Preparation and cytotoxic evaluation of new steroidal oximes and aza-homosteroids from diosgenin and cholesterol.

Using cholesterol and diosgenin as starting materials, we have designed a straightforward methodology to prepare in a reduced number of steps a novel series of steroidal oximes and their aza-homolactam analogs with four types of side chains: cholestane, spirostane, 22-oxocholestane and 22,26-epoxycholestene. The products were evaluated for their cytotoxic activity against the MCF-7 breast cancer cell line. Moreover, the selectivity of the most active compounds was determined against peripheral blood lymphocytes. Compounds 5, 8 and 13 were found to be the most active derivatives, exhibiting IC50 values in the low micromolar range (7.9-9.5 µM) and excellent selectivities (IC50 > 100 µM) against the non-tumor cell line.

Scalaradial Is a Potent Inhibitor of Transient Receptor Potential Melastatin 2 (TRPM2) Ion Channels.

TRPM2 is a Ca2+-permeable, nonselective cation channel that plays a role in oxidant-induced cell death, insulin secretion, and cytokine release. Few TRPM2 inhibitors have been reported, which hampers the validation of TRPM2 as a drug target. While screening our in-house marine-derived chemical library, we identified scalaradial and 12-deacetylscalaradial as the active components within an extract of an undescribed species of Cacospongia (class Demospongiae, family Thorectidae) that strongly inhibited TRPM2-mediated Ca2+ influx in TRPM2-overexpressing HEK293 cells. In whole-cell patch-clamp experiments, scalaradial (and similarly 12-deacetylscalaradial) inhibited TRPM2-mediated currents in a concentration- and time-dependent manner (∼20 min to full onset; IC50 210 nM). Scalaradial inhibited TRPM7 with less potency (IC50 760 nM) but failed to inhibit CRAC, TRPM4, and TRPV1 currents in whole-cell patch clamp experiments. Scalaradial's effect on TRPM2 channels was shown to be independent of its well-known ability to inhibit secreted phospholipase A2 (sPLA2) and its reported effects on extracellular signal-regulated kinases (ERK) and Akt pathways. In addition, scalaradial was shown to inhibit endogenous TRPM2 currents in a rat insulinoma cell line (IC50 330 nM). Based on its potency and emerging specificity profile, scalaradial is an important addition to the small number of known TRPM2 inhibitors.

Anti-Tumoral Effects of Anti-Progestins in a Patient-Derived Breast Cancer Xenograft Model.

Breast cancer is a hormone-dependent disease in which estrogen signaling targeting drugs fail in about 10 % due to resistance. Strong evidences highlighted the mitogen role of progesterone, its ligands, and the corresponding progesterone receptor (PR) isoforms in mammary carcinoma. Several PR antagonists have been synthesized; however, some of them are non-selective and led to side or toxic effects. Herein, we evaluated the anti-tumor activity of a commercially available PR modulator, ulipristal acetate (UPA), and a new selective and passive PR antagonist "APR19" in a novel preclinical approach based on patient-derived breast tumor (HBCx-34) xenografted in nude mice. As opposed to P4 that slightly reduces tumor volume, UPA and APR19 treatment for 42 days led to a significant 30 % reduction in tumor weight, accompanied by a significant 40 % retardation in tumor growth upon UPA exposure while a 1.5-fold increase in necrotic areas was observed in APR19-treated tumors. Interestingly, PR expression was upregulated by a 2.5-fold factor in UPA-treated tumors while APR19 significantly reduced expression of both PR and estrogen receptor α, indicating a potential distinct molecular mechanism among PR antagonists. Cell proliferation was clearly reduced in UPA group compared to vehicle conditions, as revealed by the significant reduction in Ki-67, Cyclin D1, and proliferating cell nuclear antigen (PCNA) expression. Likewise, an increase in activated, cleaved poly(ADP-ribose) polymerase (PARP) expression was also demonstrated upon UPA exposure. Collectively, our findings provide direct in vivo evidence for anti-progestin-mediated control of human breast cancer growth, given their anti-proliferative and pro-apoptotic activities, supporting a potential role in breast cancer therapy.

Antihormonal potential of selected D-homo and D-seco estratriene derivatives.

