Expression of Luteinizing Hormone-Releasing Hormone (LHRH) and Type-I LHRH Receptor in Transitional Cell Carcinoma Type of Human Bladder Cancer.

Bladder cancer (BC) is the tenth most frequently detected cancer in both sexes. Type-I luteinizing hormone-releasing hormone (LHRH) receptor (LHRH-R-I) is expressed not only in the pituitary, but also in several types of cancer disease. There are few data about LHRH-R-I expression in human BC. This study aimed to investigate the expression of LHRH and LHRH-R-I in the transitional cell carcinoma (TCC) type of human BC. RNA was extracted from 24 human bladder tumor specimens and three BC cell lines. RT-PCR was performed to detect mRNA for LHRH and LHRH-R-I. The protein of LHRH-R-I was further studied by immunohistochemistry (IHC), ligand competition assay, and Western Blot. PCR products of LHRH were found in 19 of 24 (79%) specimens and mRNA of LHRH-R-I was detected in 20 of 24 specimens (83%). Positive immunostaining for LHRH-R-I with different expression intensity was found in all samples examined, showing negative correlation with TCC grade. Radioligand binding studies also showed the presence of specific LHRH-R-I and high affinity binding of LHRH analogs. The high incidence of LHRH-R in BC suggests that it could serve as a molecular target for therapy of human BC with cytotoxic LHRH analogs or modern powerful antagonistic analogs of LHRH.

Somatostatin Receptors as Molecular Targets in Human Uveal Melanoma.

Uveal melanoma (UM) is the most common primary intraocular malignancy in adults, with an incidence of 4⁻5 cases per million. The prognosis of UM is very poor. In the present study, our aim was to investigate the expression of mRNA and protein for somatostatin receptor types-1, -2, -3, -4, -5 (SSTR-1⁻5) in human UM tissue samples and in OCM-1 and OCM-3 human UM cell lines by qRT-PCR, western blot and ligand competition assay. The mRNA for SSTR-2 showed markedly higher expression in UM tissues than SSTR-5. The presence of SSTRs was demonstrated in 70% of UM specimens using ligand competition assay and both human UM models displayed specific high affinity SSTRs. Among the five SSTRs, the mRNA investigated for SSTR-2 and SSTR-5 receptors was strongly expressed in both human UM cell lines, SSTR-5 showing the highest expression. The presence of the SSTR-2 and SSTR-5 receptor proteins was confirmed in both cell lines by western blot. In summary, the expression of somatostatin receptors in human UM specimens and in OCM-1 and OCM-3 human UM cell lines suggests that they could serve as a potential molecular target for therapy of UM using modern powerful cytotoxic SST analogs targeting SSTR-2 and SSTR-5 receptors.

Cystathionine-beta-synthase inhibition for colon cancer: Enhancement of the efficacy of aminooxyacetic acid via the prodrug approach.

Colon cancer cells contain high levels of cystathionine-beta-synthase (CBS). Its product, hydrogen sulfide (H2S) promotes the growth and proliferation of colorectal tumor cells. In order to improve the antitumor efficacy of the prototypical CBS inhibitor aminooxyacetic acid (AOAA), we have designed and synthesized YD0171, a methyl ester derivative of AOAA. The antiproliferative effect of YD0171 exceeded the antiproliferative potency of AOAA in HCT116 human colon cancer cells. The esterase inhibitor paraoxon prevented the cellular inhibition of CBS activity by YD0171. YD0171 suppressed mitochondrial respiration and glycolytic function and induced G0/G1 arrest, but did not induce tumor cell apoptosis or necrosis. Metabolomic analysis in HCT116 cells showed that YD0171 affects multiple pathways of cell metabolism. The efficacy of YD0171 as an inhibitor of tumor growth was also tested in nude mice bearing subcutaneous HCT116 cancer cell xenografts. Animals were treated via subcutaneous injection of vehicle, AOAA (1, 3 or 9 mg/kg/day) or YD0171 (0.1, 0.5 or 1 mg/kg/day) for 3 weeks. Tumor growth was significantly reduced by 9 mg/kg/day AOAA, but not at the lower doses. YD0171 was more potent: tumor volume was significantly inhibited at 0.5 and 1 mg/kg/day. Thus, the in vivo efficacy of YD0171 is 9-times higher than that of AOAA. YD0171 (1 mg/kg/day) attenuated tumor growth and metastasis formation in the intracecal HCT116 tumor model. YD0171 (3 mg/kg/day) also reduced tumor growth in patient-derived tumor xenograft (PDTX) bearing athymic mice. YD0171 (3 mg/kg/day) induced the regression of established HCT116 tumors in vivo. A 5-day safety study in mice demonstrated that YD0171 at 20 mg/kg/day (given in two divided doses) does not increase plasma markers of organ injury, nor does it induce histological alterations in the liver or kidney. YD0171 caused a slight elevation in plasma homocysteine levels. In conclusion, the prodrug approach improves the pharmacological profile of AOAA; YD0171 represents a prototype for CBS inhibitory anticancer prodrugs. By targeting colorectal cancer bioenergetics, an emerging important hallmark of cancer, the approach exemplified herein may offer direct translational opportunities.

