ) was firstly cloned from Acinetobacter radioresistens a2 with high transphosphatidylation activity and no hydrolysis activity. In the PLD-catalyzed synthesis process (12h), both the transphosphatidylation conversion rate and selectivity of PS and DHA-PS were about 100%, which is the only PLD enzyme reported with this superiority up till now. In comparison with the majority of other known PLDs, PLD a2 exerted the highest activity at neutral pH, and it was stable from pH 4.0 to pH 9.0. In addition, PLD a2 had excellent thermal stability, with an optimum reaction temperature of 40°C and keeping more than 80% activity from 20°C to 60°C. The high catalytic selectivity mechanism of PLD a2 was explained by utilizing homology modeling, two-step docking, and binding energy and conformation analysis. PLD a2 ensured a stable supply of the biocatalyst with its most preponderant transphosphatidylation activity and PS selectivity, and had great potential in phospholipids industrial production. Copyright © 2017 Elsevier B.V. All rights reserved.

Cervical cancer cells (HeLa) response to photodynamic therapy using a zinc phthalocyanine photosensitizer.

PubMed

Hodgkinson, Natasha; Kruger, Cherie Ann; Mokwena, Mpho; Abrahamse, Heidi

2017-12-01

Cervical cancer is the most common gynecological malignancy worldwide, and the leading cause of cancer related deaths among females. Conventional treatment for early cervical cancer is radical hysterectomy. In locally advanced cancer the treatment of choice is concurrent chemo radiation. Although such treatment methods show promise, they do have adverse side effects. To minimize these effects, as well as prevent cancer re-occurrence, new treatment methods are being investigated. Photodynamic therapy (PDT) involves the selective uptake of a photosensitizer (PS) by cancer cells, illumination with light of an appropriate wavelength that triggers a photochemical reaction leading to the generation of reactive oxygen and subsequent tumor regression. The effect of PDT on a cervical cancer cell line (HeLa) was assessed by exposing cultured cells to a sulphonated zinc phthalocyanine PS (ZnPcS mix ) and irradiating the cells using a 673nm diode laser. The effects were measured using the Trypan blue viability assay, adenosine triphosphate assay (ATP) luminescence assay for proliferation, Lactate Dehydrogenase (LDH) membrane integrity cytotoxicity assay, and fluorescent microscopy to assess PS cellular localization and nuclear damage. Fluorescent microscopy revealed localization of the PS in the cytoplasm and perinuclear region of HeLa cells. PDT treated cellular responses showed dose dependent structural changes, with decreased cell viability and proliferation, as well as considerable membrane damage. Hoechst stained cells also revealed DNA damage in PDT treated cells. The final findings from this study suggest that ZnPcS mix is a promising PS for the PDT treatment of cervical cancer in vitro, where a significant 85% cellular cytotoxicity with only 25% cellular viability was noted in cells which received 1μM ZnPcS mix when an 8J/cm 2 fluence was applied. Copyright © 2017 Elsevier B.V. All rights reserved.

Whole-Cell Electrical Activity Under Direct Mechanical Stimulus by AFM Cantilever Using Planar Patch Clamp Chip Approach