Since many estrogen derivatives exhibit anti-hormone or enzyme inhibition potential, a large number of steroidal derivatives have been synthesised from appropriate precursors, in order to obtain potential therapeutics for the treatment of hormone-dependent cancers. In molecular docking studies, based on X-ray crystallographic analysis, selected D-homo and D-seco estratriene derivatives were predicted to bind strongly to estrogen receptor α (ERα), aromatase and 17,20 lyase, suggesting they could be good starting compounds for antihormonal studies. Test results in vivo suggest that these compounds do not possess estrogenic activity, while some of them showed weak anti-estrogenic properties. In vitro anti-aromatase and anti-lyase assays showed partial inhibition of these two enzymes, while some compounds activated aromatase. Aromatase activators are capable of promoting estrogen synthesis for treatment of pathological conditions caused by estrogen depletion, e.g. osteopenia or osteoporosis.

In cell scalaradial interactome profiling using a bio-orthogonal clickable probe.

A bio-orthogonal click-chemistry procedure was developed to allow the in cell interactome profiling of scalaradial, an anti-inflammatory marine natural product. The results were validated through the application of the classical in vitro chemical proteomics and several bio-physical methods; peroxiredoxins, 14-3-3 isoforms and proteasomes were recognized as main scalaradial targets.

Synthesis and cytotoxic activity of some 4,6-diaza-A,B-dihomo-steroid bilactams.

Using cholesterol, stigmasterol and sitosterol as starting materials, some 4,6-diaza-A,B-dihomo-steroid bilactams were synthesized via two different synthetic routes by oxidation, reduction, oximation, Beckman rearrangement, etc. The cytotoxic activity of the synthesized compounds against SGC 7901 (human ventriculi carcinoma), Bel-7404 (human liver carcinoma), HeLa (human cervical carcinoma) and HT-29 (colonic carcinoma) cancer cells were investigated. The results showed that compounds 2 and 7b displayed a good cytotoxic activity to the SGC 7901, Bel 7404 and HeLa tumor cell lines with the IC50 values of 11.6, 16.4, 13.9 and 13.1, 21.8, 13.1 µmol/L, respectively. Their cytotoxic activity is almost same as cisplatin to these cells. The information obtained from the studies may be useful for the design of novel chemotherapeutic drugs.

A new strategy for selective targeting of progesterone receptor with passive antagonists.

Currently available progesterone (P4) receptor (PR) antagonists, such as mifepristone (RU486), lack specificity and display partial agonist properties, leading to potential drawbacks in their clinical use. Recent x-ray crystallographic studies have identified key contacts involved in the binding of agonists and antagonists with PR opening the way for a new rational strategy for inactivating PR. We report here the synthesis and characterization of a novel class of PR antagonists (APRn) designed from such studies. The lead molecule, the homosteroid APR19, displays in vivo endometrial anti-P4 activity. APR19 inhibits P4-induced PR recruitment and transactivation from synthetic and endogenous gene promoters. Importantly, it exhibits high PR selectivity with respect to other steroid hormone receptors and is devoid of any partial agonist activity on PR target gene transcription. Two-hybrid and immunostaining experiments reveal that APR19-bound PR is unable to interact with either steroid receptor coactivators 1 and 2 (SRC1 and SCR2) or nuclear receptor corepressor (NcoR) and silencing mediator of retinoid acid and thyroid hormone receptor (SMRT), in contrast to RU486-PR complexes. APR19 also inhibits agonist-induced phosphorylation of serine 294 regulating PR transcriptional activity and turnover kinetics. In silico docking studies based on the crystal structure of the PR ligand-binding domain show that, in contrast to P4, APR19 does not establish stabilizing hydrogen bonds with the ligand-binding cavity, resulting in an unstable ligand-receptor complex. Altogether, these properties highly distinguish APR19 from RU486 and likely its derivatives, suggesting that it belongs to a new class of pure antiprogestins that inactivate PR by a passive mechanism. These specific PR antagonists open new perspectives for long-term hormonal therapy.

The inactivation mechanism of human group IIA phospholipase A(2) by Scalaradial.

Secretory phospholipases A(2) (sPLA(2)s) are implicated in the pathogenesis of several inflammation diseases, such as rheumatoid arthritis, septic shock, psoriasis, and asthma. Thus, an understanding of their inactivation mechanisms could be useful for the development of new classes of chemical selective inhibitors. In the marine environment, several bioactive terpenoids possess interesting anti-inflammatory activity, often through covalent and/or noncovalent inactivation of sPLA(2). Herein, we report the molecular mechanism of human group IIA phospholipase A(2) (sPLA(2)-IIA) inactivation by Scalaradial (SLD), a marine 1,4-dialdehyde terpenoid isolated from the sponge Cacospongia mollior and endowed with a significant anti-inflammatory profile. Our results have been collected by a combination of biochemical approaches, advanced mass spectrometry, surface plasmon resonance, and molecular modeling. These suggest that SLD acts as a competitive inhibitor. Indeed, the sPLA(2)-IIA inactivation process seems to be driven by the noncovalent recognition process of SLD in the enzyme active site and by chelation of the catalytic calcium ion. In contrast, covalent modification of the enzyme by the SLD dialdehyde moiety emerges as only a minor side event in the ligand-enzyme interaction. These results could be helpful for the rational design of new PLA(2) inhibitors that would be able to selectively target the enzyme active site.