Screening of a composite library of clinically used drugs and well-characterized pharmacological compounds for cystathionine ß-synthase inhibition identifies benserazide as a drug potentially suitable for repurposing for the experimental therapy of colon cancer.

Cystathionine-ß-synthase (CBS) has been recently identified as a drug target for several forms of cancer. Currently no potent and selective CBS inhibitors are available. Using a composite collection of 8871 clinically used drugs and well-annotated pharmacological compounds (including the LOPAC library, the FDA Approved Drug Library, the NIH Clinical Collection, the New Prestwick Chemical Library, the US Drug Collection, the International Drug Collection, the 'Killer Plates' collection and a small custom collection of PLP-dependent enzyme inhibitors), we conducted an in vitro screen in order to identify inhibitors for CBS using a primary 7-azido-4-methylcoumarin (AzMc) screen to detect CBS-derived hydrogen sulfide (H2S) production. Initial hits were subjected to counterscreens using the methylene blue assay (a secondary assay to measure H2S production) and were assessed for their ability to quench the H2S signal produced by the H2S donor compound GYY4137. Four compounds, hexachlorophene, tannic acid, aurintricarboxylic acid and benserazide showed concentration-dependent CBS inhibitory actions without scavenging H2S released from GYY4137, identifying them as direct CBS inhibitors. Hexachlorophene (IC50: ∼60µM), tannic acid (IC50: ∼40µM) and benserazide (IC50: ∼30µM) were less potent CBS inhibitors than the two reference compounds AOAA (IC50: ∼3µM) and NSC67078 (IC50: ∼1µM), while aurintricarboxylic acid (IC50: ∼3µM) was equipotent with AOAA. The second reference compound NSC67078 not only inhibited the CBS-induced AzMC fluorescence signal (IC50: ∼1µM), but also inhibited with the GYY4137-induced AzMC fluorescence signal with (IC50 of ∼6µM) indicative of scavenging/non-specific effects. Hexachlorophene (IC50: ∼6µM), tannic acid (IC50: ∼20µM), benserazide (IC50: ∼20µM), and NSC67078 (IC50: ∼0.3µM) inhibited HCT116 colon cancer cells proliferation with greater potency than AOAA (IC50: ∼300µM). In contrast, although a CBS inhibitor in the cell-free assay, aurintricarboxylic acid failed to inhibit HCT116 proliferation at lower concentrations, and stimulated cell proliferation at 300µM. Copper-containing compounds present in the libraries, were also found to be potent inhibitors of recombinant CBS; however this activity was due to the CBS inhibitory effect of copper ions themselves. However, copper ions, up to 300µM, did not inhibit HCT116 cell proliferation. Benserazide was only a weak inhibitor of the activity of the other H2S-generating enzymes CSE and 3-MST activity (16% and 35% inhibition at 100µM, respectively) in vitro. Benserazide suppressed HCT116 mitochondrial function and inhibited proliferation of the high CBS-expressing colon cancer cell line HT29, but not the low CBS-expressing line, LoVo. The major benserazide metabolite 2,3,4-trihydroxybenzylhydrazine also inhibited CBS activity and suppressed HCT116 cell proliferation in vitro. In an in vivo study of nude mice bearing human colon cancer cell xenografts, benserazide (50mg/kg/days.q.) prevented tumor growth. In silico docking simulations showed that benserazide binds in the active site of the enzyme and reacts with the PLP cofactor by forming reversible but kinetically stable Schiff base-like adducts with the formyl moiety of pyridoxal. We conclude that benserazide inhibits CBS activity and suppresses colon cancer cell proliferation and bioenergetics in vitro, and tumor growth in vivo. Further pharmacokinetic, pharmacodynamic and preclinical animal studies are necessary to evaluate the potential of repurposing benserazide for the treatment of colorectal cancers.

