Rat Pheochromocytoma PC12 Research Articles

Abstract 4874: Receptor pharmacology and anti-cancer activity of selective DRD2/3 antagonist imipridone ONC206

Abstract Dopamine receptor D2 (DRD2) is a G protein-coupled receptor (GPCR) overexpressed in many cancers and its antagonism causes anti-tumor effects. ONC201, founding member of the imipridone class of small molecules, is a DRD2 antagonist in Phase II advanced cancer clinical trials. We evaluated the binding target and anti-tumor activity of ONC206, an ONC201 analog. An orphan small molecule target prediction algorithm revealed that ONC206, like ONC201, antagonizes DRD2. Experimental GPCR profiling using the PathHunter® β-Arrestin assay, confirmed that ONC206 selectively antagonizes D2-like (DRD2/3) but not D1-like (DRD 1/5) dopamine receptors. In this assay, ONC206 possesses a ~10-fold increased potency for DRD2 compared to ONC201 with a Ki of ~320nM with selectivity that was superior to approved antipsychotics. Shotgun mutagenesis across 350 amino acids of DRD2 identified 7 residues critical for ONC206-mediated antagonism of DRD2-induced calcium flux. Consistent with competitive inhibition, mutated residues were within the orthosteric binding site. While 6 mutated residues were also critical for ONC201 activity, one of the mutated residues was unique to ONC206, suggesting differentiated receptor pharmacology. TCGA analysis and immunohistochemistry of patient-derived tissue microarrays revealed DRD2 was overexpressed in neuroblastoma, sarcoma and pheochromocytoma specimens relative to normal tissues. In vitro profiling of ONC206 in the Genomic of Drug Sensitivity in Cancer collection of cell lines revealed broad nanomolar efficacy across most tumor types (GI50 <78-889nM) and ~5 to 20-fold improved potency relative to ONC201. Bone cancer and neuroblastoma were identified as the most ONC206-responsive solid tumor types that were comparatively less responsive to ONC201. Within bone cancer, Ewing's sarcoma (n=16) was most sensitive to ONC206 with nanomolar sensitivity (GI50 168-303nM) that was superior to ONC201. ONC206 was highly efficacious in neuroblastoma (n=35, GI50 87-589nM) including cell lines derived from metastatic sites and with MYCN amplification associated with poor prognosis. In the PC12 rat pheochromocytoma cell line ONC206 (GI50 200nM) was superior to ONC201. ONC206 time-course experiments revealed anti-cancer effects occurring at 48-72 post-treatment, similar to ONC201. In support of a wide therapeutic window, ONC206 reduced the viability of normal human fibroblasts at relatively high doses (GI50 > 5µM). Efficacy evaluation in the MHH-ES-1 Ewing's sarcoma xenograft model demonstrated that ONC206 (100 mg/kg PO every 10 days) causes significant tumor growth inhibition that was comparable to methotrexate (400 mg/kg, IP) while being better tolerated. Thus, imipridone ONC206 acts as a selective antagonist of DRD2/3 at nanomolar concentrations and may address tumor types where the properties of ONC201 do not permit for complete therapeutic engagement in vivo. Citation Format: Varun Vijay Prabhu, Neel Madhukar, Rohinton Tarapore, Mathew Garnett, Ultan McDermott, Cyril Benes, Neil Charter, Sean Deacon, Alexander VanEngelenburg, Joseph Rucker, Benjamin Doranz, Olivier Elemento, Wolfgang Oster, Martin Stogniew, Joshua Allen. Receptor pharmacology and anti-cancer activity of selective DRD2/3 antagonist imipridone ONC206 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4874.

3,4-Dihydroxyphenylacetaldehyde-Induced Protein Modifications and Their Mitigation by N-Acetylcysteine.

