Mechanistic evaluation of a novel cyclohexenone derivative's functionality against nociception and inflammation: An in-vitro, in-vivo and in-silico approach.

The synthesis of a novel cyclohexanone derivative (CHD; Ethyl 6-(4-metohxyphenyl)-2-oxo-4-phenylcyclohexe-3-enecarboxylate) was described and the subsequent aim was to perform an in vitro, in vivo and in silico pharmacological evaluation as a putative anti-nociceptive and anti-inflammatory agent in mice. Initial in vitro studies revealed that CHD inhibited both cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX) enzymes and it also reduced mRNA expression of COX-2 and the pro-inflammatory cytokines TNF-α and IL-1ß. It was then shown that CHD dose dependently inhibited chemically induced tonic nociception in the abdominal constriction assay and also phasic thermal nociception (i.e. anti-nociception) in the hot plate and tail immersion tests in comparison with aspirin and tramadol respectively. The thermal test outcomes indicated a possible moderate centrally mediated anti-nociception which, in the case of the hot plate test, was pentylenetetrazole (PTZ) and naloxone reversible, implicating GABAergic and opioidergic mechanisms. CHD was also effective against both the neurogenic and inflammatory mediator phases induced in the formalin test and it also disclosed anti-inflammatory activity against the phlogistic agents, carrageenan, serotonin, histamine and xylene compared with standard drugs in edema volume tests. In silico studies indicated that CHD possessed preferential affinity for GABAA, opioid and COX-2 target sites and this was supported by molecular dynamic simulations where computation of free energy of binding also favored the formation of stable complexes with these sites. These findings suggest that CHD has prospective anti-nociceptive and anti-inflammatory properties, probably mediated through GABAergic and opioidergic interactions supplemented by COX-2 and 5-LOX enzyme inhibition in addition to reducing pro-inflammatory cytokine expression. CHD may therefore possess potentially beneficial therapeutic effectiveness in the management of inflammation and pain.

Design, synthesis, and biological evaluation of new pyrazoloquinazoline derivatives as dual COX-2/5-LOX inhibitors.

A new series of pyrazoloquinazoline derivatives equipped with different chalcones was designed, synthesized, and identified through 1 H nuclear magnetic resonance (NMR), 13 C NMR, and infrared spectroscopic techniques. Our design strategy of the quinazolinone-privileged scaffold as a new scaffold was based on merging pharmacophores previously reported to exhibit cyclooxygenase-2 (COX-2)/5-lipoxygenase (5-LOX) inhibitory activity. All the newly synthesized derivatives were biologically evaluated for COX and 5-LOX inhibitory activity and COX-2 selectivity, using celecoxib and zileuton as reference drugs, as they exhibited promising anti-inflammatory activity. Compound 3j was found to be the most promising derivative, with IC50 values of 667 and 47 nM against COX-1 and COX-2, respectively, which are superior to that of celecoxib (IC50 value against COX-2 = 95 nM), showing an SI of 14.2 that was much better than celecoxib. Compounds 3f and 3h exhibited COX-1 inhibition, with IC50 values of 1,485 and 684 nM, respectively. The synthesized compounds showed a significant inhibitory activity against 5-LOX, with IC50 values ranging from 0.6 to 4.3 µM, where compounds 3f and 3h were found to be the most potent derivatives, with IC50 values of 0.6 and 1.0 µM, respectively, in comparison with that of zileuton (IC50 = 0.8 µM). These promising derivatives, 3f, 3h, and 3j, were further investigated in vivo for anti-inflammatory, gastric ulcerogenic effects, and prostaglandin production (PGE2) in rat serum. The molecular docking studies concerning the binding sites of COX-2 and 5-LOX revealed similar orientation, compared with reported inhibitors, which encouraged us to design new leads targeting COX-2 and 5-LOX as dual inhibitors, as a new avenue in anti-inflammatory therapy.

Diclofenac conjugates with biocides through silver(I) ions (CoMeD's); Development of a reliable model for the prediction of anti-proliferation of NSAID's-silver formulations.

