Antiangiogenic and antitumor activities of cyclooxygenase-2 inhibitors.

We provide evidence that cyclooxygenase (COX)-2-derived prostaglandins contribute to tumor growth by inducing newly formed blood vessels (neoangiogenesis) that sustain tumor cell viability and growth. COX-2 is expressed within human tumor neovasculature as well as in neoplastic cells present in human colon, breast, prostate, and lung cancer biopsy tissue. COX-1 is broadly distributed in normal, as well as in neoplastic, tissues. The contribution of COX-2 to human tumor growth was indicated by the ability of celecoxib, an agent that inhibits the COX-2 enzyme, to suppress growth of lung and colon tumors implanted into recipient mice. Mechanistically, celecoxib demonstrated a potent antiangiogenic activity. In a rat model of angiogenesis, we observe that corneal blood vessel formation is suppressed by celecoxib, but not by a COX-1 inhibitor. These and other data indicate that COX-2 and COX-2-derived prostaglandins may play a major role in development of cancer through numerous biochemical mechanisms, including stimulation of tumor cell growth and neovascularization. The ability of celecoxib to block angiogenesis and suppress tumor growth suggests a novel application of this anti-inflammatory drug in the treatment of human cancer.

Synthesis and activity of sulfonamide-substituted 4,5-diaryl thiazoles as selective cyclooxygenase-2 inhibitors.

A series of sulfonamide-substituted 4,5-diarylthiazoles was prepared via three synthetic routes as selective COX-2 inhibitors. Recently in the synthesis of selective COX-2 inhibitors we have discovered that the sulfonamide moiety is a suitable replacement for the methylsulfonyl moiety yielding compounds with activity both in vitro and in vivo.

Inhibitory effects of caffeic acid phenethyl ester on the activity and expression of cyclooxygenase-2 in human oral epithelial cells and in a rat model of inflammation.

We investigated the mechanisms by which caffeic acid phenethyl ester (CAPE), a phenolic antioxidant, inhibited the stimulation of prostaglandin (PG) synthesis in cultured human oral epithelial cells and in an animal model of acute inflammation. Treatment of cells with CAPE (2.5 microg/ml) suppressed phorbol ester (12-O-tetradecanoylphorbol-13-acetate; TPA) and calcium ionophore (A23187)-mediated induction of PGE2 synthesis. This relatively low concentration of CAPE did not affect amounts of cyclooxygenase (COX) enzymes. CAPE nonselectively inhibited the activities of baculovirus-expressed hCOX-1 and hCOX-2 enzymes. TPA- and A23187-stimulated release of arachidonic acid from membrane phospholipids was also suppressed by CAPE (4-8 microg/ml). Higher concentrations of CAPE (10-20 microg/ml) suppressed the induction of COX-2 mRNA and protein mediated by TPA. Transient transfections using human COX-2 promoter deletion constructs were performed; the effects of TPA and CAPE were localized to a 124-bp region of the COX-2 promoter. In the rat carrageenan air pouch model of inflammation, CAPE (10-100 mg/kg) caused dose-dependent suppression of PG synthesis. Amounts of COX-2 in the pouch were markedly suppressed by 100 mg/kg CAPE but were unaffected by indomethacin. These data are important for understanding the anticancer and anti-inflammatory properties of CAPE.

Pharmacological analysis of cyclooxygenase-1 in inflammation.

The enzymes cyclooxygenase-1 and cyclooxygenase-2 (COX-1 and COX-2) catalyze the conversion of arachidonic acid to prostaglandin (PG) H2, the precursor of PGs and thromboxane. These lipid mediators play important roles in inflammation and pain and in normal physiological functions. While there are abundant data indicating that the inducible isoform, COX-2, is important in inflammation and pain, the constitutively expressed isoform, COX-1, has also been suggested to play a role in inflammatory processes. To address the latter question pharmacologically, we used a highly selective COX-1 inhibitor, SC-560 (COX-1 IC50 = 0.009 microM; COX-2 IC50 = 6.3 microM). SC-560 inhibited COX-1-derived platelet thromboxane B2, gastric PGE2, and dermal PGE2 production, indicating that it was orally active, but did not inhibit COX-2-derived PGs in the lipopolysaccharide-induced rat air pouch. Therapeutic or prophylactic administration of SC-560 in the rat carrageenan footpad model did not affect acute inflammation or hyperalgesia at doses that markedly inhibited in vivo COX-1 activity. By contrast, celecoxib, a selective COX-2 inhibitor, was anti-inflammatory and analgesic in this model. Paradoxically, both SC-560 and celecoxib reduced paw PGs to equivalent levels. Increased levels of PGs were found in the cerebrospinal fluid after carrageenan injection and were markedly reduced by celecoxib, but were not affected by SC-560. These results suggest that, in addition to the role of peripherally produced PGs, there is a critical, centrally mediated neurological component to inflammatory pain that is mediated at least in part by COX-2.