PubMed Central

Upadhye, Kalpesh V.; Candiello, Joseph E.; Davidson, Lance A.; Lin, Hai

2011-01-01

Patch clamp is a powerful tool for studying the properties of ion-channels and cellular membrane. In recent years, planar patch clamp chips have been fabricated from various materials including glass, quartz, silicon, silicon nitride, polydimethyl-siloxane (PDMS), and silicon dioxide. Planar patch clamps have made automation of patch clamp recordings possible. However, most planar patch clamp chips have limitations when used in combination with other techniques. Furthermore, the fabrication methods used are often expensive and require specialized equipments. An improved design as well as fabrication and characterization of a silicon-based planar patch clamp chip are described in this report. Fabrication involves true batch fabrication processes that can be performed in most common microfabrication facilities using well established MEMS techniques. Our planar patch clamp chips can form giga-ohm seals with the cell plasma membrane with success rate comparable to existing patch clamp techniques. The chip permits whole-cell voltage clamp recordings on variety of cell types including Chinese Hamster Ovary (CHO) cells and pheochromocytoma (PC12) cells, for times longer than most available patch clamp chips. When combined with a custom microfluidics chamber, we demonstrate that it is possible to perfuse the extra-cellular as well as intra-cellular buffers. The chamber design allows integration of planar patch clamp with atomic force microscope (AFM). Using our planar patch clamp chip and microfluidics chamber, we have recorded whole-cell mechanosensitive (MS) currents produced by directly stimulating human keratinocyte (HaCaT) cells using an AFM cantilever. Our results reveal the spatial distribution of MS ion channels and temporal details of the responses from MS channels. The results show that planar patch clamp chips have great potential for multi-parametric high throughput studies of ion channel proteins. PMID:22174731

Screen-Printed Carbon Electrodes Modified with Cobalt Phthalocyanine for Selective Sulfur Detection in Cosmetic Products

PubMed Central

Chen, Pei-Yen; Luo, Chin-Hsiang; Chen, Mei-Chin; Tsai, Feng-Jie; Chang, Nai-Fang; Shih, Ying

2011-01-01

Cobalt phthalocyanine (CoPc) films were deposited on the surface of a screen-printed carbon electrode using a simple drop coating method. The cyclic voltammogram of the resulting CoPc modified screen-printed electrode (CoPc/SPE) prepared under optimum conditions shows a well-behaved redox couple due to the (CoI/CoII) system. The CoPc/SPE surface demonstrates excellent electrochemical activity towards the oxidation of sulfur in a 0.01 mol·L−1 NaOH. A linear calibration curve with the detection limit (DL, S/N = 3) of 0.325 mg·L−1 was achieved by CoPc/SPE coupled with flow injection analysis of the sulfur concentration ranging from 4 to 1120 mg·L−1. The precision of the system response was evaluated (3.60% and 3.52% RSD for 12 repeated injections), in the range of 64 and 480 mg·L−1 sulfur. The applicability of the method was successfully demonstrated in a real sample analysis of sulfur in anti-acne creams, and good recovery was obtained. The CoPc/SPE displayed several advantages in sulfur determination including easy fabrication, high stability, and low cost. PMID:21747708

Pregnancy alters choline dynamics: results of a randomized trial using stable isotope methodology in pregnant and nonpregnant women.

PubMed

Yan, Jian; Jiang, Xinyin; West, Allyson A; Perry, Cydne A; Malysheva, Olga V; Brenna, J Thomas; Stabler, Sally P; Allen, Robert H; Gregory, Jesse F; Caudill, Marie A

2013-12-01

Although biomarkers of choline metabolism are altered by pregnancy, little is known about the influence of human pregnancy on the dynamics of choline-related metabolic processes. This study used stable isotope methodology to examine the effects of pregnancy on choline partitioning and the metabolic activity of choline-related pathways. Healthy third-trimester pregnant (n = 26; initially week 27 of gestation) and nonpregnant (n = 21) women consumed 22% of their total choline intake (480 or 930 mg/d) as methyl-d9-choline for the final 6 wk of a 12-wk feeding study. Plasma d9-betaine:d9-phosphatidylcholine (PC) was lower (P ≤ 0.04) in pregnant than in nonpregnant women, suggesting greater partitioning of choline into the cytidine diphosphate-choline (CDP-choline) PC biosynthetic pathway relative to betaine synthesis during pregnancy. Pregnant women also used more choline-derived methyl groups for PC synthesis via phosphatidylethanolamine N-methyltransferase (PEMT) as indicated by comparable increases in PEMT-PC enrichment in pregnant and nonpregnant women despite unequal (pregnant > nonpregnant; P < 0.001) PC pool sizes. Pregnancy enhanced the hydrolysis of PEMT-PC to free choline as shown by greater (P < 0.001) plasma d3-choline:d3-PC. Notably, d3-PC enrichment increased (P ≤ 0.011) incrementally from maternal to placental to fetal compartments, signifying the selective transfer of PEMT-PC to the fetus. The enhanced use of choline for PC production via both the CDP-choline and PEMT pathways shows the substantial demand for choline during late pregnancy. Selective partitioning of PEMT-PC to the fetal compartment may imply a unique requirement of PEMT-PC by the developing fetus.