The conjugation of diclofenac (DICLH), a Non-Steroidal Anti-inflammatory Drug (NSAID), with biocides such as dimethyl sulfoxide (DMSO) and triphenylphosphine (TPP), through silver(I) ions, results into the chemical [Agn(DICL)n(L)m]k (L = DMSO and n = 2, m = 2, k = infinite (1); L = TPP and n = 1, m = 2, k = 1 (2)). The compounds were characterized by m.p., FT-IR, UV-vis and 1H NMR spectroscopic techniques. The crystal and molecular structures of 1-2 were determined by X-ray crystallography. The in vitro cytotoxic activity of 1-2 against the human breast adenocarcinoma cancer cells MCF-7 (hormone dependent) and MDA-MB-231 (hormone independent) reveals that the 1 inhibits the MCF-7 rather than the MDA-MB-231 cells, suggesting hormone mimetic behaviour. Compound 2 inhibits both cancerous cell lines, stronger than cisplatin. Both compounds inhibit MCF-7 cells migration. Compounds 1-2, exhibit, lower toxicity against fetal lung fibroblast (MRC-5) cells than cisplatin. Their genotoxicity was evaluated on MRC-5 cells. The molecular mechanism of 1-2 against MCF-7 cells was clarified by (i) their cell cycle arrest study (ii) their mitochondrial membrane permeability (iii) their binding affinity towards Calf Thymus (CT)-DNA and (iv) their inhibitory activity against the enzyme lipoxygenase (LOX). Regression analysis of the data obtained for [Ag(NSAID)(Ar3P)m] (NSAID = p­hydroxy­benzoic acid (p-HO-BZAH), salicylic acid (SALH2), aspirin (ASPH), naproxen (NAPRH), nimesulide (NIMH); L = TPP, Tri(p­tolyl)phosphine (TPTP), Tri(o­tolyl)phosphine (TOTP), Tri(m­tolyl)phosphine (TMTP); m = 2 or 3) and [Ag(DICL)2(DMSO)2]k (k = infinite) was performed. Considering the biological results (IC50) as dependent variable a theoretical equation is obtained for these compounds. The calculated IC50 values are compared satisfactorily with the corresponding experimental inhibitory activity of the complexes.

CarbORev-5901: The First Carborane-Based Inhibitor of the 5-Lipoxygenase Pathway.

The progression of cancer is accelerated by increased proliferation, angiogenesis, and inflammation. These processes are mediated by leukotrienes. Several cancer cell lines overexpress 5-lipoxygenase, an enzyme that converts arachidonic acid into leukotrienes. An early inhibitor of the 5-lipoxygenase pathway is Rev-5901, which, however, lacks in in vivo efficacy, as it is rapidly metabolized. We investigated the introduction of carboranes as highly hydrophobic and metabolically stable pharmacophores into lipoxygenase inhibitors. Carboranes are icosahedral boron clusters that are remarkably stable and used to increase the metabolic stability of unstable pharmaceutics without changing their biological activity. By introduction of meta-carborane into Rev-5901, the first carborane-based inhibitor of the 5-lipoxygenase pathway was obtained. We report the synthesis and inhibitory and cytotoxic behavior of these compounds toward several melanoma and colon cancer cell lines and their related anticancer mechanisms.

Inhibition of 12/15-LO ameliorates CVB3-induced myocarditis by activating Nrf2.

Cardiac 12/15-lipoxygenase (12/15-LO) was reported to be markedly up-regulated and involved in the development of heart failure. Nuclear factor E2-related factor 2 (Nrf2) plays anti-inflammatory and anti-oxidation roles in response to oxidative stress. However, the role of 12/15-LO in viral myocarditis (VMC) and its underlying molecular mechanism have not yet been elucidated. Here, we demonstrated that 12/15-LO was up-regulated and Nrf2 was down-regulated in coxsackievirus B3 (CVB3)-infected mice and cardiac myocytes. Baicalein, the specific inhibitor of 12/15-LO, was employed to investigate the role of 12/15-LO and its underlying mechanism in VMC. We found that baicalein treatment alleviated CVB3-induced VMC mouse models, as demonstrated by less inflammatory lesions in the heart tissues and less CK-MB level. Moreover, baicalein treatment attenuated CVB3-induced inflammatory cytokine production and oxidative stress. Mechanistic analysis suggested that baicalein treatment relieved CVB3-induced reduction of Nrf2 and heme oxygenase-1 (HO-1) expressions. Taken together, our study indicated that inhibition of 12/15-LO ameliorates VMC by activating Nrf2, providing a new therapeutic strategy for the therapy of VMC.

