Differential distribution of glucocorticoid and estrogen receptor isoforms: localization of GRbeta and ERalpha in nucleoli and GRalpha and ERbeta in the mitochondria of human osteosarcoma SaOS-2 and hepatocarcinoma HepG2 cell lines

The localization of glucocorticoid and estrogen receptors alpha (GRalpha, ERalpha) and beta (GRbeta, ERbeta) in osteosarcoma SaOS-2 and hepatocarcinoma HepG2 cells was studied by immunofluorescence labelling and confocal laser scanning microscopy, as well as by subcellular fractionation and immunoblotting of the proteins of the fractions with respective antibodies. In HepG2 and SaOS-2 cells GRbeta and ERalpha were localized mainly in the nucleus, particularly concentrated in nuclear structures, which on the basis of their staining with antibody against C23-nucleolin, were characterized as nucleoli. A faint, diffuse GRbeta and ERalpha staining was also observed in the cytoplasm. GRalpha and ERbeta were specifically enriched at the site of cell mitochondria, which were visualized by labelling with the vital dye CMX. Immunoblotting experiments corroborated the immunofluorescence labelling distribution of glucocorticoid and estrogen receptor isoforms in the cell lines studied. These findings support the concept of a direct action of steroid/thyroid hormones on mitochondrial functions by way of their cognate receptors and also suggest a direct involvement of GRbeta and ERalpha in nucleolar-related processes in HepG2 and SaOS-2 cells.     

Differential subcellular distribution of estrogen receptor isoforms: localization of ERalpha in the nucleoli and ERbeta in the mitochondria of human osteosarcoma SaOS-2 and hepatocarcinoma HepG2 cell lines

The localization of estrogen receptors alpha (ERalpha) and beta (ERbeta) in osteosarcoma SaOS-2 and hepatocarcinoma HepG2 cells was studied by immunofluorescence labelling and confocal laser scanning microscopy, as well as by subcellular fractionation and immunoblotting of the proteins of the fractions with respective antibodies. In both cell types, ERalpha was localized mainly in the nucleus, particularly concentrated on nuclear structures, which on the basis of their staining with pyronin and with antibodies against the nucleoli-specific Ki67 antigen and C23-nucleolin, were characterized as nucleoli. A faint, diffuse ERalpha staining was also observed in the cytoplasm. ERbeta was specifically enriched at the site of the mitochondria, visualized by labelling with the vital dye CMX and antibody against the mitochondrial-specific cytochrome oxidase subunit I. Immunoblotting experiments corroborated the immunofluorescence labelling distribution of ERalpha and ERbeta. These findings support the concept of a direct action of steroid/thyroid hormones on mitochondrial functions by way of their cognate receptors and also suggest a direct involvement of ERalpha in nucleolar-related processes.     

Estrogen receptor alpha negative breast cancer patients: estrogen receptor beta as a therapeutic target

Clinical management of breast cancer is increasingly guided by assessment of tumor phenotypic parameters. One of these is estrogen receptor (ER) status, currently defined by ERalpha expression. However with the discovery of a second ER, ERbeta and its variant isoforms, the definition of ER status is potentially more complex. In breast tumors there are two ERbeta expression cohorts. One where ERbeta is co-expressed with ERalpha and the other expressing ERbeta alone. In the latter subgroup of currently defined ER negative patients ERbeta has the potential to be a therapeutic target. Characterization of the nature and role of ERbeta in ERalpha negative tumors is essentially unexplored but available data suggest that the role of ERbeta may be different when co-expressed with ERalpha and when expressed alone. This review summarizes available data and explores the possibility that ERbeta signaling may be a therapeutic target in these tumors. Evidence so far supports the idea that the role of ERbeta in breast cancer is different in ERalpha negative compared to ERalpha positive tumors. However, cohort size and numbers of independent studies are small to date, and more studies are needed with better standardization of antibodies and protocols. Also, the ability to determine the role of ERbeta in ERalpha negative breast cancer and therefore assess ERbeta signaling pathways as therapeutic targets would be greatly facilitated by identification of specific downstream markers of ERbeta activity in breast cancer.     

