Substance P-Evoked Calcium Mobilization and Ionic Current Activation in the Human Astrocytoma Cell Line U 373 MG: Pharmacological Characterization

Abstract— In the human astrocytoma cell line U 373 MG, application of substance P (SP) leads to a transient increase in cytosolic calcium concentration and to a biphasic current response in voltage-clamped cells. Using these two functional assays we have characterized pharmacologically the SP response in U 373 MG cells. SP and [l -Pro⁹]SP displayed high potencies in both assays with EC₅₀values of 2.5 ± 10^?9M and 1 ± 10^?9M on calcium responses and 110^?9M and 510^?9M on ion current responses, respectively. The high potency of SP and [l -Pro⁹]SP as well as the low potency of [Lys⁵,MeLeu⁹,N-Leu¹⁰]neurokinin A_(4-10) and the inactivity of senktide demonstrate the NK₁-type pharmacology of these responses. Furthermore, the NK₁ antagonists (±)-CP 96,345, its chloro analogue, (±)-cis-3-(2-chlorobenzylamino)-2-benz-hydrylquinuclidine, and RP 67580 were potent antagonists of both SP responses. For the calcium mobilization induced by SP (1 (10^?7M), the IC₅₀ values for the three antagonists were 4 ± 10^?10M, 4 ± 10^?9M, and 9 ± 10^?9M, respectively, whereas on the current response evoked by SP 10^?8M), the IC₅₀ values were 8 ± 10^?9M, 2.4 ± 10^?8M, and 1.2 10^?7M, respectively. Despite differences in the absolute IC₅₀ values obtained with both techniques, the relative potencies of the three antagonists correlate fairly well. The U 373 MG cell line provides a useful model system for studies of the pharmacology of the human NK₁receptor and its transduction mechanisms at the level of second messengers and modulation of ion currents.     

Identification of glyoxalase 1 polymorphisms associated with enzyme activity

The glyoxalase system and its main enzyme, glyoxalase 1 (GLO1), protect cells from advanced glycation end products (AGEs), such as methylglyoxal (MG) and other reactive dicarbonyls, the formation of which is increased in diabetes patients as a result of excessive glycolysis. MG is partly responsible for harmful protein alterations in living cells, notably in neurons, leading to their dysfunction, and recent studies have shown a negative correlation between GLO1 expression and tissue damage. Neuronal dysfunction is a common diabetes complication due to elevated blood sugar levels, leading to high levels of AGEs. The aim of our study was to determine whether single nucleotide polymorphisms (SNPs) in the GLO1 gene influence activity of the enzyme. In total, 125 healthy controls, 101 type 1 diabetes, and 100 type 2 diabetes patients were genotyped for three common SNPs, rs2736654 (A111E), rs1130534 (G124G), and rs1049346 (5′-UTR), in GLO1. GLO1 activity was determined in whole blood lysates for all participants of the study.     

Cathepsin L silencing enhances arsenic trioxide mediated in vitro cytotoxicity and apoptosis in glioblastoma U87MG spheroids

Despite improved treatment options, glioblastoma multiforme (GBM) remains the most aggressive brain tumour with the shortest post-diagnostic survival. Arsenite (As₂O₃) is already being used in the treatment of acute promyelocytic leukaemia (APL), yet its effects on GBM have not been evaluated in detail. In U87MG cell monolayers, we have previously shown that arsenite cytotoxicity significantly increases upon transient inhibition of lysosomal protease Cathepsin L (CatL). As multicellular spheroids more closely represent in vivo tumours, we aimed to evaluate the impact of permanent CatL silencing on arsenite treatment in U87MG spheroids. CatL was stably silenced using shRNA expression plasmid packed lentiviruses. By using metabolic- and cell viability assays, we demonstrated that long-term CatL silencing significantly increased arsenite cytotoxicity in U87MG spheroids. Silenced CatL also increased arsenite-mediated apoptosis in spheroids via elevated p53 expression, Bax/Bcl2 ratio and caspase 3/7 activity, though with lower efficacy than in monolayers. Arsenite cytotoxicity was enhanced by lower CatL activity, since similar cytotoxicity increase was also observed using the novel CatL inhibitor AT094. The results have significant translational impact, since stable CatL silencing would enable the application of lower systemic doses of arsenite to achieve the desired cytotoxic effects on GBMs in vivo.     