The Role of Capase-8 in Breast Carcinoma Cells

DTIC Science & Technology

2006-04-01

and exposed to film. pcDNA3 or pcDNA3-huSNARK-transfected 293T cell extracts (100 #g of protein) were used to determine the phosphorylation of the...proteins (Itoh and Nagata, 1993; Boldin et al, 1995; Chinnaiyan et al, 1995; Siegel et al, 2000). Additional interactions mediated through the N...lanes 5–7) fractions were separated from total cellular membrane extract (lane 1). Following fractionation, association of FADD and caspase-8 with

Immunoprecipitation of PDE2 phosphorylated and inactivated by an associated protein kinase.

PubMed

Bentley, J Kelley

2005-01-01

A PDE2A2-associated protein kinase phosphorylates PDE2A2 in vivo and in vitro to inhibit its catalytic activity. Rat brain PDE2A2 may be solubilized using nona (ethylene glycol) mono dodecyl ether (Lubrol 12A9). PDE2A2 exists in a complex with a protein kinase regulating its activity in an adenosine triphosphate-dependent manner. When native or recombinant PDE2 is immunoprecipitated from PC12 cells using an antibody to the amino terminus in a buffer containing Lubrol 12A9, protease inhibitors, and phosphatase inhibitors, a coimmunoprecipitating nerve growth factor-stimulated protein kinase acts to phosphorylate it. PDE2A2 phosphoryla-tion occurs optimally at pH 6.5 in a sodium 2-(4-morpholino)-ethane sulfonate buffer with 5 mM MgCl2 and 1 mM Na3VO4. I describe protocols for producing an antibody to an amino-terminal bacterial fusion protein encoding amino acids 1-251 of PDE2A2 as well as the use of this antibody in immunoprecipitating a PDE2: tyrosine protein-kinase complex from rat brain or PC12 cells.

Minocycline attenuates both OGD-induced HMGB1 release and HMGB1-induced cell death in ischemic neuronal injury in PC12 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kikuchi, Kiyoshi; Department of Neurosurgery, Omuta City General Hospital, 2-19-1 Takarazaka, Omuta-City, Fukuoka 836-8567; Kawahara, Ko-ichi

2009-07-24

High mobility group box-1 (HMGB1), a non-histone DNA-binding protein, is massively released into the extracellular space from neuronal cells after ischemic insult and exacerbates brain tissue damage in rats. Minocycline is a semisynthetic second-generation tetracycline antibiotic which has recently been shown to be a promising neuroprotective agent. In this study, we found that minocycline inhibited HMGB1 release in oxygen-glucose deprivation (OGD)-treated PC12 cells and triggered the activation of p38mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinases (ERK1/2). The ERK kinase (MEK)1/2 inhibitor U-0126 and p38MAPK inhibitor SB203580 blocked HMGB1 release in response to OGD. Furthermore, HMGB1 triggered cell death inmore » a dose-dependent fashion. Minocycline significantly rescued HMGB1-induced cell death in a dose-dependent manner. In light of recent observations as well as the good safety profile of minocycline in humans, we propose that minocycline might play a potent neuroprotective role through the inhibition of HMGB1-induced neuronal cell death in cerebral infarction.« less

Hepatoprotectant Ursodeoxycholyl Lysophosphatidylethanolamide Increasing Phosphatidylcholine Levels as a Potential Therapy of Acute Liver Injury

PubMed Central

Chamulitrat, Walee; Zhang, Wujuan; Xu, Weihong; Pathil, Anita; Setchell, Kenneth; Stremmel, Wolfgang