Development of novel aminothiazole-comprising 5-LO inhibitors.

BACKGROUND: Leukotrienes are pivotal lipid mediators in various immune and inflammatory reactions. Herein, 5-LO is a validated target. 2-Aminothiazoles, as a privileged structure, implicate known 5-LO inhibitors like ST-1083 (IC50 [polymorphonuclear leukocytes (PMNL)] = 0.68 µM), yet deep structure-activity relationships (SAR) have not been established. MATERIALS & METHODS: Compounds were synthesized via Hantzsch thiazole synthesis. Inhibitory activities were evaluated using intact PMNL and purified 5-LO together with cytotoxicity measurements in U937 cells. RESULTS: We introduced novel functionalities at 2-, 3-, 4- and 5-position of the 2-aminothiazole scaffold and conducted bioisosteric replacement to optimize the parent scaffold. SARs of the 2-aminothiazole scaffold were deduced and extended primarily for inhibition of the 5-LO enzyme. CONCLUSION: SAR studies provided at least two optimized leads (ST-1853, ST-1906) with high potency (IC50 [polymorphonuclear leukocytes] = 0.05 µM), specificity and noncytotoxic behavior.

Triblock Conjugates: Identification of a Highly Potent Antiinflammatory Agent.

Rationally designed conjugates of chrysin, indole, and barbituric acid were synthesized and screened for their antiinflammatory activities through in vitro and in vivo experiments. Improved over the previously reported chrysin-indole-pyrazole conjugates and also in comparison to the chrysin, indole, and barbituric acid based COX-2 inhibitors, the new compounds have displayed significantly better IC50 for COX-2 and some of them also exhibited inhibition of 5-LOX enzyme. For one of the test compounds, IC50 for COX-2 and 5-LOX was 1 and 1.5 nM, respectively. Investigations of Swiss Albino mice through capsaicin induced paw lickings and dextran induced inflammation showed that these compounds possess appreciable analgesic and antiinflammatory activities. Ki, Ka, and ΔG for the enzyme-compound interaction were calculated and found to be in agreement with the biological data. The experimental results were supported by the molecular docking studies of the compounds in the active site of COX-2 and 5-LOX. Overall, a highly promising antiinflammatory agent was identified.

α-Glucosidase inhibition, 15-lipoxygenase inhibition, and brine shrimp toxicity of extracts and isolated compounds from Terminalia macroptera leaves.

CONTEXT: Terminalia macroptera Guill. & Perr. (Combretaceae), a tree that grows in West Africa, has been used in traditional medicine against a variety of diseases such as hepatitis, gonorrhea, skin diseases, and diabetes. OBJECTIVE: To investigate enzyme inhibitory activity against α-glucosidase and 15-lipoxygenase (15-LO) and toxicity against brine shrimp of extracts and compounds from T. macroptera leaves. MATERIALS AND METHODS: Methanol extract, ethyl acetate, and butanol extracts obtained from the methanol extract, six isolated polyphenols (chebulagic acid, chebulic acid trimethyl ester, corilagin, methyl gallate, narcissin, and rutin), and shikimic acid were evaluated for enzyme inhibition and toxicity. RESULTS: In enzyme inhibition assays, all extracts showed high or very high activity. Chebulagic acid showed an IC50 value of 0.05 µM towards α-glucosidase and 24.9 ± 0.4 µM towards 15-LO, in contrast to positive controls (acarbose: IC50 201 ± 28 µM towards α-glucosidase, quercetin: 93 ± 3 µM towards 15-LO). Corilagin and narcissin were good 15-LO and α-glucosidase inhibitors, as well, while shikimic acid, methyl gallate, and chebulic acid trimethyl ester were less active or inactive. Rutin was a good α-glucosidase inhibitor (IC50 ca. 3 µM), but less active towards 15-LO. None of the extracts or the isolated compounds seemed to be very toxic in the brine shrimp assay compared with the positive control podophyllotoxin. CONCLUSION: Inhibition of α-glucosidase in the gastrointestinal tract may be a rationale for the medicinal use of T. macroptera leaves against diabetes in traditional medicine in Mali. The plant extracts and its constituents show strong inhibition of the peroxidative enzyme 15-LO.