The catecholaldehyde hypothesis posits that 3,4-dihydroxyphenylacetaldehyde (DOPAL), an obligate intermediary metabolite of dopamine, is an autotoxin that challenges neuronal homeostasis in catecholaminergic neurons. DOPAL toxicity may involve protein modifications, such as oligomerization of α-synuclein (AS). Potential interactions between DOPAL and other proteins related to catecholaminergic neurodegeneration, however, have not been systemically explored. This study examined DOPAL-induced protein-quinone adduct formation ("quinonization") and protein oligomerization, ubiquitination, and aggregation in cultured MO3.13 human oligodendrocytes and PC12 rat pheochromocytoma cells and in test tube experiments. Using near-infrared fluorescence spectroscopy, we detected spontaneous DOPAL oxidation to DOPAL-quinone, DOPAL-induced quinonization of intracellular proteins in both cell lines, and DOPAL-induced quinonization of several proteins related to catecholaminergic neurodegeneration, including AS, the type 2 vesicular monoamine transporter, glucocerebrosidase, ubiquitin, and l-aromatic-amino-acid decarboxylase (LAAAD). DOPAL also oligomerized AS, ubiquitin, and LAAAD; inactivated LAAAD (IC50 54 μM); evoked substantial intracellular protein ubiquitination; and aggregated intracellular AS. Remarkably, N-acetylcysteine, which decreases DOPAL-quinone formation, attenuated or prevented all of these protein modifications and functional changes. The results fit with the proposal that treatments based on decreasing the formation and oxidation of DOPAL may slow or prevent catecholaminergic neurodegeneration.

Resveratrol protects neuronal cells from isoflurane-induced inflammation and oxidative stress-associated death by attenuating apoptosis via Akt/p38 MAPK signaling.

The aim of the present study was to determine whether resveratrol protects neuronal cells from inflammation and isoflurane-induced oxidative stress-associated death via attenuating apoptosis via Akt/p38 mitogen-activated protein kinase (MAPK) signaling. The PC12 rat pheochromocytoma cell line was treated with 2% isoflurane + 21% O2 + 5% CO2 for 6 h and pre-treated with resveratrol (0-1,000 µM) for 0, 24 or 48 h prior to isoflurane treatment. An MTT assay, flow cytometry and ELISA of tumor necrosis factor-α, interleukin-6, malondialdehyde and superoxide dismutase revealed that resveratrol reduced growth inhibition, restrained apoptosis and suppressed inflammation and oxidative stress induced by isoflurane in PC12 cells. Pretreatment with resveratrol effectively reduced caspase-3 activity and inducible nitric oxide synthase protein expression in isoflurane-induced PC12 cells. In addition, western blot analysis demonstrated that resveratrol treatment significantly attenuated isoflurane-induced decreases in the activated phosphorylated (p)-Akt/Akt ratio and increases in the p-p38/p38 MAPK protein ratio in PC12 cells. These findings indicated that resveratrol was able to protect neuronal cells from isoflurane-induced inflammation and oxidative stress-associated death by attenuating apoptosis via Akt/p38 MAPK signaling.

Abstract 4147A: Potent anti-cancer activity of the imipridone ONC206: A selective dopamine D2-like receptor antagonist

