Dietary supplementation of Capsicum powder affects the growth, immunoglobulins, pro-inflammatory cytokines, adipokines, meat, and liver histology of aflatoxin B1 exposed broiler chickens.

The effects of dietary supplementation with Capsicum annuum fruit pericarp powder (CPP) and Capsicum annuum fruit seed powder (CSP) on the health and performance of broiler chickens exposed to aflatoxin B1 (AFB1) was investigated. Four dietary groups were established: CON (control), AFT (0.5 mg/kg AFB1), CPAF (0.5 g/kg CPP and 0.5 mg/kg AFB1), and CSAF (0.5 g/kg CSP and 0.5 mg/kg AFB1). The AFT group shows a significant (P < 0.05) reduction in the relative growth rate compared to CON, CPAF, and CSAF. In contrast, the latter two groups exhibit growth rates similar (P > 0.05) to CON. Additionally, immunoglobulin levels (IgG, IgM, and IgA) in the AFT group are significantly (P < 0.05) lower compared to the other treatment groups. Serum interleukin-6 levels in the CPAF and CSAF groups were similar (P > 0.05) to CON but higher (P < 0.05) than in AFT. Tumor necrosis factor-alpha levels were elevated (P < 0.05) in AFT compared to the other treatment groups. Interferon-gamma concentrations in AFT were significantly (P < 0.05) lower than in the other treatment groups. The liver histology reveals that the AFT treatment group has periportal hepatic inflammation. In contrast, the CPAF and CSAF treatment groups exhibit normal hepatic microanatomy. In conclusion, 0.5 g/kg CPAF dietary supplementation may help to ameliorate the adverse effects of AFB1 exposure on broiler chicken health, specifically the growth, immune parameters and liver histology.

Lyso-PAF, a biologically inactive phospholipid, contributes to RAF1 activation.

Phospholipase A2, group VII (PLA2G7) is widely recognized as a secreted, lipoprotein-associated PLA2 in plasma that converts phospholipid platelet-activating factor (PAF) to a biologically inactive product Lyso-PAF during inflammatory response. We report that intracellular PLA2G7 is selectively important for cell proliferation and tumor growth potential of melanoma cells expressing mutant NRAS, but not cells expressing BRAF V600E. Mechanistically, PLA2G7 signals through its product Lyso-PAF to contribute to RAF1 activation by mutant NRAS, which is bypassed by BRAF V600E. Intracellular Lyso-PAF promotes p21-activated kinase 2 (PAK2) activation by binding to its catalytic domain and altering ATP kinetics, while PAK2 significantly contributes to S338-phosphorylation of RAF1 in addition to PAK1. Furthermore, the PLA2G7-PAK2 axis is also required for full activation of RAF1 in cells stimulated by epidermal growth factor (EGF) or cancer cells expressing mutant KRAS. Thus, PLA2G7 and Lyso-PAF exhibit intracellular signaling functions as key elements of RAS-RAF1 signaling.

Eicosanoid Signaling in Insect Immunology: New Genes and Unresolved Issues.

This paper is focused on eicosanoid signaling in insect immunology. We begin with eicosanoid biosynthesis through the actions of phospholipase A2, responsible for hydrolyzing the C18 polyunsaturated fatty acid, linoleic acid (18:2n-6), from cellular phospholipids, which is subsequently converted into arachidonic acid (AA; 20:4n-6) via elongases and desaturases. The synthesized AA is then oxygenated into one of three groups of eicosanoids, prostaglandins (PGs), epoxyeicosatrienoic acids (EETs) and lipoxygenase products. We mark the distinction between mammalian cyclooxygenases and insect peroxynectins, both of which convert AA into PGs. One PG, PGI2 (also called prostacyclin), is newly discovered in insects, as a negative regulator of immune reactions and a positive signal in juvenile development. Two new elements of insect PG biology are a PG dehydrogenase and a PG reductase, both of which enact necessary PG catabolism. EETs, which are produced from AA via cytochrome P450s, also act in immune signaling, acting as pro-inflammatory signals. Eicosanoids signal a wide range of cellular immune reactions to infections, invasions and wounding, including nodulation, cell spreading, hemocyte migration and releasing prophenoloxidase from oenocytoids, a class of lepidopteran hemocytes. We briefly review the relatively scant knowledge on insect PG receptors and note PGs also act in gut immunity and in humoral immunity. Detailed new information on PG actions in mosquito immunity against the malarial agent, Plasmodium berghei, has recently emerged and we treat this exciting new work. The new findings on eicosanoid actions in insect immunity have emerged from a very broad range of research at the genetic, cellular and organismal levels, all taking place at the international level.