Regulation of prostaglandin biosynthesis in vivo by glutathione.

Intraperitoneal administration of urate crystals to mice reduced subsequent macrophage conversion of arachidonic acid (AA) to prostaglandins (PGs) and 12-hydroxyeicosatetraenoic acid for up to 6 h. In contrast, levels of 12-hydroxyheptadecatrienoic acid (12-HHT) were markedly elevated. This metabolic profile was previously observed in vitro when recombinant cyclooxygenase (COX) enzymes were incubated with reduced glutathione (GSH). Analysis of peritoneal GSH levels revealed a fivefold elevation after urate crystal administration. The GSH synthesis inhibitor L-buthionine-[S,R]-sulfoximine partially reversed the urate crystal effect on both GSH elevation and PG synthesis. Moreover, addition of exogenous GSH to isolated peritoneal macrophages shifted AA metabolism from PGs to 12-HHT. Urate crystal administration reduced COX-1, but induced COX-2 expression in peritoneal cells. The reduction of COX-1 may contribute to the attenuation of PG synthesis after 1 and 2 h, but PG synthesis remained inhibited up to 6 h, when COX-2 levels were high. Overall, our results indicate that elevated GSH levels inhibit PG production in this model and provide in vivo evidence for the role of GSH in the regulation of PG biosynthesis.

Novel terphenyls as selective cyclooxygenase-2 inhibitors and orally active anti-inflammatory agents.

A novel series of terphenyl methyl sulfones and sulfonamides have been shown to be highly potent and selective cyclooxygenase-2 (COX-2) inhibitors. The sulfonamide analogs 17 and 21 were found to be much more potent COX-2 inhibitors and orally active anti-inflammatory agents than the corresponding methyl sulfone analogs 16 and 20, respectively, albeit with some decrease in COX-2 selectivity. Structure-activity relationship studies have determined that incorporation of two fluorine atoms in the central phenyl group, as in 20 and 21, is extremely advantageous for both in vitro COX-2 potency and selectivity as well as in vivo activity. Several noticeable examples in the 1,2-diaryl-4,5-difluorobenzenesulfonamide series are 21a-c,k,l,n (COX-2, IC50 = 0.002-0.004 microM), in which all have in vitro COX-1/COX-2 selectivity > 1000. In addition, sulfonamides 21a,b,d,g,j,m,n,q were shown to have greatly enhanced oral activity with more than 90% inhibition of prostaglandin E2 production in the air pouch model of inflammation. Furthermore, sulfonamide 21b was found to be very active in the rat adjuvant-induced arthritis model (ED50 = 0.05 mg/kg) and carrageenan-induced hyperalgesia assay (ED50 = 38.7 mg/kg) with no indication of gastrointestinal toxicity in rats at doses as high as 200 mg/kg.

Dual inhibition of nitric oxide and prostaglandin production contributes to the antiinflammatory properties of nitric oxide synthase inhibitors.

We have recently put forward the hypothesis that the dual inhibition of proinflammatory nitric oxide (NO) and prostaglandins (PG) may contribute to the antiinflammatory properties of nitric oxide synthase (NOS) inhibitors. This hypothesis was tested in the present study. A rapid inflammatory response characterized by edema, high levels of nitrites (NO2-, a breakdown product of NO), PG, and cellular infiltration into a fluid exudate was induced by the administration of carrageenan into the subcutaneous rat air pouch. The time course of the induction of inducible nitric oxide synthase (iNOS) protein in the pouch tissue was found to coincide with the production of NO2-. Dexamethasone inhibited both iNOS protein expression and NO2- synthesis in the fluid exudate (IC50 = 0.16 mg/kg). Oral administration of N-iminoethyl-L-lysine (L-NIL) or NG-nitro-L-arginine methyl ester (NO2Arg) not only blocked nitrite accumulation in the pouch fluid in a dose-dependent fashion but also attenuated the elevated release of PG. Finally, carrageenan administration produced a time-dependent increase in cellular infiltration into the pouch exudate that was inhibited by dexamethasone and NOS inhibitors. At early times, i.e., 6 h, the cellular infiltrate is composed primarily of neutrophils (98%). Pretreatment with colchicine reduced both neutrophil infiltration and leukotriene B4 accumulation in the air pouch by 98% but did not affect either NO2- or PG levels. In conclusion, the major findings of this paper are that (a) selective inhibitors of iNOS are clearly antiinflammatory agents by inhibiting not only NO but also PG and cellular infiltration and (b) that neutrophils are not responsible for high levels of NO and PG produced.

Poly(ADP-ribose) quantification at the femtomole level in mammalian cells.

