Differentiation of normal and cancer cells induced by sulfhydryl reduction: biochemical and molecular mechanisms

We examined the morphological, biochemical and molecular outcome of a nonspecific sulfhydryl reduction in cells, obtained by supplementation of N-acetyl-L-cysteine (NAC) in a 0.1-10 mM concentration range. In human normal primary keratinocytes and in colon and ovary carcinoma cells we obtained evidences for: (i) a dose-dependent inhibition of proliferation without toxicity or apoptosis; (ii) a transition from a proliferative mesenchymal morphology to cell-specific differentiated structures; (iii) a noticeable increase in cell-cell and cell-substratum junctions; (iv) a relocation of the oncogenic beta-catenin at the cell-cell junctions; (v) inhibition of microtubules aggregation; (vi) upregulation of differentiation-related genes including p53, heat shock protein 27 gene, N-myc downstream-regulated gene 1, E-cadherin, and downregulation of cyclooxygenase-2; (vii) inhibition of c-Src tyrosine kinase. In conclusion, a thiol reduction devoid of toxicity as that operated by NAC apparently leads to terminal differentiation of normal and cancer cells through a pleiade of converging mechanisms, many of which are targets of the recently developed differentiation therapy

Global gene expression analysis in time series following N-acetyl L-cysteine induced epithelial differentiation of human normal and cancer cells in vitro

BACKGROUND: Cancer prevention trials using different types of antioxidant supplements have been carried out at several occasions and one of the investigated compounds has been the antioxidant N-acetyl-L-cysteine (NAC). Studies at the cellular level have previously demonstrated that a single supplementation of NAC induces a ten-fold more rapid differentiation in normal primary human keratinocytes as well as a reversion of a colon carcinoma cell line from neoplastic proliferation to apical-basolateral differentiation [1]. The investigated cells showed an early change in the organization of the cytoskeleton, several newly established adherens junctions with E-cadherin/β-catenin complexes and increased focal adhesions, all features characterizing the differentiation process. METHODS: In order to investigate the molecular mechanisms underlying the proliferation arrest and accelerated differentiation induced by NAC treatment of NHEK and Caco-2 cells in vitro, we performed global gene expression analysis of NAC treated cells in a time series (1, 12 and 24 hours post NAC treatment) using the Affymetrix GeneChip™ Human Genome U95Av2 chip, which contains approximately 12,000 previously characterized sequences. The treated samples were compared to the corresponding untreated culture at the same time point. RESULTS: Microarray data analysis revealed an increasing number of differentially expressed transcripts over time upon NAC treatment. The early response (1 hour) was transient, while a constitutive trend was commonly found among genes differentially regulated at later time points (12 and 24 hours). Connections to the induction of differentiation and inhibition of growth were identified for a majority of up- and down-regulated genes. All of the observed transcriptional changes, except for seven genes, were unique to either cell line. Only one gene, ID-1, was mutually regulated at 1 hour post treatment and might represent a common mediator of early NAC action. The detection of several genes that previously have been identified as stimulated or repressed during the differentiation of NHEK and Caco-2 provided validation of results. In addition, real-time kinetic PCR analysis of selected genes also verified the differential regulation as identified by the microarray platform. CONCLUSION: NAC induces a limited and transient early response followed by a more consistent and extensively different expression at later time points in both the normal and cancer cell lines investigated. The responses are largely related to inhibition of proliferation and stimulation of differentiation in both cell types but are almost completely lineage specific. ID-1 is indicated as an early mediator of NAC action

Prodan as a membrane surface fluorescence probe: partitioning between water and phospholipid phases.