Cacospongionolide and scalaradial, two marine sesterterpenoids as potent apoptosis-inducing factors in human carcinoma cell lines.

Apoptosis, a form of programmed cell death, is a critical defence mechanism against the formation and progression of cancer and acts by eliminating potentially deleterious cells without causing such adverse effects, as inflammatory response and ensuing scar formation. Therefore, targeting apoptotic pathways becomes an intriguing strategy for the development of chemotherapeutic agents. In last decades, marine natural products, such as sesterterpenoids, have played an important role in the discovery and development of new drugs. Interestingly, many of these compounds have a strong potential as anticancer drugs by inhibiting cell proliferation and/or inducing cell death. In the present study, we investigated the effects of scalaradial and cacospongionolide, two sesterterpenoids from Cacospongia scalaris and Fasciospongia cavernosa marine sponges, on the apoptotic signalling pathway in three different human tumoral cells. Results were obtained by using DNA fragmentation, comet and viability assays, quantification of the mitochondrial transmembrane potential and Western blot. The T47D (human breast carcinoma), A431 (human epidermoid carcinoma), HeLa (human cervix carcinoma) and HCT116 (human colon carcinoma) cells were incubated for 24 h with scalaradial or cacospongionolide. Treatment of T47D cells with scalaradial or cacospongionolide for 24 h brought about a significant increase in DNA migration as well as fragmentation. Moreover, incubation of HCT116 and HeLa cells with scalaradial or cacospongionolide for 24 h caused an increased expression of pro-apoptotic proteins. Furthermore, scalaradial or cacospongionolide, added to HCT116 and HeLa cells overnight, induced a significant and concentration-dependent loss of mitochondrial transmembrane potential, an early apoptosis signalling event. These effects paralleled with those achieved with p50 and p65, NF-κB subunits, nuclear level. In conclusion, scalaradial and cacospongionolide, by determining human cancer cell apoptosis, may represent new promising compounds to inhibit cancer cell proliferation.

Synthesis and antiproliferative activity of some steroidal lactone compounds.

Using cholesterol as starting material, some steroidal lactone compounds with the structures of 3-substituted-6-oxo-7-oxa-B-homo-cholestane or 3-substituted-7-oxo-6-oxa-B-homo-cholestane were synthesized by oxidation, reduction, Baeyer-Villiger reaction and condensation reaction. The cytotoxicity of these compounds against MGC 7901 (human gastric carcinoma), HeLa (human cervical carcinoma) and SMMC 7404 (human liver carcinoma) cells was investigated. Our results showed that the synthesized compounds displayed a distinct cytotoxicity against these cancer cells. In particular, compounds 8 and 9 have similar cytotoxic capability as cisplatin does. The information obtained from the studies may be useful for the design of novel chemotherapeutic drugs.

Nakiterpiosin targets tubulin and triggers mitotic catastrophe in human cancer cells.

Agents that interfere with mitotic progression by perturbing microtubule dynamics are commonly used for cancer chemotherapy. Here, we identify nakiterpiosin as a novel antimitotic drug that targets microtubules. Nakiterpiosin induces mitotic arrest and triggers mitotic catastrophe in human cancer cells by impairing bipolar spindle assembly. At higher concentration, it alters the interphase microtubule network and suppresses microtubule dynamics. In the presence of nakiterpiosin, microtubules are no longer arranged in a centrosomal array and centrosome-mediated microtubule regrowth after cold depolymerization is inhibited. However, centrosome organization, the ultrastructure of Golgi stacks, and protein secretion are not affected, suggesting that the drug has minimal toxicity toward other cellular functions. Nakiterpiosin interacts directly with tubulin, inhibits microtubule polymerization in vitro, and decreases polymer mass in cells. Furthermore, it enhances tubulin acetylation and reduces viability of paclitaxel-resistant cancer cells. In conclusion, nakiterpiosin exerts antiproliferative activity by perturbing microtubule dynamics during mitosis that activates the spindle assembly checkpoint and triggers cell death. These findings suggest the potential use of nakiterpiosin as a chemotherapeutic agent.