2012-01-01

It has been long known that hepatic synthesis of phosphatidylcholine (PC) is depressed during acute such as carbon tetrachloride-induced liver injury. Anti-hepatotoxic properties of PC as liposomes have been recognized for treatment of acute liver damage. Ursodeoxycholate (UDCA) is a known hepatoprotectant in stabilizing cellular membrane. For therapeutic management of liver injury, we coupled UDCA with a phospholipid known as ursodeoxycholyl lysophosphatidylethanolamide (UDCA-LPE). UDCA-LPE has been shown to first-in-class hepatoprotectant being superior to UDCA or PC. It inhibits mitochondrial damage and apoptosis, elicits survival signaling pathway, and promotes regeneration of hepatocytes. We herein report that a unique contribution of UDCA-LPE in increasing concentrations of PC in vitro and in vivo. UDCA-LPE-treated hepatocytes contained significantly increased PC levels. UDCA-LPE underwent the hydrolysis to LPE which was not the precursor of the increased PC. The levels of PC in the liver and blood were increased rapidly after intraperitoneally administration UDCA-LPE, and were found to be sustained even after 24 h. Among PC synthesis genes tested, UDCA-LPE treatment of mouse hepatocytes increased transcription of CDP-diacylglycerol synthase 1 which is an enzyme catalyzing phosphatidic acid to generate intermediates for PC synthesis. Thus, UDCA-LPE as a hepatoprotectant was able to induce synthesis of protective PC which would supplement for the loss of PC occurring during acute liver injury. This property has placed UDCA-LPE as a candidate agent for therapy of acute hepatotoxicity such as acetaminophen poisoning. PMID:22363296

Identification and characterization of intracellular proteins that bind oligonucleotides with phosphorothioate linkages

PubMed Central

Liang, Xue-hai; Sun, Hong; Shen, Wen; Crooke, Stanley T.

2015-01-01

Although the RNase H-dependent mechanism of inhibition of gene expression by chemically modified antisense oligonucleotides (ASOs) has been well characterized, little is known about the interactions between ASOs and intracellular proteins that may alter cellular localization and/or potency of ASOs. Here, we report the identification of 56 intracellular ASO-binding proteins using multi-step affinity selection approaches. Many of the tested proteins had no significant effect on ASO activity; however, some proteins, including La/SSB, NPM1, ANXA2, VARS and PC4, appeared to enhance ASO activities, likely through mechanisms related to subcellular distribution. VARS and ANXA2 co-localized with ASOs in endocytic organelles, and reduction in the level of VARS altered lysosome/ASO localization patterns, implying that these proteins may facilitate ASO release from the endocytic pathway. Depletion of La and NPM1 reduced nuclear ASO levels, suggesting potential roles in ASO nuclear accumulation. On the other hand, Ku70 and Ku80 proteins inhibited ASO activity, most likely by competition with RNase H1 for ASO/RNA duplex binding. Our results demonstrate that phosphorothioate-modified ASOs bind a set of cellular proteins that affect ASO activity via different mechanisms. PMID:25712094

Design and synthesis of a series of bioavailable fatty acid synthase (FASN) KR domain inhibitors for cancer therapy.

PubMed

Lu, Tianbao; Schubert, Carsten; Cummings, Maxwell D; Bignan, Gilles; Connolly, Peter J; Smans, Karine; Ludovici, Donald; Parker, Michael H; Meyer, Christophe; Rocaboy, Christian; Alexander, Richard; Grasberger, Bruce; De Breucker, Sabine; Esser, Norbert; Fraiponts, Erwin; Gilissen, Ron; Janssens, Boudewijn; Peeters, Danielle; Van Nuffel, Luc; Vermeulen, Peter; Bischoff, James; Meerpoel, Lieven

