Aligned copper nanowires as a cut-and-paste exclusive electrochemical transducer for free-enzyme highly selective quantification of intracellular hydrogen peroxide in cisplatin-treated cells.

The role and reliable quantification of intracellular hydrogen peroxide during cancer therapy constitutes an unexplored and fascinating application. In this work, we report the fabrication of vertically aligned copper nanowires (v-CuNWs) using electrosynthesis on templates, and their application as a cut-and-paste exclusive and flexible electrochemical transducer. This easily adaptable electrodic platform is demonstrated for a fast, simple and free-enzyme selective quantification of intracellular hydrogen peroxide in Cisplatin-treated human renal HK-2 cells. The v-CuNWs sensor was compared with an HRP-enzyme-based biosensor showing excellent correlation and indicates the good selectivity and analytical performance of the v-CuNWs. This sensing approach opens novel avenues for monitoring cell death processes and shows the potential of H2O2 as a cellular damage biomarker, with a clear potency for further developments for in vitro diagnosis and its implication in cancer therapy.

Intracellular EP2 prostanoid receptor promotes cancer-related phenotypes in PC3 cells.

Prostaglandin E2 (PGE2) and hypoxia-inducible factor-1α (HIF-1α) affect many mechanisms that have been involved in the pathogenesis of prostate cancer (PC). HIF-1α, which is up-regulated by PGE2 in LNCaP cells and PC3 cells, has been shown to contribute to metastasis and chemo-resistance of castrate-resistant PC (a lethal form of PC) and to promote in PC cells migration, invasion, angiogenesis and chemoresistance. The selective blockade of PGE2-EP2 signaling pathway in PC3 cells results in inhibition of cancer cell proliferation and invasion. PGE2 affects many mechanisms that have been shown to play a role in carcinogenesis such as proliferation, apoptosis, migration, invasion and angiogenesis. Recently, we have found in PC3 cells that most of these PGE2-induced cancer-related features are due to intracellular PGE2 (iPGE2). Here, we aimed to study in PC3 cells the role of iPGE2-intracellular EP2 (iEP2)-HIF-1α signaling in several events linked to PC progression using an experimental approach involving pharmacological inhibition of the prostaglandin uptake transporter and EGFR and pharmacological and genetic modulation of EP2 receptor and HIF-1α. We found that iPGE2 increases HIF-1α expression through iEP2-dependent EGFR transactivation and that inhibition of any of the axis iEP2-EGFR-HIF-1α in cells treated with PGE2 or EP2 agonist results in prevention of the increase in PC3 cell proliferation, adhesion, migration, invasion and angiogenesis in vitro. Of note, PGE2 induced EP2 antagonist-sensitive DNA synthesis in nuclei isolated from PC3 cells, which indicates that they have functional EP2 receptors. These results suggest that PGE2-EP2 dependent intracrine mechanisms involving EGFR and HIF-1α play a role in PC.

Omic techniques in systems biology approaches to traditional Chinese medicine research: present and future.

Omic techniques have become key tools in the development of systems biology. As the holistic approaches underlying the practice of traditional Chinese medicine (TCM) and new tendencies in Western medicine towards personalised medicine require in-depth knowledge of mechanisms of action and active compounds, the use of omic techniques is crucial for understanding and interpretation of TCM development, especially in view of its expansion in Western countries. In this short review, omic applications in TCM research are reviewed which has allowed some speculation regarding future perspectives for these approaches in TCM modernisation and standardisation. Guidelines for good practice for the application of omics in TCM research are also proposed.

15-Deoxy-Delta(12,14)-prostaglandin-J(2) reveals a new pVHL-independent, lysosomal-dependent mechanism of HIF-1alpha degradation.

Hypoxia-inducible factor-1alpha (HIF-1alpha) protein is degraded under normoxia by its association to von Hippel-Lindau protein (pVHL) and further proteasomal digestion. However, human renal cells HK-2 treated with 15-deoxy-Delta(12,14)-prostaglandin-J(2) (15d-PGJ(2)) accumulate HIF-1alpha in normoxic conditions. Thus, we aimed to investigate the mechanism involved in this accumulation. We found that 15d-PGJ(2) induced an over-accumulation of HIF-1alpha in RCC4 cells, which lack pVHL and in HK-2 cells treated with inhibitors of the pVHL-proteasome pathway. These results indicated that pVHL-proteasome-independent mechanisms are involved, and therefore we aimed to ascertain them. We have identified a new lysosomal-dependent mechanism of HIF-1alpha degradation as a target for 15d-PGJ(2) based on: (1) HIF-1alpha colocalized with the specific lysosomal marker Lamp-2a, (2) 15d-PGJ(2) inhibited the activity of cathepsin B, a lysosomal protease, and (3) inhibition of lysosomal activity did not result in over-accumulation of HIF-1alpha in 15d-PGJ(2)-treated cells. Therefore, expression of HIF-1alpha is also modulated by lysosomal degradation.