Effect of acyl and alkyl analogs of platelet-activating factor on inflammatory signaling.

Platelet-activating factor (PAF), a bioactive ether phospholipid with significant pro-inflammatory properties, was identified almost half a century ago. Despite extensive study of this autocoid, therapeutic strategies for targeting its signaling components have not been successful, including the recent clinical trials with darapladib, a drug that targets plasma PAF-acetylhydrolase (PAF-AH). We recently provided experimental evidence that the previously unrecognized acyl analog of PAF, which is concomitantly produced along with PAF during biosynthesis, dampens PAF signaling by acting both as a sacrificial substrate for PAF-AH and probably as an endogenous PAF-receptor antagonist/partial agonist. If this is the scenario in vivo, PAF-AH needs to catalyze the selective hydrolysis of alkyl-PAF and not acyl-PAF. Accordingly, different approaches are needed for treating inflammatory diseases in which PAF signaling is implicated. The interplay between acyl-PAF, alkyl-PAF, PAF-AH, and PAF-R is complex, and the outcome of this interplay has not been previously appreciated. In this review, we discuss this interaction based on our recent findings. It is very likely that the relative abundance of acyl and alkyl-PAF and their interactions with PAF-R in the presence of their hydrolyzing enzyme PAF-AH may exert a modulatory effect on PAF signaling during inflammation.

Development and validation of a UPLC-MS/MS method for simultaneous determination of LBPT and its metabolites in human plasma.

Aim: A UPLC-MS/MS method was developed to determine LBPT as well as its four metabolites in human plasma to support the clinical study aiming to evaluate the efficacy of LBPT tablet in patients undergoing hip/knee replacement. Methodology: Plasma samples were prepared by protein precipitation and then separated on a C18 analytical column using (A) acetonitrile (B) 0.1% formic acid and 10 mM ammonium formate in water. The detection was performed on a triple quadrupole tandem mass spectrometer in positive electrospray ionization using multiple reactions monitoring mode. Results & conclusion: The method has been validated in accordance with the US FDA guidelines and was applied to the measurement of five analytes in human plasma samples from a Phase II clinical trial.

Mechanisms and Treatment of Light-Induced Retinal Degeneration-Associated Inflammation: Insights from Biochemical Profiling of the Aqueous Humor.

Ocular inflammation contributes to the pathogenesis of blind-causing retinal degenerative diseases, such as age-related macular degeneration (AMD) or photic maculopathy. Here, we report on inflammatory mechanisms that are associated with retinal degeneration induced by bright visible light, which were revealed while using a rabbit model. Histologically and electrophysiologically noticeable degeneration of the retina is preceded and accompanied by oxidative stress and inflammation, as evidenced by granulocyte infiltration and edema in this tissue, as well as the upregulation of total protein, pro-inflammatory cytokines, and oxidative stress markers in aqueous humor (AH). Consistently, quantitative lipidomic studies of AH elucidated increase in the concentration of arachidonic (AA) and docosahexaenoic (DHA) acids and lyso-platelet activating factor (lyso-PAF), together with pronounced oxidative and inflammatory alterations in content of lipid mediators oxylipins. These alterations include long-term elevation of prostaglandins, which are synthesized from AA via cyclooxygenase-dependent pathways, as well as a short burst of linoleic acid derivatives that can be produced by both enzymatic and non-enzymatic free radical-dependent mechanisms. The upregulation of all oxylipins is inhibited by the premedication of the eyes while using mitochondria-targeted antioxidant SkQ1, whereas the accumulation of prostaglandins and lyso-PAF can be specifically suppressed by topical treatment with cyclooxygenase inhibitor Nepafenac. Interestingly, the most prominent antioxidant and anti-inflammatory benefits and overall retinal protective effects are achieved by simultaneous administrating of both drugs indicating their synergistic action. Taken together, these findings provide a rationale for using a combination of mitochondria-targeted antioxidant and cyclooxygenase inhibitor for the treatment of inflammatory components of retinal degenerative diseases.