Abstract DRD2 is a G protein-coupled receptor (GPCR) that is overexpressed in many cancers, controls an array of pro-survival signaling pathways, and its antagonism causes anti-cancer effects. ONC201, the founding member of the imipridone class of anti-cancer compounds, is a small molecule DRD2 antagonist that is in Phase I/II advanced cancer clinical trials. In this study, we evaluated the binding target and antitumor activity of ONC206, a chemical analogue of ONC201. An orphan small molecule target prediction algorithm revealed that ONC206, like ONC201, antagonizes DRD2. Experimental GPCR profiling using the PathHunter® β-Arrestin assay, determined that ONC206 selectively antagonizes the D2-like (DRD2/3/4), but not the D1-like (DRD 1/5), subfamily of dopamine receptors. ONC206 possesses a ~10-fold increased affinity for DRD2 compared to ONC201 with a Ki of ~320nM with selectivity that was superior to approved antipsychotics. The increased association rate for the ONC206-DRD2 interaction was responsible for the increased affinity, whereas the dissociation rate was similar to ONC201 and atypical antipsychotics that are well tolerated. TCGA analysis and immunohistochemistry of patient-derived tissue microarrays revealed DRD2 was overexpressed in neuroblastoma, sarcoma and pheochromocytoma specimens relative to normal tissues. In vitro efficacy profiling of ONC206 in the Genomic of Drug Sensitivity in Cancer collection of cell lines revealed broad efficacy across most tumor types (GI50 <78-889nM). Bone cancer and neuroblastoma were identified as the most ONC206-responsive solid tumor types that were comparatively less responsive to ONC201. Within bone cancer cell lines, Ewing’s sarcoma (n=16) was the most sensitive to ONC206 with nanomolar sensitivity (GI50 168-303nM) that was superior to ONC201. ONC206 was highly efficacious in neuroblastoma (n=35, GI50 87-589nM) including cell lines derived from metastatic sites and with MYCN amplification associated with poor prognosis. In the PC12 rat pheochromocytoma cell line ONC206 (GI50 200nM) was superior to ONC201. ONC206 time-course experiments revealed anti-cancer effects occurring at 48-72 post-treatment, similar to ONC201. In support of a wide therapeutic window, ONC206 reduced the viability of normal fibroblasts (HFF-1) at relatively high doses (GI50 > 5µM). Efficacy evaluations in MHH-ES-1 athymic nude mice xenografts demonstrated that ONC206 (100 mg/kg PO every 10 days) causes significant tumor growth inhibition that was comparable to methotrexate (400 mg/kg, IP) while being better tolerated. In summary, ONC206 is an imipridone that acts as a selective antagonist of DRD2 at nanomolar concentrations and has broad-spectrum anti-tumor activity. ONC206 may address tumor types where the properties of ONC201 do not permit for complete therapeutic engagement in vivo. Citation Format: Varun Vijay Prabhu, Neel Madhukar, Jessica Wagner, Rohinton Tarapore, Mathew Garnett, Ultan McDermott, Cyril Benes, Neil Charter, Sean Deacon, Alexander VanEngelenburg, Olivier Elemento, Wafik El-Deiry, Martin Stogniew, Wolfgang Oster, Joshua Allen. Potent anti-cancer activity of the imipridone ONC206: A selective dopamine D2-like receptor antagonist [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4147A. doi:10.1158/1538-7445.AM2017-4147A

Synthesis, characterization and in vitro antitumor behavior of a vanadium(V) complex with 4′-(3-methoxyphenyl)-2,2′:6′2″-terpyridine

A dioxovanadium(V) complex, [VO2(moptpy)](ClO4) (1, moptpy = 4′-(3-methoxyphenyl)2,2′:6′2″-terpyridine), was synthesized and characterized by elemental analysis, ESI-MS, UV–vis, IR, and 1H, 13C, and 51V NMR. The cytotoxicity in vitro of 1 was evaluated against cancer cell lines HepG-2 (hepatocellular carcinoma), SGC-7901 (gastric carcinoma), SiHa (cervical cancer), BEL-7402 (hepatocellular), and rat PC-12 (pheochromocytoma) by the MTT method. The results demonstrated that 1 exhibits superior anticancer activity in vitro with much lower values of 50% inhibitive concentration (IC50) against the selected cell lines than cisplatin, and 1 shows the highest cytotoxic activity toward SGC-7901 cells (IC50 = 1.3 ± 0.1 μM) among the selected cell lines. A series of cellular morphological and staining experiments were carried out, and the results indicated that 1 can effectively induce apoptosis of SGC-7901 cells through a ROS-mediated mitochondrial dysfunction pathway. In addition, cellular incorporation and cell cycle analysis were also performed, and it was concluded from the experimental observations that 1 can efficiently enter into the cell nuclei, and the complex inhibits cell growth in SGC-7901 cell at G0/G1 phase.

