Desmin Reorganization by Stimuli Inducing Oxidative Stress and Electrophiles: Role of Its Single Cysteine Residue.

The type III intermediate filament proteins vimentin and GFAP are modulated by oxidants and electrophiles, mainly through perturbation of their single cysteine residues. Desmin, the type III intermediate filament protein specific to muscle cells, is critical for muscle homeostasis, playing a key role in sarcomere organization and mitochondrial function. Here, we have studied the impact of oxidants and cysteine-reactive agents on desmin behavior. Our results show that several reactive species and drugs induce covalent modifications of desmin in vitro, of which its single cysteine residue, C333, is an important target. Moreover, stimuli eliciting oxidative stress or lipoxidation, including H2O2, 15-deoxy-prostaglandin J2, and CoCl2-elicited chemical hypoxia, provoke desmin disorganization in H9c2 rat cardiomyoblasts transfected with wild-type desmin, which is partially attenuated in cells expressing a C333S mutant. Notably, in cells lacking other cytoplasmic intermediate filaments, network formation by desmin C333S appears less efficient than that of desmin wt, especially when these proteins are expressed as fluorescent fusion constructs. Nevertheless, in these cells, the desmin C333S organization is also protected from disruption by oxidants. Taken together, our results indicate that desmin is a target for oxidative and electrophilic stress, which elicit desmin remodeling conditioned by the presence of its single cysteine residue.

TCFL5 deficiency impairs the pachytene to diplotene transition during spermatogenesis in the mouse.

Spermatogenesis is a complex, multistep process during which spermatogonia give rise to spermatozoa. Transcription Factor Like 5 (TCFL5) is a transcription factor that has been described expressed during spermatogenesis. In order to decipher the role of TCFL5 during in vivo spermatogenesis, we generated two mouse models. Ubiquitous removal of TCFL5 generated by breeding TCFL5fl/fl with SOX2-Cre mice resulted in sterile males being unable to produce spermatozoa due to a dramatic alteration of the testis architecture presenting meiosis arrest and lack of spermatids. SYCP3, SYCP1 and H1T expression analysis showed that TCFL5 deficiency causes alterations during pachytene/diplotene transition resulting in a meiotic arrest in a diplotene-like stage. Even more, TCFL5 deficient pachytene showed alterations in the number of MLH1 foci and the condensation of the sexual body. In addition, tamoxifen-inducible TCFL5 knockout mice showed, besides meiosis phenotype, alterations in the spermatids elongation process resulting in aberrant spermatids. Furthermore, TCFL5 deficiency increased spermatogonia maintenance genes (Dalz, Sox2, and Dmrt1) but also increased meiosis genes (Syce1, Stag3, and Morc2a) suggesting that the synaptonemal complex forms well, but cannot separate and meiosis does not proceed. TCFL5 is able to bind to the promoter of Syce1, Stag3, Dmrt1, and Syce1 suggesting a direct control of their expression. In conclusion, TCFL5 plays an essential role in spermatogenesis progression being indispensable for meiosis resolution and spermatids maturation.

Isoform-specific effects of transcription factor TCFL5 on the pluripotency-related genes SOX2 and KLF4 in colorectal cancer development.

Colorectal cancer (CRC) is a very common life-threatening malignancy. Transcription factor-like 5 (TCFL5) has been suggested to be involved in CRC. Here, we describe the expression of four alternative transcripts of TCFL5 and their relevance in CRC. Complete deletion of all isoforms drastically decreased pro-tumoural properties such as spheroids formation and in vivo tumour growth, although increased migration in CRC cell lines. Overexpression of the two main isoforms, TCFL5_E8 and CHA, had opposite effects: TCFL5_E8 reduced proliferation and spheroids formation, while CHA increased them. TCFL5_E8 reduced in vivo tumour formation, while CHA had no effect. In addition, TCFL5_E8 and CHA have different roles in the regulation of the pluripotency-related genes SOX2 and KLF4. Both isoforms bind directly to their promoters; however, TCFL5_E8 induced SOX2 and reduced KLF4 mRNA levels, whereas CHA did the opposite. Together, our results show that TCFL5 plays an important role in the development of CRC, being however isoform-specific. This work also points to the need to analyse separately TCFL5 isoforms in cancer, due to their different and opposite functions.