2018-05-08

We designed and synthesized a new series of fatty acid synthase (FASN) inhibitors with potential utility for the treatment of cancer. Extensive SAR studies led to highly active FASN inhibitors with good cellular activity and oral bioavailability, exemplified by compound 34. Compound 34 is a potent inhibitor of human FASN (IC 50  = 28 nM) that effectively inhibits proliferation of A2780 ovarian cells (IC 50  = 13 nM) in lipid-reduced serum (LRS). This cellular activity can be rescued by addition of palmitate, consistent with an on-target effect. Compound 34 is also active in many other cell types, including PC3M (IC 50  = 25 nM) and LnCaP-Vancouver prostate cells (IC 50  = 66 nM), and is highly bioavailable (F 61%) with good exposure after oral administration. In a pharmacodynamics study in H460 lung xenograft-bearing mice, oral treatment with compound 34 results in elevated tumor levels of malonyl-CoA and decreased tumor levels of palmitate, fully consistent with the desired target engagement. Copyright © 2018 Elsevier Ltd. All rights reserved.

Monte Carlo calculations of the cellular S-values for α-particle-emitting radionuclides incorporated into the nuclei of cancer cells of the MDA-MB231, MCF7 and PC3 lines.

PubMed

Rojas-Calderón, E L; Ávila, O; Ferro-Flores, G

2018-05-01

S-values (dose per unit of cumulated activity) for alpha particle-emitting radionuclides and monoenergetic alpha sources placed in the nuclei of three cancer cell models (MCF7, MDA-MB231 breast cancer cells and PC3 prostate cancer cells) were obtained by Monte Carlo simulation. The MCNPX code was used to calculate the fraction of energy deposited in the subcellular compartments due to the alpha sources in order to obtain the S-values. A comparison with internationally accepted S-values reported by the MIRD Cellular Committee for alpha sources in three sizes of spherical cells was also performed leading to an agreement within 4% when an alpha extended source uniformly distributed in the nucleus is simulated. This result allowed to apply the Monte Carlo Methodology to evaluate S-values for alpha particles in cancer cells. The calculation of S-values for nucleus, cytoplasm and membrane of cancer cells considering their particular geometry, distribution of the radionuclide source and chemical composition by means of Monte Carlo simulation provides a good approach for dosimetry assessment of alpha emitters inside cancer cells. Results from this work provide information and tools that may help researchers in the selection of appropriate radiopharmaceuticals in alpha-targeted cancer therapy and improve its dosimetry evaluation. Copyright © 2018 Elsevier Ltd. All rights reserved.

Crosstalk between stromal cells and cancer cells in pancreatic cancer: New insights into stromal biology.

PubMed

Zhan, Han-Xiang; Zhou, Bin; Cheng, Yu-Gang; Xu, Jian-Wei; Wang, Lei; Zhang, Guang-Yong; Hu, San-Yuan

2017-04-28

Pancreatic cancer (PC) remains one of the most lethal malignancies worldwide. Increasing evidence has confirmed the pivotal role of stromal components in the regulation of carcinogenesis, invasion, metastasis, and therapeutic resistance in PC. Interaction between neoplastic cells and stromal cells builds a specific microenvironment, which further modulates the malignant properties of cancer cells. Instead of being a "passive bystander", stroma may play a role as a "partner in crime" in PC. However, the role of stromal components in PC is complex and requires further investigation. In this article, we review recent advances regarding the regulatory roles and mechanisms of stroma biology, especially the cellular components such as pancreatic stellate cells, macrophages, neutrophils, adipocytes, epithelial cells, pericytes, mast cells, and lymphocytes, in PC. Crosstalk between stromal cells and cancer cells is thoroughly investigated. We also review the prognostic value and molecular therapeutic targets of stroma in PC. This review may help us further understand the molecular mechanisms of stromal biology and its role in PC development and therapeutic resistance. Moreover, targeting stroma components may provide new therapeutic strategies for this stubborn disease. Copyright © 2017 Elsevier B.V. All rights reserved.

A Computational Study of Laminate Transparent Armor Impacted by FSP

DTIC Science & Technology

2009-06-01

of Hsieh et al [1], on targets consisting of 3mm PC-12mm PMMA-3mm PC impacted by 17-gr, 0.22 caliber fragment simulating projectile (FSP) at impact...results from the experiments of Hsieh et al [1], on targets consisting of 3mm PC-12mm PMMA-3mm PC impacted by 17-gr, 0.22 caliber fragment simulating...investigate several different analysis techniques to qualitatively determine their accuracy when compared with experiments of Hsieh et al [1]. The

Overexpression of DJ-1/PARK7, the Parkinson's disease-related protein, improves mitochondrial function via Akt phosphorylation on threonine 308 in dopaminergic neuron-like cells.

