Javier Lucio-Cazana

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.

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 (H2O2)-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, RARα and RARγ, but not RARβ, mediated the t-RA-induced MKP-1 expression. The antiapoptotic effect of t-RA on H2O2-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 H2O2-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.

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

BackgroundMercuric 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.MethodsWe 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 α4 transfectant cells. ANOVA tests were used for statistical significance estimation.ResultsWe 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-α and IL-1β 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 (α4β1) integrin induced by mercury on peripheral blood leukocytes (PBLs). In addition, using K562 α4 stable transfectant cells, we found that at-RA inhibited VLA-4 dependent cell adhesion to VCAM-1.ConclusionHere 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.

15-Deoxy-Δ12,14-prostaglandin-J2 reveals a new pVHL-independent, lysosomal-dependent mechanism of HIF-1α degradation

Hypoxia-inducible factor-1α (HIF-1α) 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-Δ12,14-prostaglandin-J2 (15d-PGJ2) accumulate HIF-1α in normoxic conditions. Thus, we aimed to investigate the mechanism involved in this accumulation. We found that 15d-PGJ2 induced an over-accumulation of HIF-1α 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-1α degradation as a target for 15d-PGJ2 based on: (1) HIF-1α colocalized with the specific lysosomal marker Lamp-2a, (2) 15d-PGJ2 inhibited the activity of cathepsin B, a lysosomal protease, and (3) inhibition of lysosomal activity did not result in over-accumulation of HIF-1α in 15d-PGJ2-treated cells. Therefore, expression of HIF-1α is also modulated by lysosomal degradation.

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.

15-Deoxy-delta12,14-prostaglandin-J2 up-regulates cyclooxygenase-2 but inhibits prostaglandin-E2 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.

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.