Innate function of house dust mite allergens: robust enzymatic degradation of extracellular matrix at elevated pH

Background Exposure to the house dust mite Dermatophagoides pteronyssinus (D.p.) increases the risk for developing allergic diseases in humans and their best friends, the dogs. Here, we explored whether this allergenic mite via its enzymes may impact the cutaneous extracellular matrix (ECM), which critically determines epithelial barrier integrity both structurally and functionally.Methods Two extracts obtained from either dust-purified or cultured D.p. bodies were used in the present study. To assess the potential impact of D.p. on protein components of the ECM, proteolytic activity of the D.p. extracts were determined by casein and gelatin gel zymography, and their N-acetyl-β-hexosaminidase activity determined colorimetrically. In addition, IgE-dependent and innate degranulation potential of D.p. was examined in canine MPT-1 mast cells and neurite outgrowth assay using rat pheochromocytoma PC-12 cells.Results In gel zymography, both extracts digested the substrates casein and gelatin in a dose-dependent manner, especially at alkaline pH, and effective in a wide range of temperatures (30 °C−42 °C). In particular, a 25-kDa band corresponding to Der p 1, the major D.p. allergen for humans, was found enzymatically active in both casein and gelatin gels regardless of the presence of metal ions and of alkaline conditions. Besides protease activity, N-acetyl-β-hexosaminidase activity was detected in both extracts, suggesting that D.p. affects the cutaneous ECM through deteriorating both proteins and glycosaminoglycans. While both D.p. extracts induced IgE-dependent mast cell degranulation, much less innate effects on mast- and neuronal cells were observed.Conclusions Our data highlight that D.p. is a robust source of several distinct enzymes with protease- and N-acetyl-β-hexosaminidase activities. In alkaline milieu they can degrade components of the ECM. Therefore, D.p. may contribute to epithelial barrier disruption especially when the skin surface pH is elevated.

Detoxification of hydroxylated polychlorobiphenyls by Sphingomonas sp. strain N-9 isolated from forest soil

To examine the biodegradation of hydroxylated polychlorobiphenyls (OH-PCBs), we isolated Sphingomonas sp. strain N-9 from forest soil using mineral salt medium containing 4-hydroxy-3-chlorobiphenyl (4OH-3CB) at the concentration of 10 mg/L. Following incubation with strain N-9, the concentration of 4OH-3CB decreased in inverse proportion to strain N-9 proliferation, and it was converted to 3-chloro-4-hydroxybenzoic acid (4OH-3CBA) after 1 day. We observed that strain N-9 efficiently degraded lowly chlorinated OH-PCBs (1–4 Cl), while highly chlorinated OH-PCBs (5–6 Cl) were less efficiently transformed. Additionally, strain N-9 degraded PCBs and OH-PCBs with similar efficiencies, and the efficiency of OH-PCB degradation was dependent upon the positional relationships between OH-PCB hydroxyl groups and chlorinated rings. OH-PCB biodegradation may result in highly toxic products, therefore, we evaluated the cytotoxicity of two OH-PCBs [4OH-3CB and 4-hydroxy-3,5-dichlorobiphenyl (4OH-3,5CB)] and their metabolites [4OH-3CBA and 3,5-chloro-4-hydroxybenzoic acid (4OH-3,5CBA)] using PC12 rat pheochromocytoma cells. Our results revealed that both OH-PCBs induced cell membrane damage and caused neuron-like elongations in a dose-dependent manner, while similar results were not observed for their metabolites. These results indicated that strain N-9 can convert OH-PCBs into chloro-hydroxybenzoic acids having lower toxicity.

In vitro neuroprotective potential of the monoterpenes α-pinene and 1,8-cineole against H2O2-induced oxidative stress in PC12 cells.

Oxidative stress is involved in the pathogenesis of several neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Natural products are considered as therapeutically useful antioxidant agents against reactive oxygen species (ROS). We have evaluated the antioxidant and protective potential of the monoterpenes 1,8-cineole and α-pinene against H2O2-induced oxidative stress in PC12 (rat pheochromocytoma) cells. Pretreatment with these monoterpenes was found to attenuate the loss of cell viability and the changes in cell morphology. Moreover, they inhibited the intracellular ROS production and markedly enhanced the expression of antioxidant enzymes including catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GR) and heme-oxygenase 1 (HO-1). In addition, they were able to decrease apoptosis as is evident from reduced capase-3 activity. The mechanisms of their antioxidant action appear to involve ROS scavenging and induction of the nuclear Nrf2 factor. This study demonstrates the potential beneficial therapeutic effect of these common monoterpenes on the oxidant/antioxidant balance in diseases of the nervous system.