Combining individual Chlamydia trachomatis IgG antibodies MOMP, TARP, CPAF, OMP2, and HSP60 for tubal factor infertility prediction.

PROBLEM: Tubal factor infertility (TFI) is a severe complication of genital Chlamydia trachomatis infections. In fertility workup, chlamydia antibody test (CAT) is used to predict TFI. The predictive value for TFI of most commonly used CAT is moderate. METHOD OF STUDY: A total of 183 infertile Dutch Caucasian women were included in this study. All underwent tubal patency testing (hysterosalpingography [HSG] or laparoscopy). Cases had TFI, and controls had no TFI (ie normal findings during HSG or laparoscopy). TFI was categorized based on severity (TFI 1-TFI 4). This study investigated the predictive values of major outer membrane protein (MOMP), translocated actin-recruiting phosphoprotein (TARP), chlamydial protease-like activity factor (CPAF), heat shock protein-60 (HSP60) and outer membrane protein 2 (OMP2) for TFI. A predictive algorithm is developed to detect TFI with a high certainty based on combinations of antibody titres. Serum was tested with the Mikrogen recomLine immunoblot and quantified with the recomScan. A greedy algorithm that explores all possible antibody combinations was developed. RESULTS: Significant differences in the distributions of antigen titres between cases and controls were observed for CPAF (P = 0.0021), HSP60 (P = 0.0061), MOMP (P = 0.0497) and OMP2 (P = 0.0016). Single antibodies could not discriminate between TFI and controls by themselves. The greedy algorithm performs better in specificity, positive predictive value (PPV), accuracy and clinical utility index than the original Mikrogen algorithm. CPAF combined with HSP60 identified 18.2% of TFI cases with 100% certainty. Most of the TFI 4 cases were identified with cut-offs of CPAF > 10.7 or OMP2 > 3.9. CONCLUSION: This proof-of-principle study shows that combinations of antibodies in serum are predictive for TFI. A commercially available test can be adapted to predict TFI with a 100% specificity.

Miltefosina para o tratamento da leishmaniose tegumentar / Miltefosine for the treatment of cutaneous leishmaniasis