Structure-activity relationship and in vitro pharmacological evaluation of imidazo[1,2-a]pyridine-based inhibitors of 5-LO.

BACKGROUND: 5-LO is an important enzyme involved in the biosynthesis of leukotrienes, which are lipid mediators of immune and inflammation responses, with important roles in respiratory disease, cardiovascular disease, immune responses and certain types of cancer. Therefore, this enzyme has been investigated as a potential target for the treatment of these pathophysiological conditions. RESULTS: 5-LO inhibitory potential was investigated in intact polymorphonuclear leukocytes, a cell-free assay, in human whole blood and rodent cells to both elucidate structure-activity relationships and in vitro pharmacological evaluation. Chemical modifications for lead optimization via straight forward synthesis was used to combine small polar groups, which led to a suitable candidate (IC50 [polymorphonuclear leukocytes] = 1.15 µM, IC50 [S100] = 0.29 µM) with desired in vitro biopharmaceutical profiles in terms of solubility (451.9 µg/ml) and intrinsic clearance without demonstrating any cytotoxicity. CONCLUSION: Compound 9l is a novel, potent and selective 5-LO inhibitor with favorable preclinical drug-like properties.

Pro-apoptotic effects of tepoxalin, a cyclooxygenase/lipoxygenase dual inhibitor, on canine synovial fibroblasts.

Canine osteoarthritis occurs frequently and causes secondary synovitis. Administration of nonsteroidal anti-inflammatory drugs (NSAIDs) is one of the major therapeutic options for pain management of joint diseases. Tepoxalin has an unique property as an NSAIDs that suppresses both cyclooxygenase and lipoxygenase. The purpose of this study was to evaluate antiproliferative effects of tepoxalin on cultured canine synovial cells. Cytotoxic effects of tepoxalin, carprofen, meloxicam and AA-861 on cultured canine synoviocytes were evaluated by MTT colorimetric assay. Apoptosis was detected by morphological observations with Giemsa or annexin V/Hoechst 33342 staining and by the inhibition of caspase-3 activity with N-Ac-Asp-Glu-Val-Asp-CHO (Ac-DEVD-CHO). Cytotoxic effects of tepoxalin were evident in comparison with the effects of carprofen or meloxicam. The same tendency of cytotoxicity was observed when 5-lipoxygenase was inhibited by AA-861. The morphological findings and contradictory effects of Ac-DEVD-CHO with regard to the cytotoxicity proved the proapoptotic effects of tepoxalin. In conclusion, tepoxalin might control osteoarthritic synovitis by inducing apoptosis in proliferating synoviocytes, while most NSAIDs that selectively inhibit cyclooxygenase-2 most likely would not suppress synovial proliferation.

Leukotriene synthesis inhibitor decreases vasopressin release in the early phase of sepsis.

The aim was to analyze the effect of leukotriene synthesis inhibitor administered intraperitoneally in vasopressin release during sepsis. Male Wistar rats received injections of MK-886 (1.0, 2.0 or 4.0 mg/kg) or vehicle (DMSO 5%) 1 h before cecal ligation and puncture. There was some variation on the survival rate depending on the dose used but the drug did not modify the hematocrit, osmolality, serum sodium and nitrate, plasma protein, and neutrophil recruitment, in any dose. Nevertheless, vasopressin (AVP) release decreased in a dose-response manner in the early phase of sepsis. These results support the suggestion that leukotrienes (LTs) are involved in AVP release during sepsis.