Cyclooxygenase 2 Effector Genes as Potential Inflammation-Related Biomarkers for Colorectal Cancer Circulating Tumor Cells Detection by Liquid Biopsy.

Cyclooxygenase 2 (COX2) has been implicated in cancer development and metastasis. We have identified several COX2-regulated inflammation-related genes in human colorectal cancer cells and shown that some of them play important roles in tumor progression. In this work, we have studied the COX2-regulated genes in the mouse colorectal cancer cell line CT26, to find that many are also regulated by COX2 over-expression. On the other hand, we generated a CT26 cell line expressing Gfp and Luciferase, to study tumor growth and metastasis in immunocompetent Balb/c mice. We then collected solid tissue, and blood samples, from healthy and tumor-bearing mice. Using the Parsortix® cell separation system and taking advantage of the fact that the tumor cells expressed Gfp, we were able to identify circulating tumor cells (CTCs) in some of the mice. We compared the mRNA expression levels of Ptgs2 and effector genes in the samples obtained from tumor-bearing or healthy mice, namely, tumor or healthy colon, Ficoll purified buffy coat, and Parsortix-isolated cells to find different patterns between healthy, tumor-bearing mice with or without CTCs. Although for genes like Il15 we did not observe any difference between healthy and tumor-bearing mice in Ficoll or Parsortix samples; others, such as Egr1, Zc3h12a, Klf4, or Nfat5, allowed distinguishing for cancer or CTC presence. Gene expression analysis in Ficoll or Parsortix processed samples, after liquid biopsy, may offer valuable diagnostic and prognostic information and thus should be further studied.

Interaction of Signaling Lymphocytic Activation Molecule Family 1 (SLAMF1) receptor with Trypanosoma cruzi is strain-dependent and affects NADPH oxidase expression and activity.

The receptor Signaling Lymphocyte-Activation Molecule Family 1 (SLAMF1) controls susceptibility to Infection by the lethal Trypanosoma cruzi Y strain. To elucidate whether genetic diversity of the parasite was related with disease susceptibility, we further analyzed the role of SLAMF1 using 6 different Trypanosoma cruzi strains including Y. The interaction of SLAMF1 receptor with T. cruzi was evidenced by fluorescence microscopy, flow cytometry and quantitative PCR. All the strains, except VFRA, showed a decrease in parasite load in infected macrophages in Slamf1-/- compared to BALB/c. In macrophages gene expression NADPH oxidase (NOX2), and reactive oxygen species (ROS) production increased in Slamf1-/- compared to BALB/c in 5 out of 6 strains. However, Slamf1-/-macrophages infected with VFRA strain exhibited a divergent behavior, with higher parasite load, lower NOX2 expression and ROS production compared to BALB/c. Parasitological and immunological studies in vivo with Y strain showed that in the absence of SLAMF1 the immune response protected mice from the otherwise lethal Y infection favoring a proinflammatory response likely involving CD4, CD8, dendritic cells and classically activated macrophages. In the case of VFRA, no major changes were observed in the absence of SLAMF1. Thus, the results suggest that the T. cruzi affects SLAMF1-dependent ROS production, controlling parasite replication in macrophages and affecting survival in mice in a strain-dependent manner. Further studies will focus in the identification of parasite molecules involved in SLAMF1 interaction to explain the immunopathogenesis of the disease.

Coordinate ß-adrenergic inhibition of mitochondrial activity and angiogenesis arrest tumor growth.

