Carlo Angioni

Ceramide synthases and ceramide levels are increased in breast cancer tissue

Several in vitro studies have correlated dysfunction of the sphingolipid-signaling pathway with promotion of tumor cell growth as well as progression and resistance of tumors to chemotherapeutic agents. As ceramides (Cer) constitute the structural backbones of all sphingolipids, we investigated the endogenous ceramide levels in 43 malignant breast tumors and 21 benign breast biopsies and compared them with those of normal tissues using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). The total ceramide levels in malignant tumor tissue samples were statistically significantly elevated when compared with normal tissue samples. Upregulation of the total ceramide level averaged 12-fold and 4-fold higher than normal tissue samples, for malignant tumors and benign tissues, respectively. Specifically, the levels of C(16:0)-Cer, C(24:1)-Cer and C(24:0)-Cer were significantly raised in malignant tumors as compared with benign and normal tissue. The augmentation of the various ceramides could be assigned to an increase of the messenger RNA levels of ceramide synthases (CerS) LASS2 (longevity assurance), LASS4 and LASS6. Notably, elevated levels of C(16:0)-Cer were associated with a positive lymph node status, indicating a metastatic potential for this ceramide. Moreover, the levels of C(18:0)-Cer and C(20:0)-Cer were significantly higher in estrogen receptor (ER) positive tumor tissues as compared with ER negative tumor tissues. In conclusion, progression in breast cancer is associated with increased ceramide levels due to an upregulation of specific LASS genes.

5,6-EET Is Released upon Neuronal Activity and Induces Mechanical Pain Hypersensitivity via TRPA1 on Central Afferent Terminals

Epoxyeicosatrienoic acids (EETs) are cytochrome P450-epoxygenase-derived metabolites of arachidonic acid that act as endogenous signaling molecules in multiple biological systems. Here we have investigated the specific contribution of 5,6-EET to transient receptor potential (TRP) channel activation in nociceptor neurons and its consequence for nociceptive processing. We found that, during capsaicin-induced nociception, 5,6-EET levels increased in dorsal root ganglia (DRGs) and the dorsal spinal cord, and 5,6-EET is released from activated sensory neurons in vitro. 5,6-EET potently induced a calcium flux (100 nm) in cultured DRG neurons that was completely abolished when TRPA1 was deleted or inhibited. In spinal cord slices, 5,6-EET dose dependently enhanced the frequency, but not the amplitude, of spontaneous EPSCs (sEPSCs) in lamina II neurons that also responded to mustard oil (allyl isothiocyanate), indicating a presynaptic action. Furthermore, 5,6-EET-induced enhancement of sEPSC frequency was abolished in TRPA1-null mice, suggesting that 5,6-EET presynaptically facilitated spinal cord synaptic transmission by TRPA1. Finally, in vivo intrathecal injection of 5,6-EET caused mechanical allodynia in wild-type but not TRPA1-null mice. We conclude that 5,6-EET is synthesized on the acute activation of nociceptors and can produce mechanical hypersensitivity via TRPA1 at central afferent terminals in the spinal cord.