Multiple factors govern the association between pharmacology and toxicity in a class of drugs: toward a unification of class effect terminology.

The term class effect has gained in use to describe a side effect including toxicity common to a series of drugs. There is no definition of what constitutes a class effect, and it is not applied against a rigid set of criteria.Thus, the finding of toxicity in one of a series of drugs can raise the concern of a class effect, especially if one or more of the others shows findings even slightly related or at very much lower incidence. This is particularly problematic when the term is used loosely or speculatively on initial events that are themselves of low incidence and serious. This speculation exaggerates and distorts the scientific process in establishing the true benefit risk of the individual drugs and can lead to lengthy development times, or highly restrictive labeling, to the detriment of patient welfare. To provide better definition and application of the term, we suggest that the term class effect toxicity is only used when a clear mechanistic link has been established between a safety concern and drug class based on (I) where the primary pharmacology delivers a clear rationale for the observed findings and toxicities; and (II) where the secondary pharmacology is obligate to the class of the molecule and not subject to variation of structure, and the selectivity cannot be impacted significantly by variations in potency introduced by structural manipulation. With these categorizations, we believe class effect toxicity will be mainly confined to I with examples such as the tetracycline class of antibacterials which inhibit protein synthesis both as a mechanism of antibacterial activity and to produce hepatic injury by mitochondrial injury in the liver.

The sesquiterpene caulerpenyne from Caulerpa spp. is a lipoxygenase inhibitor.

Many algae contain secondary metabolites with the potential to gain importance as pharmaceutically active secretions. Caulerpa racemosa var. cylindracea and Caulerpa prolifera are very abundant on the Mediterranean coastlines. The methanolic extracts of C. racemosa and C. prolifera were tested for inhibitory effects on soybean lipoxygenase. The extract of C. prolifera showed potent inhibitory effect in a lipoxygenase enzyme activity assay. HPLC comparison revealed that C. racemosa extract contained less caulerpenyne, the major secondary metabolite of both algae. In accordance with these findings, purified caulerpenyne inhibited lipoxygenase with an IC(50) of 5.1 µM. The enzyme kinetic studies indicated that both K (M) and V (max) decreased from 0.041 to 0.019 mM and 312.5 to 151.5 U mL(-1) in the presence of 5 µM caulerpenyne, revealing an un-competitive type of inhibition kinetics. The major secondary metabolite of Caulerpa species, caulerpenyne, is thus a novel lipoxygenase inhibitor that can be easily obtained in high quantities from the abundant algae.

The anti-inflammatory effects of ZLJ-6, a novel dual cyclooxygenase/5-lipoxygenase inhibitor.

Compound ZLJ-6 [(Z)-1-methyl-1,5-dihydro-2-amino-5-[4-(mesyl)benzylidene]-4H-imi-dazole-4-one mesilate] is a potent inhibitor of cyclooxygenase (IC(50)=0.73 and 0.31 microM, for cyclooxygenase-1 and cyclooxygenase-2 respectively) in human whole blood. It also inhibited the production of thromboxane B(2) and prostaglandin E(2) in calcium ionophore A23187-induced human (IC(50)=0.50 microM) and rat whole blood (IC(50)=0.93 microM), and rat peritoneal leukocytes (IC(50)=2.27 microM). ZLJ-6 suppressed the activity of 5-lipoxygenase in the rat basophilic leukemia (RBL-1) cell lysate (IC(50)=0.32 microM) and in intact cells (IC(50)=1.06 microM) and reduced the generation of leukotriene B(4) (LTB(4)) in A23187-stimulated human (IC(50)=1.61 microM) or rat whole blood (IC(50)=0.99 microM), and rat peritoneal leukocytes (IC(50)=2.59 microM). In vivo, ZLJ-6, administered orally, demonstrated potent anti-inflammatory activity in the carrageenin-induced paw oedema model in rats and showed analgesic activity in the acetic acid-induced abdominal construction model in mice. No gastrointestinal ulcers were found with the anti-inflammatory dose (30 mg/kg) in normal rats. These results indicated that ZLJ-6 potently inhibited 5-lipoxygenase and cyclooxygenase, and blocked the production of LTB(4), TXB(2) and PGE(2). Thus ZLJ-6 is an ideal substitute for classical non-steroidal anti-inflammatory therapy.