Mitochondrial metabolism has emerged as a promising target against the mechanisms of tumor growth. Herein, we have screened an FDA-approved library to identify drugs that inhibit mitochondrial respiration. The ß1-blocker nebivolol specifically hinders oxidative phosphorylation in cancer cells by concertedly inhibiting Complex I and ATP synthase activities. Complex I inhibition is mediated by interfering the phosphorylation of NDUFS7. Inhibition of the ATP synthase is exerted by the overexpression and binding of the ATPase Inhibitory Factor 1 (IF1) to the enzyme. Remarkably, nebivolol also arrests tumor angiogenesis by arresting endothelial cell proliferation. Altogether, targeting mitochondria and angiogenesis triggers a metabolic and oxidative stress crisis that restricts the growth of colon and breast carcinomas. Nebivolol holds great promise to be repurposed for the treatment of cancer patients.

Cyclooxygenase 2-Regulated Genes an Alternative Avenue to the Development of New Therapeutic Drugs for Colorectal Cancer.

Colorectal cancer (CRC) is one of the most common and recurrent types of cancer, with high mortality rates. Several clinical trials and meta-analyses have determined that the use of pharmacological inhibitors of cyclooxygenase 2 (COX-2), the enzyme that catalyses the rate-limiting step in the synthesis of prostaglandins (PG) from arachidonic acid, can reduce the incidence of CRC as well as the risk of recurrence of this disease, when used together with commonly used chemotherapeutic agents. These observations suggest that inhibition of COX-2 may be useful in the treatment of CRC, although the current drugs targeting COX-2 are not widely used since they increase the risk of health complications. To overcome this difficulty, a possibility is to identify genes regulated by COX-2 activity that could give an advantage to the cells to form tumors and/or metastasize. The modulation of those genes as effectors of COX-2 may cancel the beneficial effects of COX-2 in tumor transformation and metastasis. A review of the available databases and literature and our own data have identified some interesting molecules induced by prostaglandins or COX-2 that have been also described to play a role in colon cancer, being thus potential pharmacological targets in colon cancer. Among those mPGES-1, DUSP4, and 10, Programmed cell death 4, Trop2, and many from the TGFß and p53 pathways have been identified as genes upregulated in response to COX-2 overexpression or PGs in colon carcinoma lines and overexpressed in colon tumor tissue. Here, we review the available evidence of the potential roles of those molecules in colon cancer in the context of PG/COX signaling pathways that could be critical mediators of some of the tumor growth and metastasis advantage induced by COX-2. At the end, this may allow defining new therapeutic targets/drugs against CRC that could act specifically against tumor cells and would be effective in the prevention and treatment of CRC, lacking the unwanted side effects of COX-2 pharmacological inhibitors, providing alternative approaches in colon cancer.

G-protein-coupled receptor kinase 2 safeguards epithelial phenotype in head and neck squamous cell carcinomas.

Head and neck squamous cell carcinoma (HNSCC) arises from the mucosal lining of the upper aerodigestive tract and display few treatment options in advanced stages. Despite increased knowledge of HNSCC molecular biology, the identification of new players involved in triggering HNSCC recurrence and metastatic disease is needed. We uncover that G-protein-coupled receptor kinase-2 (GRK2) expression is reduced in undifferentiated, high-grade human HNSCC tumors, whereas its silencing in model human HNSCC cells is sufficient to trigger epithelial-to-mesenchymal transition (EMT) phenotypic features, an EMT-like transcriptional program and enhanced lymph node colonization from orthotopic tongue tumors in mice. Conversely, enhancing GRK2 expression counteracts mesenchymal cells traits by mechanisms involving phosphorylation and decreased functionality of the key EMT inducer Snail1. Our results suggest that GRK2 safeguards the epithelial phenotype, whereas its downregulation contributes to the activation of EMT programs in HNSCC.

Exploiting the passenger ACO1-deficiency arising from 9p21 deletions to kill T-cell lymphoblastic neoplasia cells.