CONTEXTO: A leishmaniose tegumentar (LT) é uma doença infecciosa e não contagiosa, que acomete pele e mucosas, e é causada por protozoários do gênero Leshmania. A doença apresenta baixa letalidade, mas pode causar graves deformidades que impactam na autoestima dos pacientes. É considerada uma doença negligenciada por acometer principalmente populações de baixa renda e permanece como relevante problema de saúde pública, com consideráveis taxas de incidência e prevalência no País. Os medicamentos atualmente preconizados pelo Ministério da Saúde para o tratamento da LT são todos de uso sistêmico e apresentam um potencial hepato, cardio e nefrotóxico, sendo o antimoniato de meglumina, o tratamento de primeira escolha. A maior parte dos casos de LT ocorre em áreas de difícil acesso, o que dificulta tanto a aplicação parenteral da droga, como o monitoramento de seus efeitos colaterais. A miltefosina é um medicamento de uso oral e eficaz no tratamento da LT, mas ainda não é disponibilizada no SUS. A disponibilização de um medicamento de uso oral e efetivo contra a leishmaniose aumentaria a adesão ao tratamento nas áreas mais pobres e remotas do Brasil. TECNOLOGIA: Miltefosina. PERGUNTA: O uso da miltefosina é eficaz e seguro no tratamento da leishmaniose tegumentar quando comparado ao uso do medicamento recomendado como primeira linha de tratamento pelo Ministério da Saúde, o antimoniato de meglumina? EVIDÊNCIAS CIENTÍFICAS: O Parecer Técnico Científico (PTC) da Fiocruz "Miltefosina para tratamento de leishmaniose tegumentar americana: Evidências de eficácia e segurança" foi utilizado como base das evidências científicas. Na busca realizada nesse PTC, foram selecionados 6 ensaios clínicos randomizados comparando a miltefosina via oral com o antimoniato de meglumina via parenteral. Os estudos mostraram taxas similares de cura em 6 meses com os 2 medicamentos e eventos adversos de menor gravidade com a miltefosina. AVALIAÇÃO DE IMPACTO ORÇAMENTÁRIO: Estima-se que impacto orçamentário anual com a incorporação da miltefoseina varie de R$ 561.863,89 a R$ 2.809.319,45, no primeiro ano, e de R$ 8.427.958,35 a R$ 19.665.236,16, ao longo dos próximos cinco anos. O cálculo do impacto orçamentário não levou em conta os custos que seriam economizados com a incorporação da miltefosina: custos diretos, relacionados à administração parenteral dos medicamentos, e os indiretos, relacionados ao deslocamento diário do paciente, sozinho ou acompanhado de cuidadores ou outros membros da família, de sua residência até uma unidade de saúde para receber a administração parenteral dos medicamentos. Como a maioria desses pacientes residem em áreas rurais com difícil acesso aos serviços de saúde, esse deslocamento resulta em perda de trabalho. RECOMENDAÇÃO PRELIMINAR: A CONITEC, em 10/11/2016 recomendou a incorporação no SUS de miltefosina para o tratamento de pacientes com leishmaniose tegumentar. CONSULTA PÚBLICA: O tema foi colocado em consulta pública nº 40, realizada entre os dias 30/11/16 a 19/12/2016. Foram recebidas 06 contribuições de cunho técnico-científico e 3 contribuições de experiência ou opinião. Foram feitas alterações de texto e nenhuma argumentação foi considerada suficiente para alterar a recomendação preliminar. RECOMENDAÇÃO FINAL: Os membros da CONITEC em 09/05/2018 deliberaram, por unanimidade, por recomendar a incorporação no SUS de miltefosina para o tratamento de leishmaniose tegumentar em primeira linha de tratamento. DECISÃO: Incorporar a miltefosina para o tratamento da leishmaniose tegumentar em primeira linha de tratamento, no âmbito do Sistema Único de Saúde - SUS. Dada pela Portaria nº 60 de 31 de outubro de 2018, publicada no Diário Oficial da União nº 210, de 31 de outubro de 2018, seção 1, página 40.

Before platelets: the production of platelet-activating factor during growth and stress in a basal marine organism.

Corals and humans represent two extremely disparate metazoan lineages and are therefore useful for comparative evolutionary studies. Two lipid-based molecules that are central to human immunity, platelet-activating factor (PAF) and Lyso-PAF were recently identified in scleractinian corals. To identify processes in corals that involve these molecules, PAF and Lyso-PAF biosynthesis was quantified in conditions known to stimulate PAF production in mammals (tissue growth and exposure to elevated levels of ultraviolet light) and in conditions unique to corals (competing with neighbouring colonies over benthic space). Similar to observations in mammals, PAF production was higher in regions of active tissue growth and increased when corals were exposed to elevated levels of ultraviolet light. PAF production also increased when corals were attacked by the stinging cells of a neighbouring colony, though only the attacked coral exhibited an increase in PAF. This reaction was observed in adjacent areas of the colony, indicating that this response is coordinated across multiple polyps including those not directly subject to the stress. PAF and Lyso-PAF are involved in coral stress responses that are both shared with mammals and unique to the ecology of cnidarians.