Alterations in the Hippocampal Endocannabinoid System in Diet-Induced Obese Mice

The endocannabinoid (eCB) system plays central roles in the regulation of food intake and energy expenditure. Its alteration in activity contributes to the development and maintenance of obesity. Stimulation of the cannabinoid receptor type 1 (CB1 receptor) increases feeding, enhances reward aspects of eating, and promotes lipogenesis, whereas its blockade decreases appetite, sustains weight loss, increases insulin sensitivity, and alleviates dysregulation of lipid metabolism. The hypothesis has been put forward that the eCB system is overactive in obesity. Hippocampal circuits are not directly involved in the neuronal control of food intake and appetite, but they play important roles in hedonic aspects of eating. We investigated the possibility whether or not diet-induced obesity (DIO) alters the functioning of the hippocampal eCB system. We found that levels of the two eCBs, 2-arachidonoyl glycerol (2-AG) and anandamide, were increased in the hippocampus from DIO mice, with a concomitant increase of the 2-AG synthesizing enzyme diacylglycerol lipase-α and increased CB1 receptor immunoreactivity in CA1 and CA3 regions, whereas CB1 receptor agonist-induced [35S]GTPγS binding was unchanged. eCB-mediated synaptic plasticity was changed in the CA1 region, as depolarization-induced suppression of inhibition and long-term depression of inhibitory synapses were enhanced. Functionality of CB1 receptors in GABAergic neurons was furthermore revealed, as mice specifically lacking CB1 receptors on this neuronal population were partly resistant to DIO. Our results show that DIO-induced changes in the eCB system affect not only tissues directly involved in the metabolic regulation but also brain regions mediating hedonic aspects of eating and influencing cognitive processes.

Quantitative characterization of prostaglandins in the uterus of early pregnant cattle

Prostaglandins (PGs) are important regulators of reproductive processes including early embryonic development. We analyzed the most relevant PG in bovine uteri at different preimplantation pregnancy stages when compared with non-pregnant controls. Additionally, endometrium and trophoblast tissues were examined regarding specific enzymes and receptors involved in PG generation and function. Simmental heifers were artificially inseminated or received seminal plasma only. At days 12, 15, or 18, post-estrus uteri were flushed for PG determination by liquid chromatography-tandem mass spectrometry. Endometrium and trophoblast tissues were sampled for RNA extraction and quantitative real-time PCR analysis. At all days and points of time examined, the concentration of 6-keto PGF(1alpha) (stable metabolite of PGI(2)) was predominant followed by PGF(2alpha)>PGE(2)>PGD(2) approximately TXB(2) (stable metabolite of TXA(2)). At days 15 and 18, PG increased from overall low levels at day 12, with a much more pronounced increase during pregnancy. The PGF(2alpha)/PGE(2) ratio was not influenced by status. The highest PG concentration was measured at day 15 with 6-keto PGF(1alpha) (6.4 ng/ml) followed by PGF(2alpha) (1.1 ng/ml) and PGE(2) (0.3 ng/ml). Minor changes in endometrial PG biosynthesis enzymes occurred due to pregnancy. Trophoblasts revealed high transcript abundance of general and specific PG synthases contributing to uterine PG. As PGI(2) and PGF(2alpha) receptors were abundantly expressed by the trophoblast, abundant amounts of PGI(2) and PGF(2alpha) in the uterine lumen point towards an essential role of PG for the developing embryo. High amounts of PG other than PGE(2) in the preimplantation uterus may be essential rather than detrimental for successful reproduction.

Celecoxib inhibits 5-lipoxygenase

Celecoxib is a selective cyclooxygenase-2 (COX-2) inhibitor used in the therapy of inflammatory and painful conditions. Various COX-2-independent pharmacological effects, such as a chemo-preventive and tumor-regressive activity have been suggested, but the respective non-COX-2 targets of celecoxib are still a matter of research. We now demonstrate that celecoxib inhibits 5-lipoxygenase (5-LO), a key enzyme in leukotriene (LT) biosynthesis. Celecoxib suppressed 5-LO product formation in ionophore A23187-activated human polymorphonuclear leukocytes (IC(50) approximately 8 microM). Similarly, celecoxib inhibited LTB(4) formation in human whole blood (IC(50) approximately 27.3 microM). Direct interference of 5-LO with celecoxib was visualized by inhibition of enzyme catalysis both in cell homogenates and with purified 5-LO (IC(50) approximately 23.4 and 24.9 microM, respectively). Related lipoxygenases (12-LO and 15-LO) were not affected by celecoxib. Other COX-2 inhibitors (etoricoxib and rofecoxib) or unselective NSAIDs (non-steroidal anti-inflammatory drugs, diclofenac) failed to inhibit 5-LO. In rats which received celecoxib (i.p.), the blood LTB(4) levels were dose-dependently reduced with an ED(50) value approximately 35.2 mg/kg. Together, celecoxib is a direct inhibitor of 5-LO in vitro and in vivo. These findings provide a potential molecular basis for some of the described COX-2-independent pharmacological effects of celecoxib.