15-Deoxy-delta12,14-prostaglandin-J(2) up-regulates cyclooxygenase-2 but inhibits prostaglandin-E(2) production through a thiol antioxidant-sensitive mechanism.

15-Deoxy-delta12,14-prostaglandin-J(2) (15d-PGJ(2)) has potent anti-inflammatory effects including the inhibition of interleukin-1beta (IL-1beta)-induced expression of cyclooxygenase-2 (COX-2) and prostaglandin E(2) (PGE(2)) production in several cell types. 15d-PGJ(2) contains an alpha,beta-unsaturated electrophilic ketone and several evidences suggest that thiol reducing agents prevent or revert the cellular effects of 15d-PGJ(2). The present study was devoted to analyze the effect of 15d-PGJ(2) on COX-2 expression in cultured human mesangial cells (HMC). 15d-PGJ(2) induced an increase in the reduced glutathione (GSH) content and up-regulated COX-2 protein expression, but not COX-1, in a manner which was unaffected by selective peroxisome proliferator-activated receptor gamma (PPARgamma) blockade nor mimicked by ciglitazone, a PPARgamma agonist. N-acetylcysteine (NAC), a thiol reducing agent, but not reactive oxygen species scavengers, prevented 15d-PGJ(2)-induced COX-2 up-regulation. Depletion of GSH by buthionine sulfoximine, which diminishes thiol antioxidant activity, cooperated with 15d-PGJ(2) to accumulate COX-2. Therefore, 15d-PGJ(2) up-regulated COX-2 through a thiol antioxidant-sensitive mechanism. Interestingly, NAC did not inhibit the COX-2 expression induced by the electrophilic alpha,beta-unsaturated compound PGA(2). Up-regulation of COX-2 by 15d-PGJ(2) did not result in increased PGE(2) production. Furthermore, preincubation with 15d-PGJ(2) inhibited IL-1beta-induced PGE(2) production although IL-1beta-induced COX-2 expression remained unaffected by the treatment with 15d-PGJ(2). On the contrary, PGA(2) elicited an increase in PGE(2) production and it acted synergistically with IL-1beta to enhance PGE(2) production. These results indicate for the first time that 15d-PGJ(2) inhibits PGE(2) production independently of its effect on COX-2 expression.

Accumulation of hypoxia-inducible factor-1alpha through a novel electrophilic, thiol antioxidant-sensitive mechanism.

15-deoxy-Delta(12,14)-prostaglandin-J(2) (15d-PGJ(2)) is a peroxisome-activated proliferator receptor-gamma (PPARgamma) agonist which contains an alpha,beta-unsaturated electrophilic ketone involved in nucleophilic addition reactions to thiols. Here we studied its effect on hypoxia-inducible factor-1alpha (HIF-1alpha) in human proximal tubular cells HK-2. 15d-PGJ(2) induced stabilization of HIF-1alpha protein, without affecting HIF-1alpha mRNA levels or proteasome activity, leading to its nuclear accumulation and activation of HIF-induced transcription. Accumulation of HIF-1alpha was unaffected by selective PPARgamma blockade nor mimicked by the PPARgamma agonists ciglitazone and 9,10-dihydro-15d-PGJ(2). N-acetylcysteine, reduced glutathione (GSH) or dithiothreitol (i.e. agents that act as thiol reducing agents and/or increase the GSH content), but not reactive oxygen species (ROS) scavengers, prevented 15d-PGJ(2)-induced HIF-1alpha accumulation whereas the inhibitor of GSH synthesis buthionine sulfoximine cooperated with 15d-PGJ(2) to accumulate HIF-1alpha. Finally, HIF-1alpha expression was increased by the electrophilic alpha,beta-unsaturated compounds acrolein and PGA(2), but not by 9,10-dihydro-15d-PGJ(2), which lacks the electrophilic cyclopentenone moiety. Taken together, these results point out to a new mechanism to increase pharmacologically the cell levels of HIF-1alpha through the electrophilic reaction of alpha,beta-unsaturated ketones with thiol groups.