Inositol-C2-PAF acts as a biological response modifier and antagonizes cancer-relevant processes in mammary carcinoma cells.

PURPOSE: Previous studies have identified alkyl-phospholipids as promising compounds for cancer therapy by targeting constituents of the cell membrane and different signaling pathways. We previously showed that the alkylphospholipid Inositol-C2-PAF inhibits the proliferation and migration of immortalized keratinocytes and the squamous carcinoma-derived cell line SCC-25. Here, we investigated the effect of this compound on growth and motility as well as its mode of action in mammary carcinoma-derived cell lines. METHODS: Using BrdU incorporation and haptotactic cell migration assays, we assessed the effects of Inositol-C2-PAF on MCF-7 and MBA-MB-231 cell proliferation and migration. The phosphorylation status of signaling molecules was investigated by Western blotting as well as indirect immunofluorescence analysis and capillary isoelectric focusing. RESULTS: We found that Inositol-C2-PAF inhibited the growth as well as the migration in MCF-7 and MBA-MB-231 cells. Furthermore, we found that this compound inhibited phosphorylation of the protein kinase Akt at serine residue 473, but had no impact on phosphorylation at threonine 308. Phosphorylation of other kinases, such as Erk1/2, FAK and Src, which are targeted by Inositol-C2-PAF in other cells, remained unaffected by the compound in the mammary carcinoma-derived cell lines tested. In MCF-7 cells, we found that IGF-1-induced growth, as well as phosphorylation of AktS473, mTOR and the tumor suppressor pRB, was inhibited in the presence of Inositol-C2-PAF. Moreover, we found that in these cells IGF-1 had no impact on migration and did not seem to be linked to full Akt activity. Therefore, MCF-7 cell migration appears to be inhibited by Ino-C2-PAF in an Akt-independent manner. CONCLUSION: The antagonistic effects of Inositol-C2-PAF on cell migration and proliferation are indicative for its potential for breast cancer therapy, alone or in combination with other cytostatic drugs.

Peptidase Inhibitor 15 (PI15) Regulates Chlamydial CPAF Activity.

Obligate intracellular pathogenic Chlamydia trachomatis express several serine proteases whose roles in chlamydial development and pathogenicity are not completely understood. The chlamydial protease CPAF is expressed during the replicative phase of the chlamydial developmental cycle and is secreted into the lumen of the Chlamydia-containing vacuole called inclusion. How the secreted protease is activated in the inclusion lumen is currently not fully understood. We have identified human serine peptidase inhibitor PI15 as a potential host factor involved in the regulation of CPAF activation. Silencing expression as well as over expression of PI15 affected normal development of Chlamydia. PI15 was transported into the chlamydial inclusion lumen where it co-localized with CPAF aggregates. We show that PI15 binds to the CPAF zymogen and potentially induces CPAF protease activity at low concentrations. However, at high concentrations PI15 inhibits CPAF activity possibly by blocking its protease domain. Our findings shed light on a new aspect of chlamydial host co-evolution which involves the recruitment of host cell proteins into the inclusion to control the activation of bacterial proteases like CPAF that are important for the normal development of Chlamydia.

Alterations in the Plasma Levels of Specific Choline Phospholipids in Alzheimer's Disease Mimic Accelerated Aging.

Alzheimer's disease (AD) is the most common neurodegenerative disease and of continuously rising prevalence. The identification of easy-to-measure biomarkers capable to assist in the prediction and early diagnosis of AD is currently a main research goal. Lipid metabolites in peripheral blood of human patients have recently gained major attention in this respect. Here, we analyzed plasma of 174 participants (not demented at baseline; mean age: 75.70±0.44 years) of the Vienna Transdanube Aging (VITA) study, a longitudinal, population-based birth cohort study, at baseline and after 90 months or at diagnosis of probable AD. We determined the levels of specific choline phospholipids, some of which have been suggested as potential biomarkers for the prediction of AD. Our results show that during normal aging the levels of lysophosphatidylcholine, choline plasmalogen, and lyso-platelet activating factor increase significantly. Notably, we observed similar but more pronounced changes in the group that developed probable AD. Thus, our results imply that, in terms of choline-containing plasma phospholipids, the conversion to AD mimics an accelerated aging process. We conclude that age, even in the comparatively short time frame between 75 and 82.5 years, is a crucial factor in the quest for plasma lipid biomarkers for AD that must be carefully considered in future studies and trials.