Fluorescence spectral features of 6-propionyl-2-dimethylaminonaphthalene (Prodan) in phospholipid vesicles of different phase states are investigated. Like the spectra of 6-lauroyl-2-dimethylaminonaphthalene (Laurdan), the steady-state excitation and emission spectra of Prodan are sensitive to the polarity of the environment, showing a relevant shift due to the dipolar relaxation phenomenon. Because of the different lengths of their acyl residues, the partitioning of the two probes between water and the membrane bilayer differs profoundly. To account for the contribution of Prodan fluorescence arising from water, we introduce a three-wavelength generalized polarization method that makes it possible to separate the spectral properties of Prodan in the lipid phase and in water, and to determine the probe partitioning between phospholipid and water and between the gel and the liquid-crystalline phases of phospholipids. In contrast to Laurdan, Prodan preferentially partitions in the liquid-crystalline phase with respect to the gel and is sensitive to the polar head pretransition, and its partition coefficient between the membrane and water depends on the phase state, i.e., on the packing of the bilayer. Prodan is sensitive to polarity variations occurring closer to the bilayer surface than those detected by Laurdan

Estradiol enhances the resistance of LDL to oxidation by stabilizing apoB-100 conformation

Among different proposed mechanisms to account for the protection exerted by estrogens against cardiovascular diseases, the antioxidant effect has attracted considerable attention. We confirmed that 17-beta-estradiol (E2), when added to human LDL at a 6:1 ratio to apoB-100, markedly delays the phase of massive LDL lipid peroxidation induced by Cu(2+). We also observed an increased oxidative resistance of E2-treated LDL by monitoring the early phase of oxidative degradation on the basis of increased LDL surface polarity by the generalized polarization of the lipophilic fluorescent probe 2-(dimethylamino)-6-lauroylnaphthalene (Laurdan). A scavenging of free radicals by E2 is ruled out since, consistent with its structure, its rate constant for the reduction of peroxy radicals is extremely low, i.e., 0.02% of that of vitamin E. Tryptophan fluorescence lifetime and circular dichroism measurements revealed that (i) apoB-100 undergoes a conformational modification and a progressive loss of secondary structure during lipid peroxidation; (ii) E2 increases apoB-100 secondary structure and modifies its conformation; and (iii) the apoB-100 conformational change induced by E2 makes this protein resistant to modifications brought about by lipid peroxidation. We propose that E2, by affecting apoB-100 secondary structure and conformation, modifies the interaction of this protein with the outer layer of the LDL particle thus increasing its overall oxidative resistance

Thiol Redox Transitions in Cell Signaling: a Lesson from N-Acetylcysteine

Abstract The functional status of cells is under the control of external stimuli affecting the function of critical proteins and eventually gene expression. Signal sensing and transduction by messengers to specific effectors operate by post-translational modification of proteins, among which thiol redox switches play a fundamental role that is just beginning to be understood. The maintenance of the redox status is, indeed, crucial for cellular homeostasis and its dysregulation towards a more oxidized intracellular environment is associated with aberrant proliferation, ultimately related to diseases such as cancer, cardiovascular disease, and diabetes. Redox transitions occur in sensitive cysteine residues of regulatory proteins relevant to signaling, their evolution to metastable disulfides accounting for the functional redox switch. N-acetylcysteine (NAC) is a thiol-containing compound that is able to interfere with redox transitions of thiols and, thus, in principle, able to modulate redox signaling. We here review the redox chemistry of NAC, then screen possible mechanisms to explain the effects observed in NAC-treated normal and cancer cells; such effects involve a modification of global gene expression, thus of functions and morphology, with a leitmotif of a switch from proliferation to terminal differentiation. The regulation of thiol redox transitions in cell signaling is, therefore, proposed as a new tool, holding promise not only for a deeper explanation of mechanisms, but indeed for innovative pharmacological interventions