Opposing roles of mitochondrial and nuclear PARP1 in the regulation of mitochondrial and nuclear DNA integrity: implications for the regulation of mitochondrial function.

The positive role of PARP1 in regulation of various nuclear DNA transactions is well established. Although a mitochondrial localization of PARP1 has been suggested, its role in the maintenance of the mitochondrial DNA is currently unknown. Here we investigated the role of PARP1 in the repair of the mitochondrial DNA in the baseline and oxidative stress conditions. We used wild-type A549 cells or cells depleted of PARP1. Our data show that intra-mitochondrial PARP1 interacts with a key mitochondrial-specific DNA base excision repair (BER) enzymes, namely EXOG and DNA polymerase gamma (Polγ), which under oxidative stress become poly(ADP-ribose)lated (PARylated). Interaction between mitochondrial BER enzymes was significantly affected in the presence of PARP1. Moreover, the repair of the oxidative-induced damage to the mitochondrial DNA in PARP1-depleted cells was found to be more robust compared to control counterpart. In addition, mitochondrial biogenesis was enhanced in PARP1-depleted cells, including mitochondrial DNA copy number and mitochondrial membrane potential. This observation was further confirmed by analysis of lung tissue isolated from WT and PARP1 KO mice. In summary, we conclude that mitochondrial PARP1, in opposite to nuclear PARP1, exerts a negative effect on several mitochondrial-specific transactions including the repair of the mitochondrial DNA.

Upregulation and Mitochondrial Sequestration of Hemoglobin Occur in Circulating Leukocytes during Critical Illness, Conferring a Cytoprotective Phenotype.

The classical role of hemoglobin in the erythrocytes is to carry oxygen from the lungs to the tissues via the circulation. However, hemoglobin also acts as a redox regulator and as a scavenger of the gaseous mediators nitric oxide (NO) and hydrogen sulfide (H2S). Here we show that upregulation of hemoglobin (α, ß and δ variants of globin proteins) occurs in human peripheral blood mononuclear cells (PBMCs) in critical illness (patients with severe third-degree burn injury and patients with sepsis). The increase in intracellular hemoglobin concentration is a result of a combination of enhanced protein expression and uptake from the extra-cellular space via a CD163-dependent mechanism. Intracellular hemoglobin preferentially localizes to the mitochondria, where it interacts with complex I and, on the one hand, increases mitochondrial respiratory rate and mitochondrial membrane potential, and on the other hand, protects from H2O2-induced cytotoxicity and mitochondrial DNA damage. Both burn injury and sepsis were associated with increased plasma levels of H2S. Incubation of mononuclear cells with H2S induced hemoglobin mRNA upregulation in PBMCs in vitro. Intracellular hemoglobin upregulation conferred a protective effect against cell dysfunction elicited by H2S. Hemoglobin uptake also was associated with a protection from, and induced the upregulation of, HIF-1α and Nrf2 mRNA. In conclusion, PBMCs in critical illness upregulate their intracellular hemoglobin levels by a combination of active synthesis and uptake from the extracellular medium. We propose that this process serves as a defense mechanism protecting the cell against cytotoxic concentrations of H2S and other gaseous transmitters, oxidants and free radicals produced in critically ill patients.

Regulation of Vascular Tone, Angiogenesis and Cellular Bioenergetics by the 3-Mercaptopyruvate Sulfurtransferase/H2S Pathway: Functional Impairment by Hyperglycemia and Restoration by DL-α-Lipoic Acid.

Hydrogen sulfide (H2S), as a reducing agent and an antioxidant molecule, exerts protective effects against hyperglycemic stress in the vascular endothelium. The mitochondrial enzyme 3-mercaptopyruvate sulfurtransferase (3-MST) is an important biological source of H2S. We have recently demonstrated that 3-MST activity is inhibited by oxidative stress in vitro and speculated that this may have an adverse effect on cellular homeostasis. In the current study, given the importance of H2S as a vasorelaxant, angiogenesis stimulator and cellular bioenergetic mediator, we first determined whether the 3-MST/H2S system plays a physiological regulatory role in endothelial cells. Next, we tested whether a dysfunction of this pathway develops during the development of hyperglycemia and µmol/L to diabetes-associated vascular complications. Intraperitoneal (IP) 3-MP (1 mg/kg) raised plasma H2S levels in rats. 3-MP (10 1 mmol/L) promoted angiogenesis in vitro in bEnd3 microvascular endothelial cells and in vivo in a Matrigel assay in mice (0.3-1 mg/kg). In vitro studies with bEnd3 cell homogenates demonstrated that the 3-MP-induced increases in H2S production depended on enzymatic activity, although at higher concentrations (1-3 mmol/L) there was also evidence for an additional nonenzymatic H2S production by 3-MP. In vivo, 3-MP facilitated wound healing in rats, induced the relaxation of dermal microvessels and increased mitochondrial bioenergetic function. In vitro hyperglycemia or in vivo streptozotocin diabetes impaired angiogenesis, attenuated mitochondrial function and delayed wound healing; all of these responses were associated with an impairment of the proangiogenic and bioenergetic effects of 3-MP. The antioxidants DL-α-lipoic acid (LA) in vivo, or dihydrolipoic acid (DHLA) in vitro restored the ability of 3-MP to stimulate angiogenesis, cellular bioenergetics and wound healing in hyperglycemia and diabetes. We conclude that diabetes leads to an impairment of the 3-MST/H2S pathway, and speculate that this may contribute to the pathogenesis of hyperglycemic endothelial cell dysfunction. We also suggest that therapy with H2S donors, or treatment with the combination of 3-MP and lipoic acid may be beneficial in improving angiogenesis and bioenergetics in hyperglycemia.