In vitro models of TGF-beta-induced fibrosis suitable for high-throughput screening of antifibrotic agents.

Progressive fibrosis is a cause of progressive organ dysfunction. Lack of quantitative in vitro models of fibrosis accounts, at least partially, for the slow progress in developing effective antifibrotic drugs. Here, we report two complementary in vitro models of fibrosis suitable for high-throughput screening. We found that, in mesangial cells and renal fibroblasts grown in eight-well chamber slides, transforming growth factor-beta1 (TGF-beta1) disrupted the cell monolayer and induced cell migration into nodules in a dose-, time- and Smad3-dependent manner. The nodules contained increased interstitial collagens and showed an increased collagen I:IV ratio. Nodules are likely a biological consequence of TGF-beta1-induced matrix overexpression since they were mimicked by addition of collagen I to the cell culture medium. TGF-beta1-induced nodule formation was inhibited by vacuum ionized gas treatment of the plate surface. This blockage was further enhanced by precoating plates with matrix proteins but was prevented, at least in part, by poly-l-lysine (PLL). We have established two cell-based models of TGF-beta-induced fibrogenesis, using mesangial cells or fibroblasts cultured in matrix protein or PLL-coated 96-well plates, on which TGF-beta1-induced two-dimensional matrix accumulation, three-dimensional nodule formation, and monolayer disruption can be quantitated either spectrophotometrically or by using a colony counter, respectively. As a proof of principle, chemical inhibitors of Alk5 and the antifibrotic compound tranilast were shown to have inhibitory activities in both assays.

Cyclooxygenase-independent inhibition of H2O2-induced cell death by S-ketoprofen in renal cells.

The stress response of the distal tubule to oxidative attack may be relevant to recovery from acute renal failure. In distal tubular Madin-Darby cells (MDCK), H(2)O(2) induced up-regulation of cyclooxygenases (COX-1 and COX-2), prostaglandin-E(2) production and caspase-independent cell death. Cell death was inhibited by S-ketoprofen, but not by the much weaker COX inhibitor R-ketoprofen. Interestingly, we identified 15-deoxy-Delta(12,14)-prostaglandin-J(2) (15d-PGJ(2)), a peroxisome-proliferator activated receptor-gamma agonist, as a lethal prostaglandin whose effect was reproduced by the PPAR-gamma agonist ciglitazone. Nevertheless, H(2)O(2)-induced cell death was unaffected by other non-steroidal anti-inflammatory drugs (NSAIDs) or all-trans-retinoic acid. Moreover, c-Jun-N-terminal kinase inhibitor SP600125 prevented 15-deoxy-Delta(12,14)-PGJ(2)-induced cell death, but not H(2)O(2)-induced cell death. PPAR-gamma antagonist GW9662 showed no affect on the cell death. These results indicated that protection by S-ketoprofen was COX-independent and PPARgamma independent. Moreover, the IC(50) value of the action of S-ketoprofen for the inhibition of H(2)O(2)-induced MDCK cell death ( approximately equal 140microM) was much higher than the IC(50) value for the inhibition of COX-1 and COX-2 activities ( approximately equal 1microM). Further design of S-ketoprofen derivatives devoid of COX inhibitory activity will give opportunity to protect the kidney against oxidative attack while avoiding unwanted effects of NSAID.

Therapeutic effect of all-trans-retinoic acid (at-RA) on an autoimmune nephritis experimental model: role of the VLA-4 integrin.