High molecular weight of polysaccharides from Hericium erinaceus against amyloid beta-induced neurotoxicity.

BackgroundHericium erinaceus (HE) is a well-known mushroom in traditional Chinese food and medicine. HE extracts from the fruiting body and mycelia not only exhibit immunomodulatory, antimutagenic and antitumor activity but also have neuroprotective properties. Here, we purified HE polysaccharides (HEPS), composed of two high molecular weight polysaccharides (1.7 × 105 Da and 1.1 × 105 Da), and evaluated their protective effects on amyloid beta (Aβ)-induced neurotoxicity in rat pheochromocytoma PC12 cells.MethodsHEPS were prepared and purified using a 95 % ethanol extraction method. The components of HEPS were analyzed and the molecular weights of the polysaccharides were determined using high-pressure liquid chromatography (HPLC). The neuroprotective effects of the polysaccharides were evaluated through a 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay and an MTT assay and by quantifying reactive oxygen species (ROS) and mitochondrial membrane potentials (MMP) of Aβ-induced neurotoxicity in cells.ResultOur results showed that 250 μg/ml HEPS was harmless and promoted cell viability with 1.2 μM Aβ treatment. We observed that the free radical scavenging rate exceeded 90 % when the concentration of HEPS was higher than 1 mg/mL in cells. The HEPS decreased the production of ROS from 80 to 58 % in a dose-dependent manner. Cell pretreatment with 250 μg/mL HEPS significantly reduced Aβ-induced high MMPs from 74 to 51 % and 94 to 62 % at 24 and 48 h, respectively. Finally, 250 μg/mL of HEPS prevented Aβ-induced cell shrinkage and nuclear degradation of PC12 cells.ConclusionOur results demonstrate that HEPS exhibit antioxidant and neuroprotective effects on Aβ-induced neurotoxicity in neurons.

Effect of brefelamide on HGF-induced survival of 1321N1 human astrocytoma cells.

Malignant gliomas are characterized by their high level of resistance to chemo- and radiotherapy and new treatment options are urgently required. We previously demonstrated that brefelamide, an aromatic amide isolated from methanol extracts of cellular slime molds Dictyostelium brefeldianum and D. giganteum, had antiproliferative effects on 1321N1 human astrocytoma cells, a model of glioma. In this study, we investigated the mechanisms by which brefelamide inhibited 1321N1 and PC12 rat pheochromocytoma cell proliferation. When cells were cultured in serum-free medium, hepatocyte growth factor (HGF) increased survival of 1321N1 cells but not PC12 cells. HGF receptor, c-MET, was strongly expressed in 1321N1 cells, but not in PC12 cells. Pretreatment of 1321N1 cells with brefelamide inhibited both HGF-induced cell survival and expression of c-MET. Phosphorylation of extracellular signal-regulated kinase (ERK) and AKT was increased by HGF, but these changes were inhibited by brefelamide pretreatment. Moreover, HGF mRNA levels and secretion were reduced by brefelamide. These results suggest that brefelamide reduces survival of 1321N1 cells via multiple effects including suppression of HGF receptor expression and HGF secretion and inhibition of ERK and AKT phosphorylation.

Testing a cell signaling circuit

Signal Transduction It remains a challenge to understand how a limited number of signaling mechanisms encode a broad range of cellular responses. Ryu et al. explored signaling by the mitogen-activated protein kinase pathway activated by various growth factors in single PC-12 rat pheochromocytoma cells. As Bluthgen explains in a commentary, the authors tested the circuit much as an electrical engineer might test an electrical circuit. They compared effects of pulses or sustained doses of growth factor. Sustained growth factor stimulus caused a heterogeneous response of single cells—some transiently and some sustaining pathway activation. Mathematical modeling and evaluation of pulsed growth factor stimulation in cells showed that different frequencies of stimulation enabled the same growth factor to cause different cell fate decisions. Mol. Syst. Biol. 10.15252/msb.20156458 (2015). Mol. Syst. Biol. 10.15252/msb.20156642 (2015).