Effect of Cu,Zn superoxide dismutase on cholesterol metabolism in human hepatocarcinoma (HepG2) cells

The microsomal enzyme 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase and the low density lipoprotein (LDL) receptor pathway carry out a key role on cholesterol homeostasis in eucaryotic cells. The HMG-CoA reductase is sensitive to oxidative inactivation and to phosphorylation by many kinases that are able to inactivate the protein and increase its susceptibility to proteolysis. We previously demonstrated that a calf thymus Cu,Zn SOD affects cholesterol metabolism. This protein binds with rat hepatocyte cell membrane by a specific surface membrane receptor. The involvement of Cu,Zn SOD in cholesterol metabolism is confirmed further by the presence of this antioxidant enzyme in circulating serum lipoproteins. We studied the effect of native human Cu,Zn SOD, metal-free SOD (apo SOD), and SOD-inactivated with hydrogen peroxide on cholesterol metabolism in human hepatocarcinoma HepG2 cells. Results showed that all forms of SODs used, at the concentration of 150 ng/ml, are able to affect cholesterol metabolism decreasing both HMG-CoA reductase activity and its protein levels; this inhibitory effect is accompanied by reduced cholesterol synthesis measured as [14C]acetate incorporation into [14C]cholesterol and by an increased [125I]LDL binding to HepG2 cells. Furthermore, the inhibitory effect of Cu,Zn SOD on cholesterol synthesis was completely abolished when the cells were incubated with Cu,Zn SOD in the presence of bisindoilmaleimide (BDM), an inhibitor of protein kinase C (PKC); moreover, we demonstrated that Cu,Zn SOD as well as apo SOD was able to increase PKC activity. Overall, data demonstrate that Cu,Zn SOD affects cholesterol metabolism independently from its dismutase activity and its metal content and that the inhibitory action on cholesterol synthesis is mediated by an activation of protein kinase C.     

Leukotriene D4-induced adhesion of Caco-2 cells is mediated by prostaglandin E2 and upregulation of alpha2beta1-integrin

Cell-cell and extracellular matrix adhesions play important roles in the progression of cancer. We investigated the involvement of the inflammatory mediator leukotriene D4 (LTD4) in the regulation of cell-matrix adhesion of colon cancer (Caco-2) cells. We observed that LTD4 acted via its CysLT1 receptor in these cells to induce increased adhesion to collagen I. LTD4 also enhanced the activation and expression of alpha2beta1-integrins on the cell surface, which we found to be responsible for mediating the increased adhesion to collagen I. LTD4 simultaneously augmented expression of the prostaglandin-generating enzyme cyclooxygenase-2 (COX-2) and increased prostaglandin E2 (PGE2) production in Caco-2 cells. The adhesive capacity of the Caco-2 cells was reduced by specific inhibition of COX-2 and was subsequently restored by PGE2, but not by LTD4. A selective PGE2 receptor antagonist abolished the increased adhesion and the augmented alpha2beta1-integrin expression induced by both PGE2 and LTD4. Summarizing, the inflammatory mediator LTD4 regulates the adhesive properties and migration of the Caco-2 cell line by upregulating COX-2 and stimulating PGE2-induced expression of alpha2beta1-integrins. This suggests that inflammatory mediators such as LTD4 can be involved in the dissemination and survival of colon cancer cells.     

Consequence of the presence of two different beta subunit isoforms in a GABA(A) receptor

The major isoforms of GABA(A) receptors are thought to be composed of two alpha, two beta and one gamma subunit(s). GABA(A) receptors containing two beta1 subunits respond differently to the anticonvulsive compound loreclezole and the general anaesthetic etomidate than receptors containing two beta2 subunits. Receptors containing beta2 subunits show a much larger allosteric stimulation by these agents than those containing beta1 subunits. We were interested to know how receptors containing both beta1 and beta2 subunits, in different positions respond to loreclezole and etomidate. To answer this question, subunits were fused at the DNA level to form dimeric and trimeric subunits. Concatenated receptors (alpha1-beta1-alpha1/gamma2-beta1, alpha1-beta2-alpha1/gamma2-beta1, alpha1-beta1-alpha1/gamma2-beta2 and alpha1-beta2-alpha1/gamma2-beta2) were expressed in Xenopus ooctyes and functionally compared in their response to the agonist GABA and to the positive allosteric modulators, loreclezole and etomidate. We have shown that (I) in the presence of both beta1 and beta2 subunits in the same pentamer (mixed receptors) direct gating by etomidate is similar to exclusively beta1 containing receptors; (II) In mixed receptors, stimulation by etomidate assumed characteristics intermediate to exclusively beta1 or beta2 containing receptors, but the values for the concentrations < 10 microM were always much closer to those observed in alpha1-beta1-alpha1/gamma2-beta1 receptors; and (III) mixed receptors show no positional effects.     