Hydrogen sulfide and nitric oxide are mutually dependent in the regulation of angiogenesis and endothelium-dependent vasorelaxation.

Hydrogen sulfide (H(2)S) is a unique gasotransmitter, with regulatory roles in the cardiovascular, nervous, and immune systems. Some of the vascular actions of H(2)S (stimulation of angiogenesis, relaxation of vascular smooth muscle) resemble those of nitric oxide (NO). Although it was generally assumed that H(2)S and NO exert their effects via separate pathways, the results of the current study show that H(2)S and NO are mutually required to elicit angiogenesis and vasodilatation. Exposure of endothelial cells to H(2)S increases intracellular cyclic guanosine 5'-monophosphate (cGMP) in a NO-dependent manner, and activated protein kinase G (PKG) and its downstream effector, the vasodilator-stimulated phosphoprotein (VASP). Inhibition of endothelial isoform of NO synthase (eNOS) or PKG-I abolishes the H(2)S-stimulated angiogenic response, and attenuated H(2)S-stimulated vasorelaxation, demonstrating the requirement of NO in vascular H(2)S signaling. Conversely, silencing of the H(2)S-producing enzyme cystathionine-γ-lyase abolishes NO-stimulated cGMP accumulation and angiogenesis and attenuates the acetylcholine-induced vasorelaxation, indicating a partial requirement of H(2)S in the vascular activity of NO. The actions of H(2)S and NO converge at cGMP; though H(2)S does not directly activate soluble guanylyl cyclase, it maintains a tonic inhibitory effect on PDE5, thereby delaying the degradation of cGMP. H(2)S also activates PI3K/Akt, and increases eNOS phosphorylation at its activating site S1177. The cooperative action of the two gasotransmitters on increasing and maintaining intracellular cGMP is essential for PKG activation and angiogenesis and vasorelaxation. H(2)S-induced wound healing and microvessel growth in matrigel plugs is suppressed by pharmacological inhibition or genetic ablation of eNOS. Thus, NO and H(2)S are mutually required for the physiological control of vascular function.

Regulation of mitochondrial poly(ADP-Ribose) polymerase activation by the ß-adrenoceptor/cAMP/protein kinase A axis during oxidative stress.

We investigated the regulation of mitochondrial poly(ADP-ribose) polymerase 1 (PARP1) by the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) system during oxidative stress in U937 monocytes. Oxidative stress induced an early (10 minutes) mitochondrial DNA damage, and concomitant activation of PARP1 in the mitochondria. These early events were followed by a progressive mitochondrial oxidant production and nuclear PARP1 activation (by 6 hours). These processes led to a functional impairment of mitochondria, culminating in cell death of mixed (necrotic/apoptotic) type. ß-Adrenoceptor blockade with propranolol or inhibition of its downstream cAMP/PKA signaling attenuated, while ß-adrenoceptor agonists and cAMP/PKA activators enhanced, the oxidant-mediated PARP1 activation. In the presence of cAMP, recombinant PKA directly phosphorylated recombinant PARP1 on serines 465 (in the automodification domain) and 782 and 785 (both in the catalytic domain). Inhibition of the ß-adrenergic receptor/cAMP/PKA axis protected against the oxidant-mediated cell injury. Propranolol also suppressed PARP1 activation in peripheral blood leukocytes during bacterial lipopolysaccharide (LPS)-induced systemic inflammation in mice. We conclude that the activation of mitochondrial PARP1 is an early, active participant in oxidant-induced cell death, which is under the control of ß-adrenoceptor/cAMP/PKA axis through the regulation of PARP1 activity by PARP1 phosphorylation.