ADP-ribose polymers were isolated from living mammalian cells, separated by polyacrylamide gel electrophoresis, visualized in the gel with a novel silver staining agent, and quantified by computer-aided scanning densitometry. This method detects as little as approximately 40 fmol of ADP-ribose polymers of a particular size class and reduces the gel exposure times required for conventionally radiolabeled polymers from 2 months to about 1 h. The method also detects polymers with slow turnover which may be underestimated by techniques requiring metabolic radiolabeling of poly(ADP-ribose).

Interactions of poly(ADP-ribose) with nuclear proteins.

The molecular mechanisms whereby poly(ADP-ribosyl)ation primes chromatin proteins for an active role in DNA excision repair are not understood. The prevalent view is that the covalent linkage of ADP-ribose polymers is essential for the modification of target protein function. By contrast, we have focused on the possibility that ADP-ribose polymers interact non-covalently with nuclear proteins and thereby modulate their function. The results show that ADP-ribose polymers engage in highly specific and strong non-covalent interactions with a small number of nuclear proteins, predominantly histones, and among these only with specific polypeptide domains. The binding affinities were largely determined by two factors, ie the polymer sizes and the presence of branches. This provides an explanation for the target specificity of the histone shuttle mechanism that was previously reported by our laboratory. Interestingly, the polymer molecules being most effective in protein targeting in vitro, are strictly regulated in mammalian cells during DNA repair in vivo.

In vivo glucocorticoids regulate cyclooxygenase-2 but not cyclooxygenase-1 in peritoneal macrophages.

Acute inflammatory stimuli elevate both the production of prostaglandins and the synthesis and activity of prostaglandin synthase/cyclooxygenase enzyme (COX) in murine peritoneal macrophages. Adrenalectomy also elevates prostaglandin production, COX synthesis and COX activity in these cells. We have utilized cDNA probes and antisera specific for the products of the prostaglandin synthase/cyclooxygenase-1 (COX-1) and TIS10/prostaglandin synthase-2/cyclooxygenase-2 (COX-2) genes to demonstrate that adrenalectomy causes elevation of mRNA and protein from the COX-2 gene, but not from the COX-1 gene, in peritoneal macrophages. Dexamethasone replacement suppressed the elevation of COX-2 mRNA message, COX-2 protein and the increased COX enzyme activity observed in adrenalectomized animals. In contrast, both COX-1 message and COX-1 protein levels were unaffected either by adrenalectomy or by dexamethasone administration. Thus, under normal physiological conditions, tonic glucocorticoid inhibition appears to play a major role in the in vivo regulation of the COX-2 gene. These data are consistent with COX-1 being the constitutive, housekeeping enzyme in macrophages in normal physiological conditions and with the enhanced prostaglandin synthesis seen after an inflammatory stimulus resulting from the rapid induction and activity of COX-2.

Selective inhibition of inducible cyclooxygenase 2 in vivo is antiinflammatory and nonulcerogenic.

We have examined the role of cyclooxygenase 2 (COX-2) in a model of inflammation in vivo. Carrageenan administration to the subcutaneous rat air pouch induces a rapid inflammatory response characterized by high levels of prostaglandins (PGs) and leukotrienes in the fluid exudate. The time course of the induction of COX-2 mRNA and protein coincided with the production of PGs in the pouch tissue and cellular infiltrate. Carrageenan-induced COX-2 immunoreactivity was localized to macrophages obtained from the fluid exudate as well as to the inner surface layer of cells within the pouch lining. Dexamethasone inhibited both COX-2 expression and PG synthesis in the fluid exudate but failed to inhibit PG synthesis in the stomach. Furthermore, NS-398, a selective COX-2 inhibitor, and indomethacin, a nonselective COX-1/COX-2 inhibitor, blocked proinflammatory PG synthesis in the air pouch. In contrast, only indomethacin blocked gastric PG and, additionally, produced gastric lesions. These results suggest that inhibitors of COX-2 are potent antiinflammatory agents which do not produce the typical side effects (e.g., gastric ulcers) associated with the nonselective, COX-1-directed antiinflammatory drugs.

Targeting of histone tails by poly(ADP-ribose).

After Zn2+ finger-mediated binding to a DNA break, poly(ADP-ribose) polymerase becomes automodified with long polymers of ADP-ribose. These nucleic acid-like polymers may facilitate DNA repair by noncovalently interacting with neighboring proteins. Using a novel screening technique, we have identified histones as the predominant poly(ADP-ribose)-binding species in human keratinocytes, rat hepatocytes, frog eggs, and yeast. Polymer binding is confined specifically to the histone domains responsible for DNA condensation, i.e. histone tails. Our results indicate that polymers of ADP-ribose are targeted to sites of DNA strand breaks by poly(ADP-ribose) polymerase and subsequently function to alter chromatin conformation through noncovalent interactions with histone tails.

Synthesis of poly(ADP-ribose)-agarose beads: an affinity resin for studying (ADP-ribose)n-protein interactions.