Precursor T-cell lymphoblastic neoplasms are aggressive malignancies in need for more effective and specific therapeutic treatments. A significant fraction of these neoplasms harbor deletions on the locus 9p21, targeting the tumor suppressor CDKN2A but also deleting the aconitase 1 (ACO1) gene, a neighboring housekeeping gene involved in cytoplasm and mitochondrial metabolism. Here we show that reducing the aconitase activity with fluorocitrate decreases the viability of T-cell lymphoblastic neoplasia cells in correlation to the differential aconitase expression. The consequences of the treatment were evidenced in vitro using T-cell lymphoblastic neoplasia cell lines exhibiting 9p21 deletions and variable levels of ACO1 expression or activity. Similar results were observed in melanoma cell lines, suggesting a true potential for fluorocitrate in different cancer types. Notably, ectopic expression of ACO1 alleviated the susceptibility of cell lines to fluorocitrate and, conversely, knockdown experiments increased susceptibility of resistant cell lines. These findings were confirmed in vivo on athymic nude mice by using tumor xenografts derived from two T-cell lines with different levels of ACO1. Taken together, our results indicate that the non-targeted ACO1 deficiency induced by common deletions exerts a collateral cellular lethality that can be used as a novel therapeutic strategy in the treatment of several types of cancer.

DUSP10 Is a Regulator of YAP1 Activity Promoting Cell Proliferation and Colorectal Cancer Progression.

Cell contact inhibition (CCI) is deregulated in cancer. Colorectal cancer (CRC) is the third most commonly diagnosed cancer worldwide. We found that dual-specificity phosphatase 10 (DUSP10) is involved in CRC. DUSP10 overexpression increased the growth of CRC cell lines and mouse xenografts, while the opposite phenotype was observed by DUSP10 silencing. High cell density (HD) induced DUSP10 expression in CRC cell lines, particularly within the nucleus. Yes-associated protein 1 (YAP1) is activated by dephosphorylation, controlling organ growth and CCI, both processes being deregulated in CRC. Expression levels and localization of DUSP10 matched with YAP1 levels in CRC cell lines. DUSP10 and YAP1 co-immunoprecipitated and their interaction was dependent on YAP1 Ser397. The existence of DUSP10 and YAP1 pathway in vivo was confirmed by using a transgenic Drosophila model. Finally, in CRC patients' samples, high levels of nuclear DUSP10 correlated with nuclear YAP1 in epithelial tumor tissue. Strong nuclear DUSP10 staining also correlated with high tumor stage and poor survival. Overall, these findings describe a DUSP10-YAP1 molecular link in CRC cell lines promoting cell growth in HD. We present evidence suggesting a pro-tumorigenic role of nuclear DUSP10 expression in CRC patients.

Prostaglandin F2α-induced Prostate Transmembrane Protein, Androgen Induced 1 mediates ovarian cancer progression increasing epithelial plasticity.

The role of prostaglandin (PG) F2α has been scarcely studied in cancer. We have identified a new function for PGF2α in ovarian cancer, stimulating the production of Prostate Transmembrane Protein, Androgen Induced 1 (PMEPA1). We show that this induction increases cell plasticity and proliferation, enhancing tumor growth through PMEPA1. Thus, PMEPA1 overexpression in ovarian carcinoma cells, significantly increased cell proliferation rates, whereas PMEPA1 silencing decreased proliferation. In addition, PMEPA1 overexpression buffered TGFß signaling, via reduction of SMAD-dependent signaling. PMEPA1 overexpressing cells acquired an epithelial morphology, associated with higher E-cadherin expression levels while ß-catenin nuclear translocation was inhibited. Notwithstanding, high PMEPA1 levels also correlated with epithelial to mesenchymal transition markers, such as vimentin and ZEB1, allowing the cells to take advantage of both epithelial and mesenchymal characteristics, gaining in cell plasticity and adaptability. Interestingly, in mouse xenografts, PMEPA1 overexpressing ovarian cells had a clear survival and proliferative advantage, resulting in higher metastatic capacity, while PMEPA1 silencing had the opposite effect. Furthermore, high PMEPA1 expression in a cohort of advanced ovarian cancer patients was observed, correlating with E-cadherin expression. Most importantly, high PMEPA1 mRNA levels were associated with lower patient survival.