Chemical and biological studies of nakiterpiosin and nakiterpiosinone.

Nakiterpiosin and nakiterpiosinone are two related C-nor-D-homosteroids isolated from the sponge Terpios hoshinota that show promise as anticancer agents. We have previously described the asymmetric synthesis and revision of the relative configuration of nakiterpiosin. We now provide detailed information on the stereochemical analysis that supports our structure revision and the synthesis of the originally proposed and revised nakiterpiosin. In addition, we herein describe a refined approach for the synthesis of nakiterpiosin, the first synthesis of nakiterpiosinone, and preliminary mechanistic studies of nakiterpiosin's action in mammalian cells. Cells treated with nakiterpiosin exhibit compromised formation of the primary cilium, an organelle that functions as an assembly point for components of the Hedgehog signal transduction pathway. We provide evidence that the biological effects exhibited by nakiterpiosin are mechanistically distinct from those of well-established antimitotic agents such as taxol. Nakiterpiosin may be useful as an anticancer agent in those tumors resistant to existing antimitotic agents and those dependent on Hedgehog pathway responses for growth.

New-D-homoandrost-4,6-diene derivatives as potent progesterone receptor antagonist.

The aim of this study was to synthesize three different D-homoandrostadiene derivatives (2-4) and study their biological activity. We carried out in vivo and in vitro experiments using female cycling mice, which were synchronized for estrus with luteinizing hormone-releasing hormone (LHRH) and injected with the steroidal compounds. It was also determined the binding of these compounds to the progesterone receptors (PR). Since these steroids have a new D-homoandrostandienone skeleton in their molecular structure, it was of interest also to study their binding to the androgen receptors (AR). After LHRH treatment, the mice of the control group showed the presence of 14+/-4 corpus lutea in the ovary whereas the animals treated with steroids 2-4, with RBAs of 100%, exhibited 11+/-7, 12+/-2, and 10+/-4 respectively. As a result of this study, it is evident that these steroids did not inhibit the ovulation in these animals. The uterus of the control group, showed the typical progestational activity with an enlarged endometrial thickness with a secretory activity. However, the endometrium of the mice treated with steroids 2-4 did not show an enlargement of the endometrium and no secretory activity could be detected. This fact indicates that compounds 2-4 had antagonistic activity in this tissue. The overall data show that steroids 2-4 are antagonists of the PR. However, they do not bind to the AR. These results also demonstrate that 2-4 have an antiprogestational activity in vivo, but do not decrease the number of corpus lutea in the ovary of mice treated with LHRH.

Scalaradial, a dialdehyde-containing marine metabolite that causes an unexpected noncovalent PLA2 Inactivation.

Several marine terpenoids that contain at least one reactive aldehyde group, such as manoalide and its congeners, possess interesting anti-inflammatory activities that are mediated by the covalent inactivation of secretory phospholipase A(2) (sPLA(2)). Scalaradial, a 1,4-dialdehyde marine terpenoid that was isolated from the sponge Cacospongia mollior, is endowed with a relevant anti-inflammatory profile, both in vitro and in vivo, through selective sPLA(2) inhibition. Due to its peculiar dialdehyde structural feature, it has been proposed that scalaradial exerts its enzymatic inactivation by means of an irreversible covalent modification of its target. In the context of our on-going research on anti-PLA(2) natural products and their interaction at a molecular level, we studied scalaradial in an attempt to shed more light on the molecular mechanism of its PLA(2) inhibition. A detailed analysis of the reaction profile between scalaradial and bee venom PLA(2), a model sPLA(2) that shares a high structural homology with the human synovial enzyme, was performed by a combination of spectroscopic techniques, chemical reactions (selective modifications, biomimetic reactions), and classical protein chemistry (such as proteolytic digestion, HPLC and mass spectrometry), along with molecular modeling studies. Unexpectedly, our data clearly indicated the noncovalent forces to be the leading event in the PLA(2) inactivation process; thus, the covalent modification of the enzyme emerges as only a minor side event in the ligand-enzyme interaction. The overall picture might be useful in the design of SLD analogues as new potential anti-inflammatory compounds that target sPLA(2) enzymes.

Group V phospholipase A2-derived lysophosphatidylcholine mediates cyclooxygenase-2 induction in lipopolysaccharide-stimulated macrophages.