PubMed

Zhang, Yi; Gong, Xiao-Gang; Wang, Zhen-Zhen; Sun, Hong-Mei; Guo, Zhen-Yu; Hu, Jing-Hong; Ma, Ling; Li, Ping; Chen, Nai-Hong

2016-05-01

DJ-1/PARK7, the Parkinson's disease-related protein, plays an important role in mitochondrial function. However, the mechanisms by which DJ-1 affects mitochondrial function are not fully understood. Akt is a promoter of neuron survival and is partly involved in the neurodegenerative process. This research aimed at investigating a possible relationship between DJ-1 and Akt signalling in regulating mitochondrial function in the dopaminergic neuron-like cells SH-SY5Y and PC-12. Overexpression of DJ-1 was firstly validated at both the transcriptional and translational levels after transit transfection with plasmid pcDNA3-Flag-DJ-1. Confocal fluorescence microscopy demonstrated that overexpression of DJ-1 increased the mitochondrial mass, but did not disrupt the mitochondrial morphology. In addition, mitochondrial complex I activity was raised in DJ-1-overexpressing cells, and this rise occurred with an increase in cellular adenosine 5'-triphosphate content. Moreover, immunoblotting demonstrated that the levels of phosphoinositide 3-kinase and the total Akt were not altered in DJ-1-overexpressing cells, and nor was the Akt phosphorylation on serine 473 changed. By contrast, Akt phosphorylation on threonine 308 was significantly augmented by overexpression of DJ-1, and the expression of glycogen synthase kinase-3beta, a downstream effector of Akt, was suppressed. In summary, these results suggest that overexpression of DJ-1 improves the mitochondrial function, at least in part, through a mechanism involving Akt phosphorylation on threonine 308. © 2016 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

Phytosome-hyaluronic acid systems for ocular delivery of L-carnosine

PubMed Central

Abdelkader, Hamdy; Longman, Michael R; Alany, Raid G; Pierscionek, Barbara

2016-01-01

This study reports on L-carnosine phytosomes as an alternative for the prodrug N-acetyl-L-carnosine as a novel delivery system to the lens. L-carnosine was loaded into lipid-based phytosomes and hyaluronic acid (HA)-dispersed phytosomes. L-carnosine-phospholipid complexes (PC) of different molar ratios, 1:1 and 1:2, were prepared by the solvent evaporation method. These complexes were characterized with thermal and spectral analyses. PC were dispersed in either phosphate buffered saline pH 7.4 or HA (0.1% w/v) in phosphate buffered saline to form phytosomes PC1:1, PC1:2, and PC1:2 HA, respectively. These phytosomal formulations were studied for size, zeta potential, morphology, contact angle, spreading coefficient, viscosity, ex vivo transcorneal permeation, and cytotoxicity using primary human corneal cells. L-carnosine-phospholipid formed a complex at a 1:2 molar ratio and phytosomes were in the size range of 380–450 nm, polydispersity index of 0.12–0.2. The viscosity of PC1:2 HA increased by 2.4 to 5-fold compared with HA solution and PC 1:2, respectively; significantly lower surface tension, contact angle, and greater spreading ability for phytosomes were also recorded. Ex vivo transcorneal permeation parameters showed significantly controlled corneal permeation of L-carnosine with the novel carrier systems without any significant impact on primary human corneal cell viability. Ex vivo porcine lenses incubated in high sugar media without and with L-carnosine showed concentration-dependent marked inhibition of lens brunescence indicative of the potential for delaying changes that underlie cataractogenesis that may be linked to diabetic processes. PMID:27366062

MiR-103 alleviates autophagy and apoptosis by regulating SOX2 in LPS-injured PC12 cells and SCI rats.