BACKGROUND: Mercuric chloride (HgCl2) induces an autoimmune nephritis in the Brown Norway (BN) rats characterized by anti-glomerular basement membrane antibodies (anti-GBM Ab) deposition, proteinuria and a severe interstitial nephritis, all evident at day 13 of the disease. We assessed the effects of all-trans retinoic acid (at-RA) in this experimental model. At-RA is a vitamin A metabolite which has shown beneficial effects on several nephropathies, even though no clear targets for at-RA were provided. METHODS: We separated animals in four different experimental groups (HgCl2, HgCl2+at-RA, at-RA and vehicle). From each animal we collected, at days 0 and 13, numerous biological samples: urine, to measure proteinuria by colorimetry; blood to determine VLA-4 expression by flow citometry; renal tissue to study the expression of VCAM-1 by Western blot, the presence of cellular infiltrates by immunohistochemistry, the IgG deposition by immunofluorescence, and the cytokines expression by RT-PCR. Additionally, adhesion assays to VCAM-1 were performed using K562 alpha4 transfectant cells. ANOVA tests were used for statistical significance estimation. RESULTS: We found that at-RA significantly decreased the serum levels of anti-GBM and consequently its deposition along the glomerular membrane. At-RA markedly reduced proteinuria as well as the number of cellular infiltrates in the renal interstitium, the levels of TNF-alpha and IL-1beta cytokines and VCAM-1 expression in renal tissue. Moreover, we reported here for the first time in an in vivo model that at-RA reduced, to basal levels, the expression of VLA-4 (alpha4beta1) integrin induced by mercury on peripheral blood leukocytes (PBLs). In addition, using K562 alpha4 stable transfectant cells, we found that at-RA inhibited VLA-4 dependent cell adhesion to VCAM-1. CONCLUSION: Here we demonstrate a therapeutic effect of at-RA on an autoimmune experimental nephritis model in rats. We report a significant reduction of the VLA-4 integrin expression on PBLs as well as the inhibition of the VLA4/VCAM1-dependent leukocyte adhesion by at-RA treatment. Thereby we point out the VLA-4 integrin as a target for at-RA in vivo.

Retinoids in nephrology: promises and pitfalls.

BACKGROUND: Retinoids, a family of vitamin A metabolites or analogs, play an important role in regulating cell proliferation, differentiation, and apoptosis. METHODS: The biological importance of retinoids in the kidney and the potential of retinoids in the treatment of renal diseases are reviewed. RESULTS: Vitamin A deficiency and mutations of retinoid nuclear receptors cause abnormalities in fetal kidneys, which might predispose to adult diseases such as hypertension. Further, the therapeutic value of retinoids in animal models of kidney diseases, such as lupus nephritis, puromycin aminonucleoside nephrosis, anti-glomerular basement membrane nephritis, mesangioproliferative nephritis, and acute renal allograft rejection has been unveiled recently. Retinoids target mesangial cells, podocytes, tubular epithelial cells, interstitial fibroblasts, as well as lymphocytes and macrophages. The anti-inflammation, anti-coagulation effects, and the proliferation- and immunity-modulating actions of retinoids, have been widely appreciated. Our recent in vitro data revealed a direct antifibrotic effect and a cytoprotective effect of retinoids in various renal cell types. In animal studies, the adverse effects of retinoids are generally minimal; however, the clinical use of retinoids in other diseases points to some major side effects. In addition, in vitro, retinoids can induce lipid accumulation in smooth muscle cells and macrophages and increase expression of some proinflammatory molecules, indicating that their clinical toxicity profile in the setting of renal diseases needs to be better understood. CONCLUSION: Retinoids not only are important in renal development, but also show promise as a new generation of renal medication and deserve to be tested in clinical trials to clarify their full potential.

Cellular defense against H2O2-induced apoptosis via MAP kinase-MKP-1 pathway.

Mitogen-activated protein (MAP) kinase phosphatase-1 (MKP-1) is an oxidative stress-inducible gene. In this study, we investigated signaling pathways involved in oxidative stress-induced MKP-1 expression and its role in apoptosis of rat mesangial cells. Northern and Western blot analyses showed that H(2)O(2) induced expression of MKP-1 mRNA and protein in a dose-dependent manner, without affecting the stability of the transcript. H(2)O(2) induced phosphorylation of extracellular signal-regulated kinase, p38 MAP kinase, and c-Jun N-terminal kinase and consequently activated activator protein 1 (AP-1). Selective inhibitors of individual MAP kinases or a dominant-negative mutant of c-jun significantly suppressed the expression of MKP-1 by H(2)O(2). Inhibition of MKP-1 by a protein tyrosine phosphatase inhibitor (vanadate) enhanced H(2)O(2)-triggered apoptosis. Consistently, transfection with a wild-type MKP-1, but not its catalytically inactive mutant MKP-1CS, attenuated H(2)O(2)-induced apoptosis. These data elucidate, for the first time, that induction of MKP-1 by H(2)O(2) is mediated by the MAP kinase-AP-1 pathway and that the induced MKP-1 is involved in cellular defense against oxidative stress-induced apoptosis of mesangial cells.