Activation of macrophages and macrophage cell lines by bacterial LPS elicits a delayed phase of PG biosynthesis that appears to be entirely mediated by cyclooxygenase-2 (COX-2). In previous work, we found that a catalytically active group V secreted phospholipase A(2) (sPLA(2)-V) was required for COX-2 induction, but the nature of the sPLA(2)-V metabolite involved was not defined. In this study, we identify lysophosphatidylcholine (lysoPC) as the sPLA(2)-V downstream mediator involved in COX-2 induction by LPS-stimulated macrophages. Inhibition of sPLA(2)-V by RNA interference or by the cell-permeable compound scalaradial blocked LPS-induced COX-2 expression, and this inhibition was overcome by incubating the cells with a nonhydrolyzable lysoPC analog, but not by arachidonic acid or oleic acid. Moreover, inhibition of sPLA(2)-V by scalaradial also prevented the activation of the transcription factor c-Rel, and such an inhibition was also selectively overcome by the lysoPC analog. Collectively, these results support a model whereby sPLA(2)-V hydrolysis of phospholipids upon LPS stimulation results in lysoPC generation, which in turn regulates COX-2 expression by a mechanism involving the transcriptional activity of c-Rel.

Biphasic regulation of extracellular signal-regulated kinases by scalaradial, a secretory phospholipase A(2) inhibitor.

The marine natural product scalaradial (SLD) is a potent inhibitor of secretory phospholipase A(2) (sPLA(2)). Our previous work has demonstrated that SLD inhibits epidermal growth factor receptor-mediated Akt phosphorylation, and this effect is independent of sPLA(2). Here we report the role of SLD in extracellular signal-regulated kinase (ERK)1/2 activation. SLD inhibited ERK1/2 phosphorylation within the first 15 min (early inhibition), then stimulated ERK1/2 phosphorylation after 15 min of SLD treatment (late stimulation) in BEL-7402 cells, displaying biphasic regulatory features. Other PLA(2) inhibitors such as the cytosolic and Ca(2+)-independent PLA(2) inhibitor methyl arachidonyl fluorophosphonate, and another sPLA(2) inhibitor, thioetheramide-phosphatidylcholine, only transiently inhibited ERK1/2 phosphorylation and did not display the stimulatory effect. The early inhibition of ERK1/2 phosphorylation by SLD was reversed by the PLA(2) metabolite arachidonic acid, while the late stimulation was abrogated by constitutively active myristolated-Akt. Furthermore, SLD dose- and time-dependently inhibited the phosphorylation of Raf-1 on Ser 259, which is an established event by which Akt inhibits ERK1/2 activation. Taken together, these data demonstrate a biphasic regulation of ERK1/2 phosphorylation by SLD in a time-dependent manner, i.e., early inhibition and late stimulation. The early inhibition of ERK1/2 phosphorylation is mediated by sPLA(2), at least in part, and the late stimulation is effected through SLD inhibition of Akt. These findings provide further insight into the mechanisms underlying the pharmacological effect of SLD.

Scalaradial inhibition of epidermal growth factor receptor-mediated Akt phosphorylation is independent of secretory phospholipase A2.

The marine natural product 12-epi-scalaradial (SLD) is a specific secretory phospholipase A(2) (sPLA(2)) inhibitor. However, little is known about whether this compound has other pharmacological effects. Here, we revealed a novel effect of SLD on epidermal growth factor receptor (EGFR)-mediated Akt phosphorylation. SLD dose- and time-dependently inhibited epidermal growth factor (EGF)-stimulated Akt phosphorylation, which is required for Akt activation. SLD also blocked the EGF-stimulated membrane translocation of 3-phosphoinositide-dependent protein kinase 1 and inhibited phosphatidylinositol 3-kinase activity. This inhibition is specific for SLD because other phospholipase inhibitors, including sPLA(2) inhibitor thioetheramide-phosphatidylcholine, cytosolic PLA(2) inhibitor arachidonyl trifluoromethyl ketone, cytosolic PLA(2) and Ca(2+)-independent PLA(2) inhibitor methyl arachidonyl fluorophosphonate, phospholipase C inhibitor U73122, and cyclooxygenases inhibitor indomethacin, failed to inhibit EGF-stimulated Akt phosphorylation. Furthermore, arachidonic acid, the main sPLA(2)-catalyzed metabolite, was not able to rescue SLD inhibition of EGF-stimulated Akt phosphorylation. Overexpression of group IIA or group X sPLA(2) did not reverse the inhibitory effect of SLD on Akt phosphorylation, either. Our results demonstrate that SLD inhibits EGFR-mediated Akt phosphorylation, and this novel effect of SLD is independent of sPLA(2).