PubMed

Li, Guowei; Chen, Tao; Zhu, Yingxian; Xiao, Xiaoyu; Bu, Juyuan; Huang, Zongwen

2018-03-01

Recent studies revealed that microRNAs (miRNAs) may play crucial roles in the responses and pathologic processes of spinal cord injury (SCI). This study aimed to investigate the effect and the molecular basis of miR-103 on LPS-induced injuries in PC12 cells in vitro and SCI rats in vivo . PC12 cells were exposed to LPS to induce cell injuries to mimic the in vitro model of SCI. The expression of miR-103 and SOX2 in PC12 cells were altered by transient transfections. Cell viability and apoptotic cell rate were measured by CCK-8 assay and flow cytometry assay. Furthermore, Western blot analysis was performed to detect the expression levels of apoptosis- and autophagy- related proteins, MAPK/ERK pathway- and JAK/STAT pathway-related proteins. In addition, we also assessed the effect of miR-103 agomir on SCI rats. LPS exposure induced cell injuries in PC12 cells. miR-103 overexpression significantly increased cell viability, reduced cell apoptosis and autophagy, and opposite results were observed in miR-103 inhibition. miR-103 attenuated LPS-induced injuries by indirect upregulation of SOX2. SOX2 overexpression protected PC12 cells against LPS-induced injuries, while SOX2 inhibition expedited LPS-induced cell injuries. Furthermore, miR-103 overexpression inhibited MAPK/ERK pathway and JAK/STAT pathway through upregulation of SOX2. We also found that miR-103 agomir inhibited cell apoptosis and autophagy in SCI rats. This study demonstrates that miR-103 may represent a protective effect against cell apoptosis and autophagy in LPS-injured PC12 cells and SCI rats by upregulation of SOX2 expression.

Cooperative cytotoxic activity of Zn and Cu in bovine serum albumin-conjugated ZnS/CuS nano-composites in PC12 cancer cells

NASA Astrophysics Data System (ADS)

Wang, Hua-Jie; Yu, Xue-Hong; Wang, Cai-Feng; Cao, Ying

2013-11-01

Series of self-assembled and mono-dispersed bovine serum albumin (BSA)-conjugated ZnS/CuS nano-composites with different Zn/Cu ratios had been successfully synthesized by a combination method of the biomimetic synthesis and ion-exchange strategy under the gentle conditions. High-resolution transmission electron microscopy observation, Fourier transform infrared spectra and zeta potential analysis demonstrated that BSA-conjugated ZnS/CuS nano-composites with well dispersity had the hierarchical structure and BSA was a key factor to control the morphology and surface electro-negativity of final products. The real-time monitoring by atomic absorption spectroscopy and powder X-ray diffraction revealed that the Zn/Cu ratio of nano-composites could be controlled by adjusting the ion-exchange time. In addition, the metabolic and morphological assays indicated that the metabolic proliferation and spread of rat pheochromocytoma (PC12) cells could be inhibited by nano-composites, with the high anti-cancer activity at a low concentration (4 ppm). What were more important, Zn and Cu in nano-composites exhibited a positive cooperativity at inhibiting cancer cell functions. The microscope observation and biochemical marker analysis clearly revealed that the nano-composites-included lipid peroxidation and disintegration of membrane led to the death of PC12 cells. Summarily, the present study substantiated the potential of BSA-conjugated ZnS/CuS nano-composites as anti-cancer drug.

Suppression of KV7/KCNQ potassium channel enhances neuronal differentiation of PC12 cells.