Anti-inflammatory activity of four Bolivian Baccharis species (Compositae).

Hexanic, dichloromethanic, ethanolic and aqueous extracts from Baccharis obtusifolia HBK, Baccharis latifolia (R. et P.) Pers., Baccharis pentlandii D.C. and Baccharis subulata Wedd., plants used in the traditional medicine of South America have been studied for their in vitro anti-inflammatory activity in cellular systems. Calcium ionophore A23187-stimulated mouse peritoneal macrophages were validated as a source of cyclooxygenase-1 (COX-1) (prostaglandin E2, PGE2) and 5-lipoxygenase (5-LOX) (leukotriene C4, LTC4), and mouse peritoneal macrophages stimulated with Escherichia coli lipopolysaccharide (LPS) were used for testing cyclooxygenase-2 (COX-2) (PGE2), nitric oxide (NO) and tumour necrosis factor-alpha (TNF-alpha) activity. Most of the extracts tested were active in all assays.

Optimization of imidazole 5-lipoxygenase inhibitors and selection and synthesis of a development candidate.

Structural modification of imidazole 5-lipoxygenase (5-LO) inhibitors for optimizing inhibitory potency, pharmacokinetic behavior and toxicity (ocular) profile led to 4-{3-[4-(2-methyl-1H-imidazol-1-yl)phenylthio]}phenyl-3,4,5,6-tetrahydro-2H-pyran-4-carboxamide (6) with no observable ocular toxicity. The orally active and safe imidazole 5-LO inhibitor 6 was selected as a clinical candidate and advanced to clinical studies. An improved synthesis of 6 is also discussed.

Relative contribution of acetylated cyclo-oxygenase (COX)-2 and 5-lipooxygenase (LOX) in regulating gastric mucosal integrity and adaptation to aspirin.

In addition to inhibiting formation of prothrombotic eicosanoids, aspirin causes the acetylation of cyclooxygenase (COX)-2. The acetylated COX-2 remains active, and upon cell activation, initiates the generation of 15R-HETE, a lipid substrate for 5-lipoxygenase (LOX) leading to the formation of 15-epi-LXA4 (also termed "aspirin-triggered lipoxin," or ATL). Because ATL potently inhibits polymorphonuclear cell (PMN) function, we assessed the relative contribution of this lipid mediator in conjunction with another 5-LOX product, the leukotriene (LT)B4, to the pathogenesis of acute damage and gastric adaptation to aspirin. Data presented herein indicate that acute injury and gastric adaptation to aspirin is associated with ATL generation. Administration of COX inhibitors (celecoxib, indomethacin, ketoprofen) to aspirin-treated rats exacerbated acute injury and abolished adaptation to aspirin. Moreover, it inhibited ATL formation and caused a four- to fivefold increase in LTB4 synthesis. In contrast, licofelone, a COX/5-LOX inhibitor, did not exacerbate acute gastric injury nor did it interfere with gastric adaptation to aspirin. Although licofelone blocked ATL and LTB4 formation in aspirin-treated rats, it attenuated aspirin-induced gastric PMN margination. These findings indicate that the balance between the production of LTB4 and ATL modulates PMN recruitment/function and gastric mucosal responses to aspirin.

General pharmacology of the butanamide derivative S 19812, a new dual inhibitor of cyclooxygenase and lipoxygenase pathways.