Identification and functional analyses of differentially expressed metabolites in early stage endometrial carcinoma.

Diagnosis of endometrial cancer is primarily based on symptoms and imaging, with early-stage disease being difficult to diagnose. Therefore, development of potential diagnostic biomarkers is required. Metabolomics, a quantitative measurement of the dynamic metabolism in living systems, can be applied to determine metabolite profiles in different disease states. Here, serum metabolomics was performed in 46 early stage endometrial cancer patients and 46 healthy volunteers. In addition, the effect of identified metabolites on tumor cell behavior (invasion, migration, proliferation, apoptosis and autophagy) was examined in endometrial cancer cell lines. Compared with controls, phenylalanine, indoleacrylic acid (IAA), phosphocholine and lyso-platelet-activating factor-16 (lyso-PAF) were differentially detected in patients. Functional analyses demonstrated that IAA, PAF and phenylalanine all dose-dependently inhibited tumor cell invasion and migration, and suppressed cell proliferation. PAF also induced tumor cell apoptosis and autophagy, while phenylalanine had no effect on apoptosis or autophagy. IAA triggered apoptosis and had a biphasic effect on autophagy: inhibiting autophagy with doses <1 mmol/L but inducing at 1 mmol/L. Interestingly, the alterations in proliferation, apoptosis and autophagy caused by 1 mmol/L IAA, were all reversed by the concomitant treatment of tryptophan (100 µmol/L). Phosphocholine inhibited tumor cell invasion and migration, and promoted cell proliferation and autophagy, all in a dose-dependent manner. Phosphocholine also protected cells from TNF-α-induced apoptosis. In conclusion, 4 serum metabolites were identified by serum metabolomics in endometrial cancer patients and functional analyses suggested that they may play roles in modulation of tumor cell behavior, although their exact mode of action still needs to be determined.

Enzymatic methods for choline-containing water soluble phospholipids based on fluorescence of choline oxidase: Application to lyso-PAF.

In this paper we present methods to determine water soluble phospholipids containing choline (wCh-PL). The analytes were hydrolyzed by the enzyme phospholipase D and the choline formed was oxidized by the enzyme Choline Oxidase (ChOx); the fluorescence changes of the ChOx are followed during the enzymatic reaction, avoiding the necessity of an indicating step. Both reactions (hydrolysis and oxidation) can be combined in two different ways: 1) a two-step process (TSP) in which the hydrolysis reaction takes place during an incubation time and then the oxidation reaction is carried out, the analytical signal being provided by the intrinsic fluorescence of ChOx due to tryptophan; 2) a one-step process (OSP) in which both enzymatic reactions are carried out simultaneously in the same test; in this case the analytical signal is provided by the ChOx extrinsic fluorescence due to a fluorescent probe (Ru (II) chelate) linked to the enzyme (ChOx-RuC). The analytical capabilities of these methods were studied using 1,2-dioctanoyl-sn-glycero-3-phosphocholine (C8PC), a water soluble short alkyl chain Ch-PL as a substrate, and 1-O-hexadecyl-sn-glyceryl-3-phosphorylcholine (lyso-PAF). The analytical features of merit for both analytes using both methods were obtained. The TSP gave a 10-fold sensitivity and lower quantification limit (1.0*10-5 M for lyso-PAF), but OSP reduced the determination time and permitted to use the same enzyme aliquot for several measurements. Both methods gave similar precision (RSD 7%, n = 5). The TSP was applied to the determination of C8PC and lyso-PAF in spiked synthetic serum matrix using the standard addition method. The application of this methodology to PLD activity determination is also discussed.