N-acetyl-l-cysteine fosters inactivation and transfer to endolysosomes of c-Src

Abstract The non-receptor-protein tyrosine kinase c-Src is overexpressed and activated in a large number of human cancers, in which it is associated with tumor development and progression. Canonical regulation takes place by means of an alternative phosphorylation of tyrosine residues -- Tyr419 for activation and Tyr530 for inactivation. An independent redox regulation mechanism, involving cysteine residues, has also been proposed, in which oxidation activates the enzyme. Here we present a kinetic analysis of the effect of N-acetyl-l-cysteine (NAC) on c-Src, demonstrating that reduction reverts the oxidation-driven activation. In cancer cells, we show that NAC treatment produces an increase in specifically labeled reduced thiols of c-Src cysteines, thus confirming a redox transition. In addition to a decrease in Tyr419 phosphorylation, this leads to a massive shift of c-Src from plasma membranes -- where its active form is located -- to endolysosomal compartments. With the objective of deciphering the complex issue of c-Src regulation and of devising new strategies to revert its activation in cancers, redox regulation thus emerges as a promising area for study.and progression. Canonical regulation takes place by means of an alternative phosphorylation of tyrosine residues-Tyr419 for activation and Tyr530 for inactivation. An independent redox regulation mechanism, involving cysteine residues, has also been proposed, in which oxidation activates the enzyme. Here we present a kinetic analysis of the effect of N-acetyl-l-cysteine (NAC) on c-Src, demonstrating that reduction reverts the oxidation-driven activation. In cancer cells, we show that NAC treatment produces an increase in specifically labeled reduced thiols of c-Src cysteines, thus confirming a redox transition. In addition to a decrease in Tyr419 phosphorylation, this leads to a massive shift of c-Src from plasma membranes-where its active form is located-to endolysosomal compartments. With the objective of deciphering the complex issue of c-Src regulation and of devising new strategies to revert its activation in cancers, redox regulation thus emerges as a promising area for study. (C) 2008 Elsevier Inc. All rights reserved

ER stress induced by the OCH1 mutation triggers changes in lipid homeostasis in Kluyveromyces lactis

In Kluyveromyces lactis yeast, OCH1 encodes for the α-1,6-mannosyltrasferase that adds the initial α-1,6-mannose to the outer-chains of N-glycoproteins. Kloch1-1 mutant cells showed altered calcium homeostasis and endoplasmic reticulum (ER) stress. Since ER plays a major role in lipid biosynthesis and lipid droplet (LD) formation, herein the impact of Och1p depletion on lipid homeostasis was investigated. Transcriptional profiles of genes involved in biosynthesis of fatty acids, their amount and composition changed in mutant cells. An increased amount of ergosterol was determined in these cells. Enhanced transcription of genes involved in both synthesis and mobilization of LDs was also found in Kloch1-1 cells, accompanied by a reduced amount of LDs. We provide evidence that ER alterations, determined by protein misfolding as a result of reduced N-glycosylation, induced altered lipid homeostasis in Kloch1-1 cells. Chemical chaperone 4-phenyl butyrate (4-PBA) slightly alleviated the LD phenotype in cells depleted of Och1p. Remarkably, complete suppression of ER stress, via increased expression of plasma membrane calcium channel subunit Mid1, fully restored lipid homeostasis in mutant cells. To further reinforce this finding, low numbers of LDs were observed in wild type cells when ER stress was triggered by DTT treatmen

N-acetyl-l-cysteine fosters inactivation and transfer to endolysosomes of c-Src

The non-receptor-protein tyrosine kinase c-Src is overexpressed and activated in a large number of human cancers, in which it is associated with tumor development and progression. Canonical regulation takes place by means of an alternative phosphorylation of tyrosine residues -- Tyr419 for activation and Tyr530 for inactivation. An independent redox regulation mechanism, involving cysteine residues, has also been proposed, in which oxidation activates the enzyme. Here we present a kinetic analysis of the effect of N-acetyl-l-cysteine (NAC) on c-Src, demonstrating that reduction reverts the oxidation-driven activation. In cancer cells, we show that NAC treatment produces an increase in specifically labeled reduced thiols of c-Src cysteines, thus confirming a redox transition. In addition to a decrease in Tyr419 phosphorylation, this leads to a massive shift of c-Src from plasma membranes -- where its active form is located -- to endolysosomal compartments. With the objective of deciphering the complex issue of c-Src regulation and of devising new strategies to revert its activation in cancers, redox regulation thus emerges as a promising area for study