The Dual-Specificity Phosphatase 10 (DUSP10): Its Role in Cancer, Inflammation, and Immunity.

Cancer is one of the most diagnosed diseases in developed countries. Inflammation is a common response to different stress situations including cancer and infection. In those processes, the family of mitogen-activated protein kinases (MAPKs) has an important role regulating cytokine secretion, proliferation, survival, and apoptosis, among others. MAPKs regulate a large number of extracellular signals upon a variety of physiological as well as pathological conditions. MAPKs activation is tightly regulated by phosphorylation/dephosphorylation events. In this regard, the dual-specificity phosphatase 10 (DUSP10) has been described as a MAPK phosphatase that negatively regulates p38 MAPK and c-Jun N-terminal kinase (JNK) in several cellular types and tissues. Several studies have proposed that extracellular signal-regulated kinase (ERK) can be also modulated by DUSP10. This suggests a complex role of DUSP10 on MAPKs regulation and, in consequence, its impact in a wide variety of responses involved in both cancer and inflammation. Here, we review DUSP10 function in cancerous and immune cells and studies in both mouse models and patients that establish a clear role of DUSP10 in different processes such as inflammation, immunity, and cancer.

Mitochondrial ROS Production Protects the Intestine from Inflammation through Functional M2 Macrophage Polarization.

Mitochondria are signaling hubs in cellular physiology that play a role in inflammatory diseases. We found that partial inhibition of the mitochondrial ATP synthase in the intestine of transgenic mice triggers an anti-inflammatory response through NFκB activation mediated by mitochondrial mtROS. This shielding phenotype is revealed when mice are challenged by DSS-induced colitis, which, in control animals, triggers inflammation, recruitment of M1 pro-inflammatory macrophages, and the activation of the pro-oncogenic STAT3 and Akt/mTOR pathways. In contrast, transgenic mice can polarize macrophages to the M2 anti-inflammatory phenotype. Using the mitochondria-targeted antioxidant MitoQ to quench mtROS in vivo, we observe decreased NFκB activation, preventing its cellular protective effects. These findings stress the relevance of mitochondrial signaling to the innate immune system and emphasize the potential role of the ATP synthase as a therapeutic target in inflammatory and other related diseases.

Coffee Silverskin Extract Protects against Accelerated Aging Caused by Oxidative Agents.

Nowadays, coffee beans are almost exclusively used for the preparation of the beverage. The sustainability of coffee production can be achieved introducing new applications for the valorization of coffee by-products. Coffee silverskin is the by-product generated during roasting, and because of its powerful antioxidant capacity, coffee silverskin aqueous extract (CSE) may be used for other applications, such as antiaging cosmetics and dermaceutics. This study aims to contribute to the coffee sector's sustainability through the application of CSE to preserve skin health. Preclinical data regarding the antiaging properties of CSE employing human keratinocytes and Caenorhabditis elegans are collected during the present study. Accelerated aging was induced by tert-butyl hydroperoxide (t-BOOH) in HaCaT cells and by ultraviolet radiation C (UVC) in C. elegans. Results suggest that the tested concentrations of coffee extracts were not cytotoxic, and CSE 1 mg/mL gave resistance to skin cells when oxidative damage was induced by t-BOOH. On the other hand, nematodes treated with CSE (1 mg/mL) showed a significant increased longevity compared to those cultured on a standard diet. In conclusion, our results support the antiaging properties of the CSE and its great potential for improving skin health due to its antioxidant character associated with phenols among other bioactive compounds present in the botanical material.

Prostaglandins induce early growth response 1 transcription factor mediated microsomal prostaglandin E2 synthase up-regulation for colorectal cancer progression.