Partial Protection of PC12 Cells from Cellular Stress by Low-Dose Sodium Nitroprusside Pre-treatment.

The PC12 rat pheochromocytoma cell line is an in vitro model system widely used for the investigation of intracellular signaling events contributing to neuronal differentiation and cell death. We found earlier that the nitric oxide donor compound sodium nitroprusside (SNP) induced apoptosis of PC12 cells if it was applied in high concentration (400µM). Yoshioka et al. (J Pharmacol Sci 101:126-134, 2006) reported that cell death evoked by cytotoxic concentrations of SNP could be prevented by a 100µM SNP pre-treatment in a murine macrophage cell line. The apoptosis caused by toxic-dose SNP treatment (400µM) could be partially overcome in PC12 cells as well by the low-dose SNP pre-treatment. The partial inhibition of apoptosis was accompanied by reduced phosphorylation of certain proteins (such as stress-activated protein kinases, the p53, and the eIF2α proteins), decreased caspase activation, and less intense internucleosomal DNA fragmentation. The 100µM SNP pre-treatment reduced the pro-apoptotic potential of certain other stress stimuli (serum withdrawal, cisplatin and tunicamycin treatments) as well, although the underlying biochemical changes were not entirely uniform. On the contrary, the 100µM SNP pre-treatment was unable to prevent cell death caused by the protein synthesis inhibitor anisomycin. Further clarification of the above-mentioned processes may be important in understanding the mechanisms by which mild nitrosative stress protects cells against certain forms of cellular stress conditions.

Essential Oils from the Medicinal Herbs Upregulate Dopamine Transporter in Rat Pheochromocytoma Cells.

The dopamine transporter (DAT) protein, a component of the dopamine system, undergoes adaptive neurobiological changes from drug abuse. Prevention of relapse and reduction of withdrawal symptoms are still the major limitations in the current pharmacological treatments of drug addiction. The present study aimed to investigate the effects of essential oils extracted from Elsholtzia ciliata, Shinchim, Angelicae gigantis Radix, and Eugenia caryophyllata, well-known traditional Korean medicines for addiction, on the modulation of dopamine system in amphetamine-treated cells and to explore the possible mechanism underlying its therapeutic effect. The potential cytotoxic effect of essential oils was evaluated in PC12 rat pheochromocytoma cells using cell viability assays. Quantification of DAT, p-CREB, p-MAPK, and p-Akt was done by immunoblotting. DAT was significantly reduced in cells treated with 50 μM of amphetamine in a time-dependent manner. No significant toxicity of essential oils from Elsholtzia ciliata and Shinchim was observed at doses of 10, 25, and 50 μg/mL. However, essential oils from A. gigantis Radix at a dose of 100 μg/mL and E. caryophyllata at doses of 50 and 100 μg/mL showed cytotoxicity. Treatment with GBR 12909, a highly selective DAT inhibitor, significantly increased DAT expression compared with that of amphetamine only by enhancing phosphorylation of mitogen-activated protein kinases (MAPK) and Akt. In addition, essential oils effectively induced hyperphosphorylation of cyclic-AMP response element-binding protein (CREB), MAPK, and Akt, which resulted in DAT upregulation. Our study implies that the essential oils may rehabilitate brain dopamine function through increased DAT availability in abstinent former drug users.