Metabolomics of reef benthic interactions reveals a bioactive lipid involved in coral defence.

Holobionts are assemblages of microbial symbionts and their macrobial host. As extant representatives of some of the oldest macro-organisms, corals and algae are important for understanding how holobionts develop and interact with one another. Using untargeted metabolomics, we show that non-self interactions altered the coral metabolome more than self-interactions (i.e. different or same genus, respectively). Platelet activating factor (PAF) and Lyso-PAF, central inflammatory modulators in mammals, were major lipid components of the coral holobionts. When corals were damaged during competitive interactions with algae, PAF increased along with expression of the gene encoding Lyso-PAF acetyltransferase; the protein responsible for converting Lyso-PAF to PAF. This shows that self and non-self recognition among some of the oldest extant holobionts involve bioactive lipids identical to those in highly derived taxa like humans. This further strengthens the hypothesis that major players of the immune response evolved during the pre-Cambrian.

Platelet-activating factor induces cell cycle arrest and disrupts the DNA damage response in mast cells.

Platelet-activating factor (PAF) is a potent phospholipid modulator of inflammation that has diverse physiological and pathological functions. Previously, we demonstrated that PAF has an essential role in ultraviolet (UV)-induced immunosuppression and reduces the repair of damaged DNA, suggesting that UV-induced PAF is contributing to skin cancer initiation by inducing immune suppression and also affecting a proper DNA damage response. The exact role of PAF in modulating cell proliferation, differentiation or transformation is unclear. Here, we investigated the mechanism(s) by which PAF affects the cell cycle and impairs early DNA damage response. PAF arrests proliferation in transformed and nontransformed human mast cells by reducing the expression of cyclin-B1 and promoting the expression of p21. PAF-treated cells show a dose-dependent cell cycle arrest mainly at G2-M, and a decrease in the DNA damage response elements MCPH1/BRIT-1 and ataxia telangiectasia and rad related (ATR). In addition, PAF disrupts the localization of p-ataxia telangiectasia mutated (p-ATM), and phosphorylated-ataxia telangiectasia and rad related (p-ATR) at the site of DNA damage. Whereas the potent effect on cell cycle arrest may imply a tumor suppressor activity for PAF, the impairment of proper DNA damage response might implicate PAF as a tumor promoter. The outcome of these diverse effects may be dependent on specific cues in the microenvironment.

Systemic chemotherapy is modulated by platelet-activating factor-receptor agonists.

Chemotherapy is used to treat numerous cancers including melanoma. However, its effectiveness in clinical settings is often hampered by various mechanisms. Previous studies have demonstrated that prooxidative stressor-mediated generation of oxidized lipids with platelet-activating factor-receptor (PAF-R) agonistic activity induces systemic immunosuppression that augments the growth of experimental melanoma tumors. We have recently shown that treatment of murine B16F10 melanoma cells in vitro or tumors implanted into syngeneic mice and treated intratumorally with various chemotherapeutic agents generated PAF-R agonists in a process blocked by antioxidants. Notably, these intratumoral chemotherapy-generated PAF-R agonists augmented the growth of secondary (untreated) tumors in a PAF-R dependent manner. As both localized and systemic chemotherapies are used based on tumor localization/stage and metastases, the current studies were sought to determine effects of PAF-R agonists on systemic chemotherapy against experimental melanoma. Here, we show that systemic chemotherapy with etoposide (ETOP) attenuates the growth of melanoma tumors when given subsequent to the tumor cell implantation. Importantly, this ETOP-mediated suppression of melanoma tumor growth was blocked by exogenous administration of a PAF-R agonist, CPAF. These findings indicate that PAF-R agonists not only negatively affect the ability of localized chemotherapy but also compromise the efficacy of systemic chemotherapy against murine melanoma.