Stimulation of peroxisomal palmitoyl-CoA oxidase activity by ciprofibrate in hepatic cell lines: Comparative studies in Fao,MH1C1 and HepG2 cells

Summary— The response of two rat cell lines, Fao and MH₁C₁, and one human cell line, HepG2, to the peroxisome proliferator ciprofibrate, was studied. Using a fluorometric assay for palmitoyl-CoA oxidase, the dose- and time-dependent increase of this enzymatic activity was determined. From the lowest concentration (100 μM) stimulation is evident in the two rat cell lines. In the Fao line, the activity was stimulated reaching a seven-fold increase over the control level at 250 μM after 72 h of treatment. In the MH₁C₁ line, the maximum stimulation, four- to five-fold, was obtained at 250 and 500 μM after 72 h. In the HepG2 cell line, activity increased two-fold at 250 μM after 72 h reaching a three-fold increase at 1000 μM after 48 h. Ciprofibrate was more toxic to Fao cells than to MH₁C₁ and HepG2 cells which is also the order of the acyl-CoA oxidase stimulation by ciprofibrate. These preliminary results suggest that the two rat cell lines are appropriate for investigating the induction of peroxisomal β-oxidation enzymes and the expression of their genes. The HepG2 cell line is a complementary model for the study of interspecies differences in the response to peroxisomal proliferators and of the peroxisomal functions implied in the lipid metabolism of human liver.     

The interaction of CK2alpha and CK2beta, the subunits of protein kinase CK2, requires CK2beta in a preformed conformation and is enthalpically driven

The protein kinase CK2 (former name: "casein kinase 2") predominantly occurs as a heterotetrameric holoenzyme composed of two catalytic chains (CK2alpha) and two noncatalytic subunits (CK2beta). The CK2beta subunits form a stable dimer to which the CK2alpha monomers are attached independently. In contrast to the cyclins in the case of the cyclin-dependent kinases CK2beta is no on-switch of CK2alpha; rather the formation of the CK2 holoenzyme is accompanied with an overall change of the enzyme's profile including a modulation of the substrate specificity, an increase of the thermostability, and an allocation of docking sites for membranes and other proteins. In this study we used C-terminal deletion variants of human CK2alpha and CK2beta that were enzymologically fully competent and in particular able to form a heterotetrameric holoenzyme. With differential scanning calorimetry (DSC) we confirmed the strong thermostabilization effect of CK2alpha on CK2beta with an upshift of the CK2alpha melting temperature of more than 9 degrees . Using isothermal titration calorimetry (ITC) we measured a dissociation constant of 12.6 nM. This high affinity between CK2alpha and CK2beta is mainly caused by enthalpic rather than entropic contributions. Finally, we determined a crystal structure of the CK2beta construct to 2.8 A resolution and revealed by structural comparisons with the CK2 holoenzyme structure that the CK2beta conformation is largely conserved upon association with CK2alpha, whereas the latter undergoes significant structural adaptations of its backbone.     

HIF-2alpha expression in human fetal paraganglia and neuroblastoma: relation to sympathetic differentiation, glucose deficiency, and hypoxia

Solid tumors are frequently necrotic and hypoxic due to poor vascularization. Tumor cells adapt to hypoxia by modulating their phenotype. Key players in this process are the hypoxia-inducible factors (HIF-1alpha to 3alpha). HIFs are also expressed during normal development; for example, HIF-2alpha is specifically expressed and appears to be involved in the development of the murine sympathetic nervous system (SNS). Here, we demonstrate that HIF-2alpha protein is selectively present in human fetal week 8.5 SNS paraganglia. Neuroblastoma is derived from SNS precursors. In a subset of neuroblastomas, a spontaneous neuronal to neuroendocrine differentiation occurs in areas adjacent to necrotic zones. As HIF-2alpha activity has been associated not only with hypoxic but also with hypoglycemic conditions, we have investigated putative effects of hypoxia, glucose depletion, and HIF-2alpha on the neuroblastoma phenotype. HIF-2alpha was detected in hypoxic and in well-oxygenized neuroblastoma cells and tissue, presumably reflecting their embryonic features. With regard to differentiation, hypoxic cells lost their neuronal/neuroendocrine features and gained marker gene expression associated with an immature, neural crest-like phenotype. Low glucose potentiated the effect of hypoxia. These findings suggest that poorly vascularized neuroblastomas become immature and maintain a more aggressive phenotype, which possibly could involve a sustained stabilization and activation of HIF-2alpha.     