Luminescence Lifetime Imaging Microscopy For Analysis Of Oxygen In The Cancer Cell

Molecular oxygen (O2), the key component of aerobic metabolism, plays important roles in normal and cancer cells and tissues. A real‐time and quantitative O2 imaging in 3D cell models would allow to discriminate complex effects of hypoxia on cell function in complex (heterogeneous) cell populations at subcellular resolution. Here, we used luminescence lifetime imaging (FLIM, PLIM) to develop and evaluate phosphorescent O2 probes with 3D spheroid and multi‐cellular aggregate models. We tested rat embryonic neurosphere culture with cationic nanoparticle O2 probes (MM2, NanO2) and developed O2 imaging protocol, multiplexed with analysis of distribution of specific protein markers such as HIF‐2α. To address the problem of efficient in‐depth staining, we designed two novel intracellular O2 probes: small molecule Pt‐Glc and nanoparticle PA2. Both probes displayed high analytical performance (brightness, photostability, O2 calibration) and were evaluated with rat pheochromocytoma PC12 and human colon cancer HCT116 cell aggregates. Resting cells cultured in 3D aggregates of >50 μm size displayed significant deoxygenation even under ambient atmospheric O2 levels. Still, they showed high viability and experienced different levels of oxygenation, upon treatment with mitochondrial uncouplers, inhibitors or pharmacological drugs. This data confirms the importance of probing of cellular O2 for experiments with 3D cell models. When combined with other live cell imaging dyes, such as markers of necrosis and apoptosis, the method allows for multi‐parameter quantitative assessment of cell physiology within relevant 3D micro‐environment.Supported by Science Foundation Ireland (SFI) grant 13/SIRG/2144.

Effect of L-lysine alpha-oxidase from Trichoderma cf. aureoviride Rifai ВКМF-4268D on pheochromocytoma PC12 cell line

L-Amino acid oxidases (L-ААО, EC 1.4.3.2) comprise a group of flavoproteins, catalyzing oxidative deamination of L-alpha amino acids to the corresponding alpha-keto acids, NH3 and Н2О2. In most cases these enzymes present homodimeric molecules with a molecular mass of 100-150 kDa, which were shown to possess antiviral, antifungal and antitumor activity. L-lysine alpha-oxidase (LO) holds an outstanding place among this group of enzymes and its biological role may differ significantly from the other L-AAO, because it cleaves an essential amino acid - L-lysine without significant action on the other amino acids. Although much research has examined LO effects in the organism, the molecular basis of these effects is yet to be identified. To fill this gap, the present work addressed one of hypothetical mechanisms of LO biological action using the enzyme from Trichoderma cf. aureoviride Rifai ВКМF-4268D and rat pheochromocytoma PC-12 as a model cell line. Using flow cytometry a dose-dependent cytotoxicity of LO was shown. The significant growth of intracellular reactive oxygen species levels, detected by 2,7-dichlorodihydrofluorescein assay, implies generation of peroxide as one of the molecular mechanisms of LO cytotoxic action, although this does not rule out other probable ways of LO action in the organizm.

Synthetic chalcones with potent antioxidant ability on H₂O₂-induced apoptosis in PC12 cells.

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.

The role of the p53 protein in nitrosative stress-induced apoptosis of PC12 rat pheochromocytoma cells.

PC12 rat pheochromocytoma cells are widely used to investigate signaling pathways. The p143p53PC12 cell line expresses a Val143Ala mutant p53 protein that is less capable of binding to the p53 consensus site in DNA than its wild-type counterpart. Nitric oxide (NO), depending on its concentration, is able to activate several signal transduction pathways. We used sodium nitroprusside (SNP), an NO donor compound, to analyze NO-induced cellular stress in order to clarify the mechanism and role of nitrosative stress in pathological processes, including inflammation and cancer. SNP caused cell death when applied at a concentration of 400μM, p143p53PC12 cells showing higher sensitivity than wild-type PC12 cells. The mechanisms leading to the increased SNP-sensitivity of p143p53PC12 cells were then investigated. The 400-μM SNP treatment caused stress kinase activation, phosphorylation of the eukaryotic initiation factor eIF2α and p53 protein, proteolytic activation of protein kinase R, caspase-9, and caspase-3, p53 stabilization, CHOP induction, cytochrome c release from mitochondria, and a decline in the level of the Bcl-2 protein in both cell lines. All these SNP-induced changes were more robust and/or permanent in cells with the mutant p53 protein. We thus conclude that (1) the main cause of the SNP-induced apoptosis of PC12 cells is the repression of the bcl-2 gene, evoked through p53 stabilization, stress kinase activation, and CHOP induction; (2) the higher SNP sensitivity of p143p53PC12 cells is the consequence of the stronger and earlier activation of the intrinsic apoptotic pathway.