Expression of the platelet-activating factor receptor enhances benzyl isothiocyanate-induced apoptosis in murine and human melanoma cells.

Melanoma cells often express platelet-activating factor receptor (PAF-R), which has been demonstrated to increase metastatic behavior. However, the effect of PAF-R on the responsiveness of melanoma to naturally occurring cytotoxic agents remains to be elucidated. The present study aimed to determine the relative cytotoxicity and mechanism of benzyl isothiocyanate (BITC), a component of cruciferous vegetables, in melanoma cells expressing PAF-R. To evaluate the importance of PAF-R signaling in melanoma cell growth, PAF-R-negative murine B16F10 cells were transduced with a retrovirus containing the cDNA for PAF-R to generate cells stably expressing PAF-R (B16-PAF-R) or an empty vector (MSCV) to generate PAF-R-deficient B16-MSCV control cells. Activation of PAF-R, using the PAF-R agonist, 1-hexadecyl-2-N-methylcarbamoyl-3-glycerophosphocholine, induced an increase in the proliferation of B16-PAF-R cells compared with the B16-MSCV cells. Reverse transcription quantitative polymerase chain reaction revealed the presence of functional PAF-R in human melanoma SK23MEL cells, but not in SK5MEL cells. The present study investigated the effect of BITC treatments on the survival of murine and human melanoma cells, in the presence or absence of functional PAF-R. The results revealed that treatment with BITC decreased the survival rate of the PAF-R-positive and negative murine and human melanoma cells. However, the expression of PAF-R substantially augmented BITC-mediated cytotoxicity in the PAF-R-positive cells at lower concentrations compared with the PAF-R-negative cells. In order to determine the underlying mechanism, flow cytometric analysis was used, which demonstrated a significant increase in the generation of reactive oxygen species (ROS) in the B16-PAF-R cells compared with the B16-MSCV cells, which enhanced apoptosis by BITC, as measured by increased caspase-3/7 luminescence. Notably, the BITC-mediated decreased cell survival rate, increased ROS and increased apoptosis in the B16-PAF-R cells were significantly attenuated by the antioxidant, vitamin C, indicating ROS involvement. Additionally, the WEB2086 PAF-R antagonist, inhibited the BITC-mediated enhancement of apoptosis in the B16-PAF-R cells, indicating a role for PAF-R-signaling in the BITC-mediated effects. These findings indicated that the selectivity of BITC towards PAF­R in melanoma offers a promising chemopreventive agent for PAF-R-positive melanoma treatment.

New members of the mammalian glycerophosphodiester phosphodiesterase family: GDE4 and GDE7 produce lysophosphatidic acid by lysophospholipase D activity.

The known mammalian glycerophosphodiester phosphodiesterases (GP-PDEs) hydrolyze glycerophosphodiesters. In this study, two novel members of the mammalian GP-PDE family, GDE4 and GDE7, were isolated, and the molecular basis of mammalian GP-PDEs was further explored. The GDE4 and GDE7 sequences are highly homologous and evolutionarily close. GDE4 is expressed in intestinal epithelial cells, spermatids, and macrophages, whereas GDE7 is particularly expressed in gastro-esophageal epithelial cells. Unlike other mammalian GP-PDEs, GDE4 and GDE7 cannot hydrolyze either glycerophosphoinositol or glycerophosphocholine. Unexpectedly, both GDE4 and GDE7 show a lysophospholipase D activity toward lysophosphatidylcholine (lyso-PC). We purified the recombinant GDE4 and GDE7 proteins and show that these enzymes can hydrolyze lyso-PC to produce lysophosphatidic acid (LPA). Further characterization of purified recombinant GDE4 showed that it can also convert lyso-platelet-activating factor (1-O-alkyl-sn-glycero-3-phosphocholine; lyso-PAF) to alkyl-LPA. These data contribute to our current understanding of mammalian GP-PDEs and of their physiological roles via the control of lyso-PC and lyso-PAF metabolism in gastrointestinal epithelial cells and macrophages.