S 19812 (N-hydroxy-N-methyl-4-(2,3-bis-(4-methoxyphenyl)-thiophen-5-yl) butanamide, CAS 181308-68-9), a dual inhibitor of cyclooxygenase and lipoxygenase pathways, was evaluated in different models of pain and inflammation. Its gastric tolerance was also investigated. After acute oral treatment S 19812 exhibited a non-opioid analgesic activity observed in the phenylbenzoquinone-induced writhing model in mice (ED50 = 2.1 mg/kg) and in the carrageenan-induced hyperalgesia model in rats (ED50 = 9.1 mg/kg, preventive treatment; 8.3 mg/kg, curative treatment). Anti-inflammatory activity was observed in the adjuvant-induced arthritis in rat (inhibition of edema ED50 = 11 mg/kg/day p.o., day 28). In rats and mice, S 19812 exhibited an excellent gastric tolerance at doses up to 800 mg/kg p.o.

Effect of nordihydroguaiaretic acid on intracellular Ca(2+) concentrations in hepatocytes.

The effect of nordihydroguaiaretic acid (NDGA) on Ca(2+) signaling in human hepatoma cells (HA22/VGH) has been investigated. NDGA (5-50 microM) increased [Ca(2+)](i) concentration-dependently. The [Ca(2+)](i) increase comprised an initial rise and an elevated phase over a time period of 4 min. Removal of extracellular Ca(2+) reduced 10-50 microM NDGA induced [Ca(2+)](i) signals by 45+/-5%. Consistently, the 50 microM NDGA-induced [Ca(2+)](i) increase in Ca(2+)-containing medium was reduced by 41+/-2% by 10 microM of La(3+), nifedipine or verapamil. In Ca(2+)-free medium, pretreatment with 20 microM NDGA for 6 min abolished the [Ca(2+)](i) increase induced by the endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin (1 microM). Conversely, 20 microM NDGA failed to increase [Ca(2+)](i) after 1 microM thapsigargin had depleted the endoplasmic reticulum Ca(2+) store. Inhibition of phospholipase C with 2 microM U73122 had little effect on 20 microM NDGA-induced Ca(2+) release. Several other lipoxygenase inhibitors had no effect on basal [Ca(2+)](i). Together, the data suggest that NDGA increased [Ca(2+)](i) in hepatocytes in a lipoxygenase-independent manner, by releasing Ca(2+) from the endoplasmic reticulum and causing Ca(2+) influx.

Inhibition of arachidonate 5-lipoxygenase triggers massive apoptosis in human prostate cancer cells.

Diets high in fat are associated with an increased risk of prostate cancer, although the molecular mechanism is still unknown. We have previously reported that arachidonic acid, an omega-6 fatty acid common in the Western diet, stimulates proliferation of prostate cancer cells through production of the 5-lipoxygenase metabolite, 5-HETE (5-hydroxyeicosatetraenoic acid). We now show that 5-HETE is also a potent survival factor for human prostate cancer cells. These cells constitutively produce 5-HETE in serum-free medium with no added stimulus. Exogenous arachidonate markedly increases the production of 5-HETE. Inhibition of 5-lipoxygenase by MK886 completely blocks 5-HETE production and induces massive apoptosis in both hormone-responsive (LNCaP) and -nonresponsive (PC3) human prostate cancer cells. This cell death is very rapid: cells treated with MK886 showed mitochondrial permeability transition between 30 and 60 min, externalization of phosphatidylserine within 2 hr, and degradation of DNA to nucleosomal subunits beginning within 2-4 hr posttreatment. Cell death was effectively blocked by the thiol antioxidant, N-acetyl-L-cysteine, but not by androgen, a powerful survival factor for prostate cancer cells. Apoptosis was specific for 5-lipoxygenase-programmed cell death was not observed with inhibitors of 12-lipoxygenase, cyclooxygenase, or cytochrome P450 pathways of arachidonic acid metabolism. Exogenous 5-HETE protects these cells from apoptosis induced by 5-lipoxygenase inhibitors, confirming a critical role of 5-lipoxygenase activity in the survival of these cells. These findings provide a possible molecular mechanism by which dietary fat may influence the progression of prostate cancer.