Effect of sodium ferulate on delayed rectifier K+ currents in PC12 cells.

In order to investigate the effect of sodium ferulate (SF) on voltage-activated K+ channels, the delayed rectifier K+ current (Ik) in PC12 rat pheochromocytoma cells was recorded using the automated patch-clamp method. The results indicated that following the application of SF, the Ik in PC12 cells was significantly decreased in a concentration-dependent manner. The analysis of activation kinetic curves and inactivation kinetic curves of Ik showed that SF had an effect on the activation and inactivation kinetics. Following the application of 15.3 μM SF, the activation curve of the Ik of PC12 cells was shifted to positive potentials and the inactivation curve of the Ik of PC12 cells was shifted to negative potentials. This study revealed that the delayed rectifier K+ currents of PC12 cells were inhibited following SF treatment in a concentration-dependent manner. The mechanism may be associated with the delayed activation and enhanced inactivation of Ik-associated channels.

Liquiritin modulates ERK‑ and AKT/GSK‑3β‑dependent pathways to protect against glutamate‑induced cell damage in differentiated PC12 cells.

Glutamate has a key role in the neuronal cell damage associated with Alzheimer’s and Parkinson’s diseases. Liquiritin (LQ), a major constituent of Glycyrrhiza Radix, possesses various pharmacological activities. The present study investigated the neuroprotective effect of LQ against glutamate-induced cell damage in the differentiated PC12 (DPC12) rat pheochromocytoma cell line. Pretreatment with 25 and 50 μM LQ for 3 h resulted in a significant increase in cell viability and inhibited excessive lactate dehydrogenase release in glutamate-exposed DPC12 cells. LQ also ameliorated glutamate-induced nuclear and mitochondrial apoptotic alterations, intracellular calcium overload and the abnormal expression of apoptosis-related proteins, including cytochrome c, B-cell lymphoma (Bcl)-2 and Bcl2-associated X protein. Treatment with LQ alone or in combination with glutamate was found to enhance the phosphoactivation of extracellular signal-regulated kinases (ERKs), AKT and its downstream element glycogen synthase kinase-3β (GSK3β), in a time-dependent manner. However, no effect was observed on the expression of total-ERKs, -AKT and -GSK3β. Furthermore, pre-incubation with 10 μM PD98059 or LY94002, inhibitors of ERK and phosphatidylinositide 3-kinase, respectively, for 30 min significantly suppressed the LQ-induced increase in glutamate-exposed DPC12 cell viability. To the best of our knowledge, the present study provides the first experimental evidence that LQ has a neuroprotective effect against glutamate toxicity in DPC12 cells, predominantly through the ERK and AKT/GSK-3β pathways. Therefore, LQ may have potential for the treatment of neurodegenerative diseases.

Phosphodiesterase 2 negatively regulates adenosine-induced transcription of the tyrosine hydroxylase gene in PC12 rat pheochromocytoma cells

Adenosine induces expression of the tyrosine hydroxylase (TH) gene in PC12 cells. However, it is suggested that atrial natriuretic peptide (ANP) inhibits expression of this gene. Using real-time PCR and luciferase reporter assays we found that ANP significantly decreases the adenosine-induced transcription of the TH gene. Results of measurements of cyclic nucleotide concentrations indicated that ANP-induced accumulation of cGMP inhibits the adenosine-induced increase in cAMP level. Using selective phosphodiesterase 2 (PDE2) inhibitors and a synthetic cGMP analog activating PDE2, we found that PDE2 is involved in coupling the ANP-triggered signal to the cAMP metabolism. We have established that ANP-induced elevated levels of cGMP as well as cGMP analog stimulate hydrolytic activity of PDE2, leading to inhibition of adenosine-induced transcription of the TH gene. We conclude that ANP mediates negative regulation of TH gene expression via stimulation of PDE2-dependent cAMP breakdown in PC12 cells.