Interferonbeta-induced changes in metallothionein expression and subcellular distribution of zinc in HepG2 cells

We evaluated the changes of metallothionein induction and cellular zinc distribution in HepG2 cells by interferonbeta treatment. Immunohistochemical staining of metallothionein was observed in the cytoplasm and nuclei of hepatocytes; which was observed predominantly in the cells treated with interferon and zinc compared to those with zinc alone, interferon alone or the no-treated control. The cellular zinc level was higher in order of the interferon- and zinc-treated cells, the zinc-alone-treated cells, and the interferon-alone-treated cells. Flow cytometry showed that S-phase population increased in interferon-alone-treated cells and interferon- and zinc-treated cells, but not in zinc-alone-treated ones. Cellular elemental distribution was analyzed using in-air micro-particle induced X-ray emission. In zinc-alone-treated sample, X-ray spectra showed good consistency between the enhanced cellular zinc distribution and the phosphorous map. Localizations of bromine followed by interferon treatment were found accompanying a spatial correlation with the phosphorous map. The samples treated with interferon and zinc showed the marked accumulation of zinc and bromine. Discrete bromine accumulation sites were clearly visible with a strong spatial correlation followed by zinc accumulation. These findings suggest that interferonbeta in combination with zinc predominantly induces metallothionein expression in HepG2 cells. In addition, interferonbeta may promote the translocation of metallothionein-bound zinc from cytoplasm to S-phase nuclei.     

Involvement of catalase in the apoptotic mechanism induced by apigenin in HepG2 human hepatoma cells

Apigenin has been reported to inhibit proliferation of cancer cells; however, the mechanism underlying its action is not completely understood. Here, we evaluated the effects of apigenin on the levels of expression and activity of antioxidant enzymes, and the involvement of ROS in the mechanism of cell death induced by apigenin in HepG2 human hepatoma cells. Upon treatment with apigenin, HepG2 cells displayed a reduction in cell viability in a dose- and time-dependent manner, and some morphological changes. In addition, apigenin treatment induced ROS generation and significantly decreased the mRNA levels and activity of catalase and levels of intracellular GSH. On the other hand, apigenin treatment did not alter the expression or activity levels of other antioxidant enzymes. Addition of exogenous catalase significantly reduced the effects of apigenin on HepG2 cell death. We also demonstrated that HepG2 cells are more sensitive to apigenin-mediated cell death than are primary cultures of mouse hepatocytes, suggesting a differential toxic effect of this agent in tumor cells. Our results suggest that apigenin-induced apoptosis in HepG2 cells may be mediated by a H₂O₂-dependent pathway via reduction of the antioxidant defenses.     

Lymphocyte senescence in COPD is associated with loss of glucocorticoid receptor expression by pro-inflammatory/cytotoxic lymphocytes

Background

Glucocorticoid (GC) resistance is a major barrier in COPD treatment. We have shown increased expression of the drug efflux pump, Pgp1 in cytotoxic/pro-inflammatory lymphocytes in COPD. Loss of lymphocyte co-stimulatory molecule CD28 (lymphocyte senescence) was associated with a further increase in their pro-inflammatory/cytotoxic potential and resistance to GC. We hypothesized that lymphocyte senescence and increased Pgp1 are also associated with down-regulation of the GC receptor (GCR).

Methods

Blood was collected from 10 COPD and 10 healthy aged-matched controls. Flow cytometry was applied to assess intracellular pro-inflammatory cytokines, CD28, Pgp1, GCR, steroid binding and relative cytoplasm/nuclear GCR by CD28+ and CD28null T, NKT-like cells. GCR localization was confirmed by fluorescent microscopy.