Endothelial dysfunction is a potential contributor to multiple organ failure and mortality in aged mice subjected to septic shock: preclinical studies in a murine model of cecal ligation and puncture.

INTRODUCTION: The goal of the current study was to investigate the effect of aging on the development of endothelial dysfunction in a murine model of sepsis, and to compare it with the effect of genetic deficiency of the endothelial isoform of nitric oxide synthase (eNOS). METHODS: Cecal ligation and puncture (CLP) was used to induce sepsis in mice. Survival rates were monitored and plasma indices of organ function were measured. Ex vivo studies included the measurement of vascular function in thoracic aortic rings, assessment of oxidative stress/cellular injury in various organs and the measurement of mitochondrial function in isolated liver mitochondria. RESULTS: eNOS deficiency and aging both exacerbated the mortality of sepsis. Both eNOS-deficient and aged mice exhibited a higher degree of sepsis-associated multiple organ dysfunction syndrome (MODS), infiltration of tissues with mononuclear cells and oxidative stress. A high degree of sepsis-induced vascular oxidative damage and endothelial dysfunction (evidenced by functional assays and multiple plasma markers of endothelial dysfunction) was detected in aortae isolated from both eNOS(-/-) and aged mice. There was a significant worsening of sepsis-induced mitochondrial dysfunction, both in eNOS-deficient mice and in aged mice. Comparison of the surviving and non-surviving groups of animals indicated that the severity of endothelial dysfunction may be a predictor of mortality of mice subjected to CLP-induced sepsis. CONCLUSIONS: Based on the studies in eNOS mice, we conclude that the lack of endothelial nitric oxide production, on its own, may be sufficient to markedly exacerbate the severity of septic shock. Aging markedly worsens the degree of endothelial dysfunction in sepsis, yielding a significant worsening of the overall outcome. Thus, endothelial dysfunction may constitute an early predictor and independent contributor to sepsis-associated MODS and mortality in aged mice.

Hydrogen sulfide replacement therapy protects the vascular endothelium in hyperglycemia by preserving mitochondrial function.

The goal of the present studies was to investigate the role of changes in hydrogen sulfide (H(2)S) homeostasis in the pathogenesis of hyperglycemic endothelial dysfunction. Exposure of bEnd3 microvascular endothelial cells to elevated extracellular glucose (in vitro "hyperglycemia") induced the mitochondrial formation of reactive oxygen species (ROS), which resulted in an increased consumption of endogenous and exogenous H(2)S. Replacement of H(2)S or overexpression of the H(2)S-producing enzyme cystathionine-γ-lyase (CSE) attenuated the hyperglycemia-induced enhancement of ROS formation, attenuated nuclear DNA injury, reduced the activation of the nuclear enzyme poly(ADP-ribose) polymerase, and improved cellular viability. In vitro hyperglycemia resulted in a switch from oxidative phosphorylation to glycolysis, an effect that was partially corrected by H(2)S supplementation. Exposure of isolated vascular rings to high glucose in vitro induced an impairment of endothelium-dependent relaxations, which was prevented by CSE overexpression or H(2)S supplementation. siRNA silencing of CSE exacerbated ROS production in hyperglycemic endothelial cells. Vascular rings from CSE(-/-) mice exhibited an accelerated impairment of endothelium-dependent relaxations in response to in vitro hyperglycemia, compared with wild-type controls. Streptozotocin-induced diabetes in rats resulted in a decrease in the circulating level of H(2)S; replacement of H(2)S protected from the development of endothelial dysfunction ex vivo. In conclusion, endogenously produced H(2)S protects against the development of hyperglycemia-induced endothelial dysfunction. We hypothesize that, in hyperglycemic endothelial cells, mitochondrial ROS production and increased H(2)S catabolism form a positive feed-forward cycle. H(2)S replacement protects against these alterations, resulting in reduced ROS formation, improved endothelial metabolic state, and maintenance of normal endothelial function.

Molecular characterization of myosin phosphatase in endothelium.