Cyclooxygenase2 (COX2) has been associated with cell growth, invasiveness, tumor progression and metastasis of colorectal carcinomas. However, the downstream prostaglandin (PG)-PG receptor pathway involved in these effects is poorly characterized.We studied the PG-pathway in gene expression databases and we found that PTGS2 (prostaglandin G/H synthase and cyclooxygenase) and PTGES (prostaglandin E synthase) are co-expressed in human colorectal tumors. Moreover, we detected that COX2 and microsomal Prostaglandin E2 synthase 1 (mPGES1) proteins are both up-regulated in colorectal human tumor biopsies.Using colon carcinoma cell cultures we found that COX2 overexpression significantly increased mPGES1 mRNA and protein. This up-regulation was due to an increase in early growth response 1 (EGR1) levels and its transcriptional activity. EGR1 was induced by COX2-generated PGF2α. A PGF2α receptor antagonist, or EGR1 silencing, inhibited the mPGES1 induction by COX2 overexpression. Moreover, using immunodeficient mice, we also demonstrated that both COX2- and mPGES1-overexpressing carcinoma cells were more efficient forming tumors.Our results describe for the first time the molecular pathway correlating PTGS2 and PTGES in colon cancer progression. We demonstrated that in this pathway mPGES1 is induced by COX2 overexpression, via autocrine PGs release, likely PGF2α, through an EGR1-dependent mechanism. This signaling provides a molecular explanation to PTGS2 and PTGES association and contribute to colon cancer advance, pointing out novel potential therapeutic targets in this oncological context.

Carcinoma-associated fibroblasts derive from mesothelial cells via mesothelial-to-mesenchymal transition in peritoneal metastasis.

Peritoneal dissemination is a frequent metastatic route for cancers of the ovary and gastrointestinal tract. Tumour cells metastasize by attaching to and invading through the mesothelial cell (MC) monolayer that lines the peritoneal cavity. Metastases are influenced by carcinoma-associated fibroblasts (CAFs), a cell population that derives from different sources. Hence, we investigated whether MCs, through mesothelial-mesenchymal transition (MMT), were a source of CAFs during peritoneal carcinomatosis and whether MMT affected the adhesion and invasion of tumour cells. Biopsies from patients with peritoneal dissemination revealed the presence of myofibroblasts expressing mesothelial markers in the proximity of carcinoma implants. Prominent new vessel formation was observed in the peritoneal areas harbouring tumour cells when compared with tumour-free regions. The use of a mouse model of peritoneal dissemination confirmed the myofibroblast conversion of MCs and the increase in angiogenesis at places of tumour implants. Treatment of omentum MCs with conditioned media from carcinoma cell cultures resulted in phenotype changes reminiscent of MMT. Adhesion experiments demonstrated that MMT enhanced the binding of cancer cells to MCs in a ß1-integrin-dependent manner. Scanning electron microscopy imaging showed that the enhanced adhesion was mostly due to increased cell-cell interaction and not to a mere matrix exposure. Invasion assays suggested a reciprocal stimulation of the invasive capacity of tumour cells and MCs. Our results demonstrate that CAFs can derive from mesothelial cells during peritoneal metastasis. We suggest that MMT renders the peritoneum more receptive for tumour cell attachment/invasion and contributes to secondary tumour growth by promoting its vascularization.

Requirements for proximal tubule epithelial cell detachment in response to ischemia: role of oxidative stress.

Sublethal renal ischemia induces tubular epithelium damage and kidney dysfunction. Using NRK-52E rat proximal tubular epithelial cells, we have established an in vitro model, which includes oxygen and nutrients deprivation, to study the proximal epithelial cell response to ischemia. By means of this system, we demonstrate that confluent NRK-52E cells lose monolayer integrity and detach from collagen IV due to: (i) actin cytoskeleton reorganization; (ii) Rac1 and RhoA activity alterations; (iii) Adherens junctions (AJ) and Tight junctions (TJ) disruption, involving redistribution but not degradation of E-cadherin, beta-catenin and ZO-1; (iv) focal adhesion complexes (FAC) disassembly, entangled by mislocalization of paxillin and FAK dephosphorylation. Reactive oxygen species (ROS) are generated during the deprivation phase and rapidly balanced at recovery involving MnSOD induction, among others. The use of antioxidants (NAC) prevented FAC disassembly by blocking paxillin redistribution and FAK dephosphorylation, without abrogating AJ or TJ disruption. In spite of this, NAC did not show any protective effect on cell detachment. H(2)O(2), as a pro-oxidant treatment, supported the contribution of ROS in tubular epithelial cell-matrix but not cell-cell adhesion alterations. In conclusion, ROS-mediated FAC disassembly was not sufficient for the proximal epithelial cell shedding in response to sublethal ischemia, which also requires intercellular adhesion disruption.