Diversity and distribution of hidden cultivable fungi associated with marine animals of Antarctica

In the present study, we surveyed the distribution and diversity of fungal assemblages associated with 10 species of marine animals from Antarctica. The collections yielded 83 taxa from 27 distinct genera, which were identified using molecular biology methods. The most abundant taxa were Cladosporium sp. 1, Debaryomyces hansenii, Glaciozyma martinii, Metschnikowia australis, Pseudogymnoascus destructans, Thelebolus cf. globosus, Pseudogymnoascus pannorum, Tolypocladium tundrense, Metschnikowia australis, and different Penicillium species. The diversity, richness, and dominance of fungal assemblages ranged among the host; however, in general, the fungal community, which was composed of endemic and cold-adapted cosmopolitan taxa distributed across the different sites of Antarctic Peninsula, displayed high diversity, richness, and dominance indices. Our results contribute to knowledge about fungal diversity in the marine environment across the Antarctic Peninsula and their phylogenetic relationships with species that occur in other cold, temperate, and tropical regions of the World. Additionally, despite their extreme habitats, marine Antarctic animals shelter cryptic and complex fungal assemblages represented by endemic and cosmopolitan cold-adapted taxa, which may represent interesting models to study different symbiotic associations between fungi and their animal hosts in the extreme conditions of Antarctica.     

Genomic analysis and lactose transporter expression in Kluyveromyces marxianus CCT 7735

Kluyveromyces marxianus CCT 7735 has been used to produce ethanol, aromatic compounds, enzymes and heterologous proteins besides assimilates lactose as carbon source. Its genome has 10.7 Mb and encodes 4787 genes distributed in 8 nuclear chromosomes and one mitochondrial. Contrary to Kluyveromyces lactis, which has a unique LAC12 gene (encodes lactose permease), K. marxianus possesses four. The presence of degenerated copies and Solo-LTRs related to retrotransposon TKM close to the LAC12 genes in K. marxianus indicates ectopic recombinations. The Lac12 permeases of K. marxianus and K. lactis are conserved, however the conservation is higher between the copy of the left side of the chromosome three and the unique copy of K. lactis, indicating that this copy is the ancestor. The expression of the four LAC12 genes occurred in aerobiosis and hypoxia. Notably, the high lactose consumption in hypoxia seems to be related to the high expression of the LAC12 genes.     

Hepatocyte susceptibility to glyoxal is dependent on cell thiamin content

Glyoxal, a reactive dicarbonyl, is detoxified primarily by the glyoxalase system utilizing glutathione (GSH) and by the aldo-keto reductase enzymes which utilizes NAD[P]H as the co-factor. Thiamin (Vitamin B(1)) is an essential coenzyme for transketolase (TK) that is part of the pentose phosphate pathway which helps maintain cellular NADPH levels. NADPH plays an intracellular role in regenerating glutathione (GSH) from oxidized GSH (GSSG), thereby increasing the antioxidant defenses of the cell. In this study we have focused on the prevention of glyoxal toxicity by supplementation with thiamin (3mM). Thiamin was cytoprotective and restored NADPH levels, glyoxal detoxification and mitochondrial membrane potential. Hepatocyte reactive oxygen species (ROS) formation, lipid peroxidation and GSH oxidation were decreased. Furthermore, hepatocytes were made thiamin deficient with oxythiamin (3mM) as measured by the decreased hepatocyte TK activity. Under thiamin deficient conditions a non-toxic dose of glyoxal (2mM) became cytotoxic and glyoxal metabolism decreased; while ROS formation, lipid peroxidation and GSH oxidation was increased.     

Overexpression of sialidase NEU3 increases the cellular radioresistance potential of U87MG glioblastoma cells

The plasma membrane-associated sialidase NEU3 is known to play important roles in different physiological and pathophysiological processes such as proliferation, cellular differentiation and tumorigenesis. Up-regulation of NEU3 has been associated to several tumors and recently it was demonstrated that its down-modulation in glioblastoma cells promotes cell invasiveness. To date, no information concerning the possible role played by NEU3 in relation to tumor radioresistance is available. Here we show that overexpression of NEU3 in glioblastoma U87MG cells activates PI3K/Akt signaling pathway resulting in an increased radioresistance capacity and in an improved efficiency of double strand DNA-repair mechanisms after irradiation. Our results demonstrate for the first time that NEU3 contributes to the radioresistance features of U87MG cells, bringing to evidence a novel rand peculiar role of the enzyme in cancer biology.     