Synthesis, X-ray crystal structures and biological activity of 16-amino-17-substituted-D-homo steroid derivatives.

D-Homo derivatives in the androstane and estrane series, 12-19, were synthesized by a fragmentation-cyclization reaction of 16-oximino-17-hydroxy-17-substituted derivatives 3-9, or by cyclization of the corresponding D-seco derivatives 20-26. The structures were confirmed by X-ray analysis of compounds 12 and 16. Preliminary assessment of inhibitory effects of D-homo derivatives from androstane series towards aromatase, 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), 17 alpha-hydroxylase/C17-20 lyase (P450c17) and 17 beta-HSD indicated much lower inhibitory potential compared to previously tested activity of another type of D-modified steroids, namely D-seco derivatives. Also, assessment of potential antiestrogenic activity of derivatives from estrane series showed absence of such an activity.

Group IIA secretory phospholipase A(2) stimulates inducible nitric oxide synthase expression via ERK and NF-kappaB in macrophages.

The mammalian group IIA secretory phospholipase A(2) (sPLA(2)) is believed to play an important role in inflammation and cell injury. The present study underlines the importance of group IIA sPLA(2) in the regulation of iNOS. Treatment of cells with sPLA(2) induced protein expression and mRNA accumulation of iNOS in a dose-dependent manner. The pretreatment of cells with rho-BPB or SCA, selective sPLA(2) inhibitors, inhibited sPLA(2)-induced iNOS expression. sPLA(2) stimulated the simultaneous activation of two classes of mitogen-activated protein kinases ERK and JNK, but did not stimulate p38 MAPK. PD98059, a selective MEK inhibitor, inhibited sPLA(2)-induced nitrite production and iNOS expression as well as ERK phosphorylation. In addition, pretreatment of rho-BPB or SCA also resulted in inhibition of sPLA(2)-induced ERK phosphorylation. The sPLA(2) signaling mechanisms involving the activation of transcription factor NF-kappaB were studied in the same cells. That stimulation of cells with sPLA(2) caused NF-kappaB activation in a time-dependent manner was shown by the detection of NF-kappaB-specific DNA-protein binding and by IkappaBalpha degradation. sPLA(2)-induced NF-kappaB activation was prevented in the presence of rho-BPB. Furthermore, the NF-kappaB inhibitor PDTC suppressed sPLA(2)-induced nitrite production and iNOS expression as well as IkappaBalpha degradation. The results strongly suggest that group IIA sPLA(2) induces iNOS in macrophages and that this induction occurs through ERK and NF-kappaB.

Selective inhibitors of cytosolic or secretory phospholipase A2 block TNF-induced activation of transcription factor nuclear factor-kappa B and expression of ICAM-1.

TNF signaling mechanisms involved in activation of transcription factor NF-kappaB were studied in the human keratinocyte cell line HaCaT. We show that TNF-induced activation of NF-kappaB was inhibited by the well-known selective inhibitors of cytosolic phospholipase A2 (cPLA2): the trifluoromethyl ketone analogue of arachidonic acid (AACOCF3) and methyl arachidonyl fluorophosphate. The trifluoromethyl ketone analogue of eicosapentaenoic acid (EPACOCF3) also suppressed TNF-induced NF-kappaB activation and inhibited in vitro cPLA2 enzyme activity with a similar potency as AACOCF3. The arachidonyl methyl ketone analogue (AACOCH3) and the eicosapentanoyl analogue (EPACHOHCF3), which both failed to inhibit cPLA2 enzyme activity in vitro, had no effect on TNF-induced NF-kappaB activation. TNF-induced NF-kappaB activation was also strongly reduced in cells stimulated in the presence of the secretory PLA2 (sPLA2) inhibitors 12-epi-scalaradial and LY311727. Addition of excess arachidonic acid suppressed the inhibitory effect of 12-epi-scalaradial and LY311727. Moreover, both methyl arachidonyl fluorophosphate and 12-epi-scalaradial blocked TNF-mediated enhancement of expression of ICAM-1. Activation of NF-kappaB by IL-1beta was markedly less sensitive to both cPLA2 and sPLA2 inhibitors. The results indicate that both cPLA2 and sPLA2 may be involved in the TNF signal transduction pathway leading to nuclear translocation of NF-kappaB and to NF-kappaB-activated gene expression in HaCaT cells.