Intervention by retinoic acid in oxidative stress-induced apoptosis.

Retinoic acid (RA) has been considered a pro-apoptotic agent, and little is known about its anti-apoptotic potential. In this article, we describe that RA strongly inhibits hydrogen peroxide (H(2)O(2))-induced apoptosis of mesangial cells by intervention in activator protein 1 (AP-1). Our data showed that: (i) H(2)O(2) induces apoptosis of mesangial cells via the AP-1 pathway; (iii) activation of AP-1 by H(2)O(2) is mediated by the c-Jun N-terminal kinase (JNK)-c-Jun/AP-1 pathway and the extracellular signal-regulated kinase-c-Fos/AP-1 pathway; (iii) RA inhibits H(2)O(2)-induced apoptosis via suppression of c-fos/c-jun expression and JNK activation; and (iv) the anti-apoptotic effect of RA is, at least in part, mediated by induction of mitogen-activated protein kinase phosphatase 1.

Transcriptional induction of mitogen-activated protein kinase phosphatase 1 by retinoids. Selective roles of nuclear receptors and contribution to the antiapoptotic effect.

All-trans-retinoic acid (t-RA) inhibits hydrogen peroxide (H(2)O(2))-induced apoptosis by inhibiting the c-Jun N-terminal kinase (JNK)-activator protein 1 (AP-1) pathway. In this report, we examined the involvement of mitogen-activated protein kinase phosphatase 1 (MKP-1) in suppression of JNK and the antiapoptotic effect of t-RA and the roles of nuclear receptors in the regulation of MKP-1 by t-RA. We found that not only t-RA, but also a selective agonist of retinoic acid receptor (RAR), a selective agonist of retinoid X receptor (RXR), and a pan-agonist of RAR and RXR all induced MKP-1 at the transcriptional level. Activation of RAR was required for all of these triggering effects, but activation of RXR was required only for the RXR agonist-induced MKP-1 expression. Among the three RAR subtypes, RARalpha and RARgamma, but not RARbeta, mediated the t-RA-induced MKP-1 expression. The antiapoptotic effect of t-RA on H(2)O(2)-induced apoptosis in several cell types was correlated with the inducibility of MKP-1 by t-RA. Inhibition of MKP-1 by vanadate enhanced JNK phosphorylation and attenuated the antiapoptotic effect of t-RA. Furthermore, overexpression of MKP-1 inhibited H(2)O(2)-induced JNK phosphorylation and apoptosis. To our knowledge, this is the first to demonstrate that 1) MKP-1 is inducible by retinoids at the transcriptional level, 2) RXR and individual RAR subtypes have different roles in this process, and 3) the induced MKP-1 plays a significant role in mediating both JNK inhibition and the antiapoptotic effect of t-RA in oxidative stress.

Suppression of constitutive but not Il-1beta-inducible expression of monocyte chemoattractant protein-1 in mesangial cells by retinoic acids: intervention in the activator protein-1 pathway.

Retinoic acid regulates a wide range of biologic processes, including inflammation. This study investigated the effect of all-trans-retinoic acid (t-RA) on the constitutive and cytokine-inducible expression of monocyte chemoattractant protein 1 (MCP-1) in rat mesangial cells. Serum-deprived mesangial cells exhibited substantial levels of MCP-1 mRNA, and the expression was markedly upregulated by interleukin-1beta (IL-1beta). Pretreatment with t-RA abrogated the constitutive mRNA expression but did not inhibit the IL-1beta-inducible expression. The similar effects were observed by 9-cis-RA. The suppressive effect of t-RA required retinoic acid receptors. t-RA did not affect the stability of MCP-1 mRNA, indicating that its suppressive effect was at the transcriptional level. Experiments that used pharmacologic and genetic inhibitors showed that the IL-1beta-inducible MCP-1 expression was dependent on nuclear factor-kappaB (NF-kappaB) and independent of activator protein 1 (AP-1). In contrast, the constitutive expression of MCP-1 was dependent on both NF-kappaB and AP-1. t-RA substantially inhibited the constitutive activity of AP-1 but did not inhibit NF-kappaB activity in mesangial cells. These data suggested that (1) constitutive and IL-1beta-inducible expression of MCP-1 was differently regulated by AP-1 and NF-kappaB and (2) t-RA inhibited selectively the constitutive expression of MCP-1 via intervention in the AP-1 pathway.