Hyperphosphorylation of extracellular regulated kinase 2 (ERK2) and inhibition of JNK2 phosphorylation are associated with increased S-phase during transformation of Syrian hamster embryo cells by Malachite Green

Malachite Green (MG), consisting of green crystals with a metallic lustre, is highly soluble in water, cytotoxic to various mammalian cells and also acts as a liver tumour promoter. In view of its industrial importance and possible exposure to human beings, MG poses a potential environmental health hazard. We have earlier reported the malignant transformation of Syrian hamster embryo (SHE) cells in primary culture by MG. In this study, we have studied the mitogen activated protein (MAP) kinase signal transduction pathway in preneoplastic cells induced by MG. Western blots of MG induced preneoplastic cells showed no phosphorylation of ERK1, an increased phosphoactive ERK2 associated with a decreased expression of phosphoactive JNK2. However, total forms of ERKs, JNKs and p38 Kinases showed similar levels of expression in control and preneoplastic SHE cells. Indirect immunofluorescence studies have shown a distinct nuclear localisation of phosphoactive ERKs in MG induced preneoplastic cells. Flow cytometric analysis showed an increase of S-phase cells in preneoplastic cells compared to control SHE cells. The present study indicates that hyperphosphorylation of ERK2, decreased JNK2 phosphorylation and an increase in S-phase cells seems to be the early changes associated with the MG induced malignant transformation of SHE cells in primary culture.     

Proteasome inhibitor-induced apoptosis of glioma cells involves the processing of multiple caspases and cytochrome c release

The proteasome is a multiprotein complex that is involved in the intracellular protein degradation in eukaryotes. Here, we show that human malignant glioma cells are susceptible to apoptotic cell death induced by the proteasome inhibitors, MG132 and lactacystin. The execution of the apoptotic death program involves the processing of caspases 2, 3, 7, 8, and 9. Apoptosis is inhibited by ectopic expression of X-linked inhibitor of apoptosis (XIAP) and by coexposure to the broad-spectrum caspase inhibitor, benzoyl-VAD-fluoromethyl ketone (zVAD-fmk), but not by the preferential caspase 8 inhibitor, crm-A. It is interesting that specific morphological alterations induced by proteasome inhibition, such as dilated rough endoplasmic reticulum and the formation of cytoplasmic vacuoles and dense mitochondrial deposits, are unaffected by zVAD-fmk. Apoptosis is also inhibited by ectopic expression of Bcl-2 or by an inhibitor of protein synthesis, cycloheximide. Further, cytochrome c release and disruption of mitochondrial membrane potential are prominent features of apoptosis triggered by proteasome inhibition. Bcl-2 is a stronger inhibitor of cytochrome c release than zVAD-fmk. XIAP and crm-A fail to modulate cytochrome c release. These data place cytochrome c release downstream of Bcl-2 activity but upstream of XIAP- and crm-A-sensitive caspases. The partial inhibition of cytochrome c release by zVAD-fmk indicates a positive feedback loop that may involve cytochrome c release and zVAD-fmk-sensitive caspases. Finally, death ligand/receptor interactions, including the CD95/CD95 ligand system, do not mediate apoptosis induced by proteasome inhibition in human malignant glioma cells.     

NAD-linked alcohol dehydrogenase 1 regulates methylglyoxal concentration in Candida albicans

We purified a fraction that showed NAD⁺-linked methylglyoxal dehydrogenase activity, directly catalyzing methylglyoxal oxidation to pyruvate, which was significantly increased in glutathione-depleted Candida albicans. It also showed NADH-linked methylglyoxal-reducing activity. The fraction was identified as a NAD⁺-linked alcohol dehydrogenase (ADH1) through mass spectrometric analyses. In ADH1-disruptants of both the wild type and glutathione-depleted cells, the intracellular methylglyoxal concentration increased significantly; defects in growth, differentiation, and virulence were observed; and G2-phase arrest was induced.