PubMed

Zhou, Najing; Huang, Sha; Li, Li; Huang, Dongyang; Yan, Yunli; Du, Xiaona; Zhang, Hailin

2016-10-01

Membrane potential shift driven by electrical activity is critical in determining the cell fate of proliferation or differentiation. As such, the ion channels that underlie the membrane electrical activity play an important role in cell proliferation/differentiation. KV7/KCNQ potassium channels are critical in determining the resting membrane potentials in many neuronal cells. However, the role of these channels in cell differentiation is not well studied. In the present study, we used PC12 cells as well as primary cultured rat cortical neurons to study the role and mechanism of KV7/KCNQ in neuronal differentiation. NGF induced PC12 cell differentiation into neuron-like cells with growth of neurites showing typical growth cone-like extensions. The Kv7/KCNQ blocker XE991 promoted NGF-induced neurite outgrowth, whereas Kv7/KCNQ opener retigabine (RTG) inhibited outgrowth. M-type Kv7 channels are likely involved in regulating neurite growth because overexpression of KCNQ2/Q3 inhibited neurite growth whereas suppression of KCNQ2/Q3 with shRNA promoted neurite growth. Membrane depolarization possibly underpins enhanced neurite growth induced by the suppression of Kv7/KCNQ. Additionally, high extracellular K(+) likely induced membrane depolarization and also promoted neurite growth. Finally, T-type Ca(2+) channels may be involved in membrane-depolarization-induced neurite growth. This study provides a new perspective for understanding neuronal differentiation as well as KV7/KCNQ channel function. Copyright © 2016 IBRO. Published by Elsevier Ltd. All rights reserved.

CGP 55398, a liposomal Ge(IV) phthalocyanine bearing two axially ligated cholesterol moieties: a new potential agent for photodynamic therapy of tumours.

PubMed Central

Segalla, A.; Milanesi, C.; Jori, G.; Capraro, H. G.; Isele, U.; Schieweck, K.

1994-01-01

Ge(IV) phthalocyanine (GePc) with two axially ligated cholesterol moieties was prepared by chemical synthesis and incorporated in a monomeric state into small unilamellar liposomes (CGP 55398). Upon photoexcitation with light wavelengths around its intense absorption peak at 680 nm, GePc shows an efficient photosensitising activity towards biological substrates through a mechanism which largely involves the intermediacy of singlet oxygen. GePc injected systemically into mice bearing an intramuscularly implanted MS-2 fibrosarcoma is quantitatively transferred to serum lipoproteins and localises in the tumour tissue with good efficiency: at 24 h post injection the GePc content in the tumour is 0.74 and 1.87 micrograms per g of tissue with a tumour/peritumoral ratio of 4.35 and 5.67 for injected doses of 0.76 and 1.52 mg kg-1 respectively. At this time the red-light irradiation of the GePc-loaded fibrosarcoma causes a fast and massive tumour necrosis involving both malignant cells and blood vessels. Images Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 PMID:8180009

Phosphatidylcholine synthesis is required for optimal function of Legionella pneumophila virulence determinants.

PubMed

Conover, Gloria M; Martinez-Morales, Fernando; Heidtman, Matthew I; Luo, Zhao-Qing; Tang, May; Chen, Cui; Geiger, Otto; Isberg, Ralph R

2008-02-01

The function of phosphatidylcholine (PC) in the bacterial cell envelope remains cryptic. We show here that productive interaction of the respiratory pathogen Legionella pneumophila with host cells requires bacterial PC. Synthesis of the lipid in L. pneumophila was shown to occur via either phospholipid N-methyltransferase (PmtA) or phosphatidylcholine synthase (PcsA), but the latter pathway was demonstrated to be of predominant importance. Loss of PC from the cell envelope caused lowered yields of L. pneumophila within macrophages as well as loss of high multiplicity cytotoxicity, while mutants defective in PC synthesis could be complemented either by reintroduction of PcsA or by overproduction of PmtA. The lowered yields and reduced cytotoxicity in mutants with defective PC biosynthesis were due to three related defects. First, there was a poorly functioning Dot/Icm apparatus, which delivers substrates required for intracellular growth into the cytosol of infected cells. Second, there was reduced bacterial binding to macrophages, possibly due to loss of PC or a PC derivative on the bacterium that is recognized by the host cell. Finally, strains lacking PC had low steady-state levels of flagellin protein, a deficit that had been previously associated with the phenotypes of lowered cytotoxicity and poor cellular adhesion.