The phosphorylation status of myosin light chain (MLC) is regulated by both MLC kinases and type 1 Ser/Thr phosphatase (PPase 1), MLC phosphatase (MLCP) activities. The activity of the catalytic subunit of MLCP (CS1ß) towards myosin depends on its associated regulatory subunit, namely myosin PPase targeting subunit 1 (MYPT1). Our previously published data strongly suggested the involvement of MLCP in endothelial cell (EC) barrier regulation. In this study, our new data demonstrate that inhibition of MLCP by either CS1ß or MYPT1 siRNA-based depletion results in significant attenuation of purine nucleotide (ATP and adenosine)-induced EC barrier enhancement. Consistent with the data, thrombin-induced EC F-actin stress fiber formation and permeability increase were attenuated by the ectopic expression of constitutively active (C/A) MYPT1. The data demonstrated for the first time direct involvement of MLCP in EC barrier enhancement/protection. Cloning of MYPT1 in human pulmonary artery EC (HPAEC) revealed the presence of two MYPT1 isoforms, long and variant 2 (V2) lacking 56 amino acids from 553 to 609 of human MYPT1 long, which were previously identified in HeLa and HEK 293 cells. Our data demonstrated that in Cos-7 cells ectopically expressed EC MYPT1 isoforms co-immunoprecipitated with intact CS1ß suggesting the importance of PPase 1 activity for the formation of functional complex of MYPT1/CS1ß. Interestingly, MYPT1 V2 shows decreased binding affinity compared to MYPT1 long for radixin (novel MLCP substrate and a member of ERM family proteins). These results suggest functional difference between EC MYPT1 isoforms in the regulation of MLCP activity and cytoskeleton.

Ultrasound Used for Diagnostic Imaging Facilitates Dendritic Branching of Developing Neurons in the Mouse Cortex.

Neuronal differentiation and synaptogenesis are regulated by precise orchestration of intrinsic and extrinsic chemical and mechanical factors throughout all developmental steps critical for the assembly of neurons into functional circuits. While ultrasound is known to alter neuronal migration and activity acutely, its chronic effect on neuronal behavior or morphology is not well characterized. Furthermore, higher-frequency (3-5 MHz) ultrasound (HFU) is extensively used in gynecological practice for imaging, and while it has not been shown harmful for the developing brain, it might be associated with mild alterations that may have functional consequences. To shed light on the neurobiological effects of HFU on the developing brain, we examined cortical pyramidal cell morphology in a transgenic mouse model, following a single and short dose of high-frequency ultrasound. Layer V neurons in the retrosplenial cortex of mouse embryos were labeled with green and red fluorescent proteins by in utero electroporation at the time of their appearance (E14.5). At the time of their presumptive arrival to layer V (E18.5), HFU stimulation was performed with parameters matched to those used in human prenatal examinations. On the third postnatal day (P3), basic morphometric analyses were performed on labeled neurons reconstructed with Neurolucida. Low-intensity HFU-treated cells showed significantly increased dendritic branching compared to control (non-stimulated) neurons and showed elevated c-fos immunoreactivity. Labeled neurons were immunopositive for the mechanosensitive receptor TRPC4 at E18.5, suggesting the role of this receptor and the associated signaling pathways in the effects of HFU stimulation.

Regulation of kinase cascade activation and heat shock protein expression by poly(ADP-ribose) polymerase inhibition in doxorubicin-induced heart failure.

Cardiomyopathy is one of the most severe side effects of the chemotherapeutic agent doxorubicin (DOX). The formation of reactive oxygen species plays a critical role in the development of cardiomyopathies, and the pathophysiological cascade activates nuclear enzyme poly(ADP-ribose) polymerase (PARP), and kinase pathways. We characterized the effects of the PARP-inhibitor and kinase-modulator compound L-2286 in DOX-induced cardiac injury models. We studied the effect of the established superoxide dismutase-mimic Tempol and compared the effects of this agent with those of the PARP inhibitor. In the rat H9C2 cardiomyocytes, in which DOX-induced poly(ADP-ribosyl)ation, L-2286 protected them from the DOX-induced injury in a concentration-dependent manner. In the in vivo studies, mice were pretreated (for 1 week) with L-2286 or Tempol before the DOX treatment. Both the agents improved the activation of cytoprotective kinases, Akt, phospho-specific protein kinase C ϵ, ζ/λ and suppressed the activity of cell death promoting kinases glycogen synthase kinase-3ß, JNK, and p38 mitogen-activated protein kinase, but the effect of PARP inhibitor was more pronounced and improved the survival as well. L-2286 activated the phosphorylation of proapoptotic transcription factor FKHR1 and promoted the expression of Hsp72 and Hsp90. These data suggest that the mode of the cytoprotective action of the PARP inhibitor may include the modulation of kinase pathways and heat shock protein expression.

The targeted LHRH analog AEZS-108 alters expression of genes related to angiogenesis and development of metastasis in uveal melanoma.