Activation of ceramide synthase 6 by celecoxib leads to a selective induction of C16:0-ceramide

Ceramides serve as bioactive molecules with important roles in cell proliferation and apoptosis. Ceramides (Cer) with different N-acyl side chains (C(14:0)-Cer-C(26:0)-Cer) possess distinctive roles in cell signaling and are differentially expressed in HCT-116 colon cancer cells. Celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor, exhibiting antiproliferative effects, activates the sphingolipid pathway. To elucidate the mechanism, HCT-116 cells were treated with 50μM celecoxib leading to a significant increase of C(16:0)-Cer. Interestingly, 50μM celecoxib resulted in a 2.8-fold increase of ceramide synthase (CerS) activity as measured by a cell-based activity assay. siRNA against several CerSs revealed that CerS6 was predominantly responsible for the increase of C(16:0)-Cer in HCT-116 cells. Moreover, the silencing of CerS6 partially protected HCT-116 cells from the toxic effects induced by celecoxib. Treatment of cells with celecoxib and fumonisin B1 (inhibitor of CerSs) or myriocin (inhibitor of l-serine palmitoyl transferase) or desipramine (inhibitor of acid sphingomyelinase and acid ceramidase) revealed that the increase of C(16:0)-Cer results predominantly from activation of the salvage pathway. Using the nude mouse model we demonstrated that celecoxib induces also in vivo a significant increase of C(16:0)-Cer in stomach, small intestine and tumor tissue. In conclusion, celecoxib causes a specific increase of C(16:0)-Cer by activating CerS6 and the salvage pathway, which contribute to the toxic effects of celecoxib.

Anti-inflammatory Role of Microsomal Prostaglandin E Synthase-1 in a Model of Neuroinflammation

A major immunological response during neuroinflammation is the activation of microglia, which subsequently release proinflammatory mediators such as prostaglandin E2 (PGE2). Besides its proinflammatory properties, cyclooxygenase-2 (COX-2)-derived PGE2 has been shown to exhibit anti-inflammatory effects on innate immune responses. Here, we investigated the role of microsomal PGE2 synthase-1 (mPGES-1), which is functionally coupled to COX-2, in immune responses using a model of lipopolysaccharide (LPS)-induced spinal neuroinflammation. Interestingly, we found that activation of E-prostanoid (EP)2 and EP4 receptors, but not EP1, EP3, PGI2 receptor (IP), thromboxane A2 receptor (TP), PGD2 receptor (DP), and PGF2 receptor (FP), efficiently blocked LPS-induced tumor necrosis factor α (TNFα) synthesis and COX-2 and mPGES-1 induction as well as prostaglandin synthesis in spinal cultures. In vivo, spinal EP2 receptors were up-regulated in microglia in response to intrathecally injected LPS. Accordingly, LPS priming reduced spinal synthesis of TNFα, interleukin 1β (IL-1β), and prostaglandins in response to a second intrathecal LPS injection. Importantly, this reduction was only seen in wild-type but not in mPGES-1-deficient mice. Furthermore, intrathecal application of EP2 and EP4 agonists as well as genetic deletion of EP2 significantly reduced spinal TNFα and IL-1β synthesis in mPGES-1 knock-out mice after LPS priming. These data suggest that initial inflammation prepares the spinal cord for a negative feedback regulation by mPGES-1-derived PGE2 followed by EP2 activation, which limits the synthesis of inflammatory mediators during chronic inflammation. Thus, our data suggest a role of mPGES-1-derived PGE2 in resolution of neuroinflammation.

Identification of human cathepsin G as a functional target of boswellic acids from the anti-inflammatory remedy frankincense.