Potentiation of tumor formation by topical administration of 15-deoxy-delta12,14-prostaglandin J2 in a model of skin carcinogenesis.

The effect of prostaglandins on the development of papillomas has been investigated in mice receiving prostaglandins E2 (PGE2) or the cyclopentenone 15-deoxy-delta(12,14)-PGJ2 (15dPGJ2) topically, using the 7,12-dimethylbenz[a]anthracene (DMBA)-induced tetradecanoylphorbol acetate (TPA)-promoted model of skin carcinogenesis. The presence of 15dPGJ2 during DMBA and TPA treatment inhibited apoptosis and increased the rate, number, size and vascularization of the papillomas, some of them progressing into carcinomas. Moreover, skin sections from mice treated for one week with DMBA and 15dPGJ2 showed a much reduced rate of apoptotic cells, and an enhanced expression of vascular epithelial growth factor when compared with animals receiving DMBA, with or without PGE2. The analysis of molecular events in the MCA3D keratinocyte cell line showed that 15dPGJ2 activated Ras and improved cell viability by inhibiting DMBA-dependent apoptosis. In addition to this, cell adhesion was impaired in MCA3D keratinocytes co-treated with 15dPGJ2 and DMBA, at the same time when the expression of cyclooxygenase-2 (COX-2) was observed under these conditions. These effects mediated by 15dPGJ2 might contribute to understand the role of COX-2 metabolites in carcinogenesis, leading to an increase of cell viability after mutagenic injury and therefore in the progression of tumors.

Identification of novel protein targets for modification by 15-deoxy-Delta12,14-prostaglandin J2 in mesangial cells reveals multiple interactions with the cytoskeleton.

The cyclopentenone prostaglandin 15-deoxy-Delta12,14-PGJ2 (15d-PGJ2) has been shown to display protective effects against renal injury or inflammation. In cultured mesangial cells (MC), 15d-PGJ2 inhibits the expression of proinflammatory genes and modulates cell proliferation. Therefore, cyclopentenone prostaglandins (cyPG) have been envisaged as a promise in the treatment of renal disease. The effects of 15d-PGJ2 may be dependent on or independent from its role as a peroxisome proliferator-activated receptor agonist. It was shown recently that an important determinant for the peroxisome proliferator-activated receptor-independent effects of 15d-PGJ2 is the capacity to modify proteins covalently and alter their function. However, a limited number of protein targets have been identified to date. Herein is shown that a biotinylated derivative of 15d-PGJ2 recapitulates the effects of 15d-PGJ2 on the stress response and inhibition of inducible nitric oxide synthase levels and forms stable adducts with proteins in intact MC. Biotinylated 15d-PGJ2 was then used to identify proteins that potentially are involved in cyPG biologic effects. Extracts from biotinylated 15d-PGJ2-treated MC were separated by two-dimensional electrophoresis, and the spots of interest were analyzed by mass spectrometry. Identified targets include proteins that are regulated by oxidative stress, such as heat-shock protein 90 and nucleoside diphosphate kinase, as well as proteins that are involved in cytoskeletal organization, such as actin, tubulin, vimentin, and tropomyosin. Biotinylated 15d-PGJ2 binding to several targets was confirmed by avidin pull-down. Consistent with these findings, 15d-PGJ2 induced early reorganization of vimentin and tubulin in MC. The cyclopentenone moiety and the presence of cysteine were important for vimentin rearrangement. These studies may contribute to the understanding of the mechanism of action and therapeutic potential of cyPG.