Uveal melanoma (UM) is the most common malignant tumor of the eye. Recently, we have established that 46% of UM specimens express LHRH receptors. This finding supports the idea of a LHRH receptor-targeted therapy of UM patients. Cytotoxic analog of LHRH, AEZS-108 exhibits effective anti-cancer activity in LHRH-receptor positive cancers. AEZS-108 is a hybrid molecule, composed of a synthetic peptide carrier and the cytotoxic doxorubicin (DOX). In the present study, we investigated AEZS-108 induced cytotoxicity and the altered mRNA expression profile of regulatory factors related to angiogenesis and metastasis in LHRH receptor positive OCM3 cells. Our results show that AEZS-108 upregulates the expression of MASPIN/SERPINB5 tumor suppressor gene, which is downregulated in normal uvea and UM specimens independently from the LHRH receptor-ligand interaction. AEZS-108 also substantially downregulates hypoxia-inducible factor 1 alpha (HIF1A) expression. In order to investigate the mechanism of the induction of MASPIN by AEZS-108, OCM3 cells were treated with free DOX, D-Lys6 LHRH analog, or AEZS-108. qRT- PCR analysis revealed in OCM3 cells that AEZS-108 is a more potent inducer of MASPIN than free DOX. In conclusion, we show for the first time that AEZS-108 has a major impact in the regulation of angiogenesis thus plays a potential role in tumor suppression. Taken together, our results support the development of novel therapeutic strategies for UM focusing on LHRH receptors.

Protein kinase C protects from DNA damage-induced necrotic cell death by inhibiting poly(ADP-ribose) polymerase-1.

The goal of the current study, conducted in freshly isolated thymocytes was (1) to investigate the possibility that the activation of poly(ADP-ribose) polymerase-1 (PARP-1) in an intact cell can be regulated by protein kinase C (PKC) mediated phosphorylation and (2) to examine the consequence of this regulatory mechanism in the context of cell death induced by the genotoxic agent. In cells stimulated by the PKC activating phorbol esters, DNA breakage was unaffected, PARP-1 was phosphorylated, 1-methyl-3-nitro-1-nitrosoguanidine-induced PARP activation and cell necrosis were suppressed, with all these effects attenuated by the PKC inhibitors GF109203X or Gö6976. Inhibition of cellular PARP activity by PKC-mediated phosphorylation may provide a plausible mechanism for the previously observed cytoprotective effects of PKC activators.

Protective effect of hydrogen sulfide in a murine model of acute lung injury induced by combined burn and smoke inhalation.

Acute lung injury results in a severe inflammatory response, which leads to priming and activation of leucocytes, release of reactive oxygen and reactive nitrogen species, destruction of pulmonary endothelium, extravasation of protein-rich fluid into the interstitium and formation of oedema. Recently, H2S (hydrogen sulfide) has been shown to decrease the synthesis of pro-inflammatory cytokines, reduce leucocyte adherence to the endothelium and subsequent diapedesis of these cells from the microvasculature in in vivo studies, and to protect cells in culture from oxidative injury. In the present study, we hypothesized that a parenteral formulation of H2S would reduce the lung injury induced by burn and smoke inhalation in a novel murine model. H(2)S post-treatment significantly decreased mortality and increased median survival in mice. H2S also inhibited IL (interleukin)-1beta levels and significantly increased the concentration of the anti-inflammatory cytokine IL-10 in lung tissue. Additionally, H2S administration attenuated protein oxidation following injury and improved the histological condition of the lung. In conclusion, these results suggest that H2S exerts protective effects in acute lung injury, at least in part through the activation of anti-inflammatory and antioxidant pathways.

Inhibition of angiogenesis by the poly(ADP-ribose) polymerase inhibitor PJ-34.