Frankincense preparations, used in folk medicine to cure inflammatory diseases, showed anti-inflammatory effectiveness in animal models and clinical trials. Boswellic acids (BAs) constitute major pharmacological principles of frankincense, but their targets and the underlying molecular modes of action are still unclear. Using a BA-affinity Sepharose matrix, a 26-kDa protein was selectively precipitated from human neutrophils and identified as the lysosomal protease cathepsin G (catG) by mass spectrometry (MALDI-TOF) and by immunological analysis. In rigid automated molecular docking experiments BAs tightly bound to the active center of catG, occupying the same part of the binding site as the synthetic catG inhibitor JNJ-10311795 (2-[3-{methyl[1-(2-naphthoyl)piperidin-4-yl]amino}carbonyl)-2-naphthyl]-1-(1-naphthyl)-2-oxoethylphosphonic acid). BAs potently suppressed the proteolytic activity of catG (IC50 of ∼600 nM) in a competitive and reversible manner. Related serine proteases were significantly less sensitive against BAs (leukocyte elastase, chymotrypsin, proteinase-3) or not affected (tryptase, chymase). BAs inhibited chemoinvasion but not chemotaxis of challenged neutrophils, and they suppressed Ca2+ mobilization in human platelets induced by isolated catG or by catG released from activated neutrophils. Finally, oral administration of defined frankincense extracts significantly reduced catG activities in human blood ex vivo vs placebo. In conclusion, we show that catG is a functional and pharmacologically relevant target of BAs, and interference with catG could explain some of the anti-inflammatory properties of frankincense.

The anti-proliferative potency of celecoxib is not a class effect of coxibs.

Celecoxib, a COX-2 (cyclooxygenase-2)-selective inhibitor (coxib), is the only NSAID (non-steroidal anti-inflammatory drug) that has been approved for adjuvant treatment of patients with familial adenomatous polyposis. To investigate if the anti-proliferative effect of celecoxib extends to other coxibs, we compared the anti-proliferative potency of all coxibs currently available (celecoxib, rofecoxib, etoricoxib, valdecoxib, lumiracoxib). Additionally, we used methylcelecoxib (DMC), a close structural analogue of celecoxib lacking COX-2-inhibitory activity. Due to the fact that COX-2 inhibition is the main characteristic of these substances (with exception of methylcelecoxib), we conducted all experiments in COX-2-overexpressing (HCA-7) and COX-2-negative (HCT-116) human colon cancer cells, in order to elucidate whether the observed effects after coxib treatment depend on COX-2 inhibition. Cell survival was assessed using the WST proliferation assay. Apoptosis and cell cycle arrest were determined using flow cytometric and Western blot analysis. The in vitro results were confirmed in vivo using the nude mouse model. Among all coxibs tested, only celecoxib and methylcelecoxib decreased cell survival by induction of cell cycle arrest and apoptosis and reduced the growth of tumor xenografts in nude mice. None of the other coxibs (rofecoxib, etoricoxib, valdecoxib, lumiracoxib) produced anti-proliferative effects, indicating the lack of a class effect and of a role for COX-2. Our data emphasize again the outstanding anti-proliferative activity of celecoxib and its close structural analogue methylcelecoxib in colon carcinoma models in vitro and in vivo.

5-Lipoxygenase inhibitors induce potent anti-proliferative and cytotoxic effects in human tumour cells independently of suppression of 5-lipoxygenase activity

BACKGROUND AND PURPOSE Certain 5‐lipoxygenase (5‐LO) inhibitors exhibit anti‐carcinogenic activities against 5‐LO overexpressing tumour types and cultured tumour cells. It has been proposed therefore that 5‐LO products significantly contribute to tumour cell proliferation. To date, the relationship between the inhibitory mechanisms of 5‐LO inhibitors, which vary widely, and tumour cell viability has not been evaluated. This study addresses the anti‐proliferative and cytotoxic potency of a number of 5‐LO inhibitors with different inhibitory mechanisms in 5‐LO‐positive and 5‐LO‐negative tumour cells.