Results

COPD was associated with increased numbers of CD28nullCD8+ T and NKT-like cells. Loss of CD28 was associated with an increased percentage of T and NKT-like cells producing IFNγ or TNFα and associated with a loss of GCR and Dex-Fluor staining but unchanged Pgp1. There was a significant loss of GCR in CD8 + CD28null compared with CD8 + CD28+ T and NKT-like cells from both COPD and controls (eg, mean ± SEM 8 ± 3% GCR + CD8 + CD28null T-cells vs 49 ± 5% GCR + CD8 + CD28+ T-cells in COPD). There was a significant negative correlation between GCR expression and IFNγ and TNFα production by T and NKT-like cells(eg, COPD: T-cell IFNγ R = −.615; ) and with FEV1 in COPD (R = −.777).

Conclusions

COPD is associated with loss of GCR in senescent CD28null and NKT-like cells suggesting alternative treatment options to GC are required to inhibit these pro-inflammatory/cytotoxic cells.     

Design, synthesis, and evaluation of a novel series of alpha-substituted phenylpropanoic acid derivatives as human peroxisome proliferator-activated receptor (PPAR) alpha/delta dual agonists for the treatment of metabolic syndrome

A series of alpha-alkyl-substituted phenylpropanoic acids was prepared as dual agonists of peroxisome proliferator-activated receptors alpha and delta (PPARalpha/delta). Structure-activity relationship studies indicated that the shape of the linking group and the shape of the substituent at the distal benzene ring play key roles in determining the potency and the selectivity of PPAR subtype transactivation. Structure-activity relationships among the amide series (10) and the reversed amide series (13) are similar, but not identical, especially in the case of the compounds bearing a bulky hydrophobic substituent at the distal benzene ring, indicating that the hydrophobic tail part of the molecules in these two series binds at somewhat different positions in the large binding pocket of PPAR. alpha-Alkyl-substituted phenylpropanoic acids of (S)-configuration were identified as potent human PPARalpha/delta dual agonists. Representative compounds exhibited marked nuclear receptor selectivity for PPARalpha and PPARdelta. Subtype-selective PPAR activation was also examined by analysis of the mRNA expression of PPAR-regulated genes.     

Inhibition of Sudan I genotoxicity in human liver-derived HepG2 cells by the antioxidant hydroxytyrosol

The chemoprotective effect of hydroxytyrosol (HT) against Sudan I-induced genotoxicity was investigated in a human hepatoma cell line, HepG2. The comet assay and micronucleus (MN) assay were used to monitor genotoxicity. Intracellular reactive oxygen species (ROS) formation was measured using a fluorescent probe, 2,7-dichlorofluorescein diacetate (DCFH-DA). The levels of oxidative DNA damage and lipid peroxidation were estimated by immunocytochemistry analysis of 8-hydroxydeoxyguanosine (8-OHdG) and by measuring levels of thiobarbituric acid-reactive substances (TBARS), respectively. Intracellular glutathione (GSH) level was estimated by fluorometric methods. The results showed that HT significantly reduced the genotoxicity caused by Sudan I. Furthermore, HT ameliorated lipid pexidation as demonstrated by a reduction in TBARS formation and attenuated GSH depletion in a concentration-dependent manner. It was also found that HT reduced intracellular ROS formation and 8-OHdG level caused by Sudan I. These results strongly suggest that HT has significant protective ability against Sudan I-induced genotoxicity.     

Role of NK cells and TGF-β in the regulation of T-cell-dependent antibody production in health and autoimmune disease

Natural killer (NK) cells are a third lymphocyte population especially important in innate immunity. NK cells may also have an important role in the regulation of acquired immunity. These lymphocytes spontaneously produce large amounts of both active and latent transforming growth factor-beta (TGF-β). NK-cell-derived TGF-β1 enabled activated CD8⁺ T cells to inhibit antibody production by blocking the induction of this response. Production of lymphocyte-derived TGF-β is decreased in systemic lupus erythematosus. Insufficient levels of this cytokine in SLE and other autoimmune diseases may contribute to defective T regulatory cell function characteristic of this and other autoimmune diseases. NK cells are found in mucosal tissues and the TGF-β spontaneously released by these cells could contribute to the usual tolerogenic response of T cells to antigens presented at these sites. Thus, in addition to its well known immunosuppressive effects, TGF-β could have an equally important role in the generation of regulatory T cells.