Angiogenesis-related treatments have a broad spectrum of potential applications ranging from cancer to macular degeneration, to wound healing. Thus, the identification of pharmacological agents that modulate new blood vessel formation has attracted much attention. In the present study, we investigated the effects of the poly(ADP-ribose) polymerase (PARP) inhibitor PJ-34 [N-(6-Oxo-5,6-dihydro-phenanthridin-2-yl)-N,N-dimethylacetamide] on angiogenesis. Treatment of chicken chorioallantoic membranes (CAM) with PJ-34 reduced vascular length in these tissues; paradoxically, lower doses of PJ-34 (0.03 or 0.3 nmol/cm2) were more effective in inhibiting neovascularisation than higher doses (3 or 30 nmol/cm2). In vitro, incubation of endothelial cells (EC) with PJ-34 (300 nM to 10 microM) inhibited their proliferation in a concentration-dependent manner with maximal inhibition of 22.3% being observed at 10 microM. Capillary morphogenesis of EC grown on Matrigel was also negatively affected by PJ-34. In addition, PJ-34 abolished the migratory response to the prototype angiogenic factor vascular endothelial growth factor (VEGF) and reduced VEGF-stimulated activation of members of the mitogen activated protein kinase family (ERK1/2, p38), as well as Akt. PJ-34 also inhibited VEGF-induced NO release and cGMP accumulation. In conclusion, we provide evidence that PARP inhibition blocks angiogenesis-related EC properties by interfering with multiple signalling pathways leading to the inhibition of new blood vessel formation.

The clinically used PARP inhibitor olaparib improves organ function, suppresses inflammatory responses and accelerates wound healing in a murine model of third-degree burn injury.

BACKGROUND AND PURPOSE: The PARP inhibitor olaparib has recently been approved for human use for the therapy of cancer. Considering the role of PARP in critical illness, we tested the effect of olaparib in a murine model of burn injury, in order to begin exploring the feasibility of repurposing olaparib for the therapy of burn patients. EXPERIMENTAL APPROACH: Mice were subjected to scald burn injury and randomized into vehicle or olaparib (10 mg·kg-1 ·day-1 i.p.) groups. Outcome variables included indices of organ injury, clinical chemistry parameters, plasma levels of inflammatory mediators (at 24 h, 7 and 21 days) and burn wound size (at 21 days). KEY RESULTS: Olaparib reduced myeloperoxidase levels in heart and lung homogenates and reduced malondialdehyde levels in all tissues 24 h post-burn. Olaparib also reduced circulating alkaline aminotransferase, amylase and blood urea nitrogen and creatinine levels, indicative of protection against hepatic, pancreatic and renal dysfunction. Pro-inflammatory mediator (TNF-α, IL-1ß, IFN-γ, GCSF, GM-CSF, eotaxin, KC, MIP-1-α and IL-3, 6 and 12) levels as well as the levels of several mediators that are generally considered anti-inflammatory (IL-4, 10 and 13) were reduced by olaparib. Plasma troponin-I levels (an indicator of skeletal muscle damage) was also attenuated by olaparib. Finally, olaparib stimulated wound healing. CONCLUSIONS AND IMPLICATIONS: The clinically approved PARP inhibitor olaparib improves organ function, suppresses inflammatory responses and accelerates wound healing in murine burn injury. The data raise the potential utility of olaparib for severe burn injury. LINKED ARTICLES: This article is part of a themed section on Inventing New Therapies Without Reinventing the Wheel: The Power of Drug Repurposing. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.2/issuetoc.

Role of endogenous and exogenous nitric oxide, carbon monoxide and hydrogen sulfide in HCT116 colon cancer cell proliferation.

The role of the three gasotransmitter systems - nitric oxide (NO), carbon monoxide (CO) and hydrogen sulfide (H2S) - in cancer cells has not yet been studied simultaneously in the same experimental system. We measured the expression of NO and CO and H2S generating enzymes in primary colon cancer tissues and HCT116 colon cancer cells, and evaluated the effect of their pharmacological inhibition or pharmacological donation on cell proliferation. Increased expression of iNOS, nNOS, HO-1, CBS and 3-MST was detected in colon cancer. Inhibitors of NOS, HO-1/2, CBS/CSE and 3-MST, at lower concentrations, slightly stimulated HCT116 cell proliferation, but inhibited proliferation at higher concentrations. Donors of NO, CO or H2S inhibited HCT116 proliferation in a concentration-dependent manner. Inhibition of the cGMP/VASP pathway, Akt and p44/42 MAPK (Erk1/2) inhibited HCT116 cell proliferation. Endogenous NO and H2S biosynthesis were found to play a role in the maintenance of the activity of the cGMP/VASP pathway in HCT116 cells. We conclude that each of the three gasotransmitters play similar, bell-shaped roles in the control of HCT116 cell proliferation: endogenously produced NO, CO and H2S, at an optimal concentration, support HCT116 proliferation; inhibition of their production (which decreases gasotransmitter levels below optimal concentrations) as well as exogenous delivery of these gasotransmitters (which increases gasotransmitter levels above optimal concentrations) suppresses colon cancer cell proliferation. The current data give a mechanistic explanation for the paradoxical finding that both inhibitors and donors of NO, CO and H2S exert anticancer actions in cancer cells.