Polymers of ADP-ribose bind chromatosomal histones in solution and may play a role in chromatin accessibility in vivo. We have enzymatically synthesized a poly(ADP-ribose) affinity resin to further characterize binding of nuclear proteins to ADP-ribose polymers. NAD+- and (ADP-ribose)-derivatized agarose beads were recognized as polymer acceptors by the nuclear enzyme poly(ADP-ribose) polymerase. This polymerase elongated the existing ligands by successive addition of exogenously available ADP-ribose residues to form polymers covalently linked to the agarose beads. Poly(ADP-ribose) formation on the beads was dependent on incubation time and the mode of ligand attachment to the agarose. The resulting poly(ADP-ribose)-derivatized agarose beads possessed polymers which closely resembled those modifying the ADP-ribose polymerase by the automodification reaction. Fractionation of rat liver nuclear lysate over the poly(ADP-ribose) resin revealed a strong affinity of H1 for ADP-ribose polymers, thereby supporting a role for poly(ADP-ribose) in chromatin functions. Poly(ADP-ribose)-agarose beads are extremely stable and will be useful not only for affinity studies, but also for mechanistic studies involving polymer elongation and catabolism.

Endogenous glucocorticoids regulate an inducible cyclooxygenase enzyme.

The effect of endogenous glucocorticoids on the expression of the cyclooxygenase enzyme was studied by contrasting cyclooxygenase expression and prostanoid synthesis in adrenalectomized and sham-adrenalectomized mice with or without the concurrent administration of endotoxin. Peritoneal macrophages obtained from adrenalectomized mice showed a 2- to 3-fold induction in cyclooxygenase synthesis and activity when compared to sham controls. Intravenous injection of a sublethal dose of endotoxin (5 micrograms/kg) further stimulated cyclooxygenase synthesis, resulting in a 4-fold increase in prostaglandin production. Similar cyclooxygenase induction can be achieved in macrophages obtained from normal mice but only after high doses of endotoxin (2.5 mg/kg) that are 100% lethal to adrenalectomized mice. Restoration of glucocorticoids in adrenalectomized animals with dexamethasone completely inhibited the elevated cyclooxygenase and protected these animals from endotoxin-induced death. In contrast, no signs of cyclooxygenase induction were observed in the kidneys of the adrenalectomized mice, even when treated with endotoxin. Dexamethasone did not affect the constitutive cyclooxygenase activity and prostaglandin production present in normal and adrenalectomized kidneys. These data indicate the existence of a constitutive cyclooxygenase that is normally present in most cells and tissues and is unaffected by steroids and of an inducible cyclooxygenase that is expressed only in the context of inflammation by proinflammatory cells, like macrophages, and that is under glucocorticoid regulation. Under normal physiological conditions glucocorticoids maintain tonic inhibition of inducible cyclooxygenase expression. Depletion of glucocorticoids or the presence of an inflammatory stimulus such as endotoxin causes rapid induction of this enzyme, resulting in an exacerbated inflammatory response that is often lethal.

The alpha-glycosidic bonds of poly(ADP-ribose) are acid-labile.

The poly(ADP-ribosyl)ation system of higher eukaryotes produces multiple ADP-ribose polymers of distinct sizes which exhibit different binding affinities for histones. Although precipitation with trichloroacetic acid (TCA) is the standard procedure for isolation of poly(ADP-ribose) from biological material, we show here that poly(ADP-ribose) is not stable under acidic conditions. Storage of poly(ADP-ribose) as TCA pellets results in acid hydrolysis of polymers, the extent of which is dependent on storage time and temperature. The alpha-glycosidic, inter-residue bonds are the preferred sites of attack, thus reducing polymer sizes by integral numbers of ADP-ribose to yield artefactually more and smaller polymers than originally present. Therefore, poly(ADP-ribosyl)ation studies involving TCA precipitation, histone extraction with acids, or acidic incubations of ADP-ribose polymers must account for the impact of acids on resulting polymer populations.

The effects of probucol on the progression of atherosclerosis in mature Watanabe heritable hyperlipidaemic rabbits.

1. Probucol was administered to mature Watanabe heritable hyperlipidaemic (WHHL) rabbits (approximately 9 months old). Groups of WHHL rabbits were randomly selected and treated as follows: Group 1 killed at 9 months (n = 9); Group II placed on sham-treated diet at 9 months and followed for 6 months (n = 8); Group III placed on probucol at 9 months and followed for 6 months (n = 8). Probucol was administered by mixing 1% wt/wt drug with standard laboratory diet. 2. Plasma concentrations of probucol increased to 93 +/- 11 micrograms ml-1 in Group III during the initial 2 weeks and increased further to 149 +/- 24 micrograms ml-1 at the end of the treatment period. 3. Plasma concentrations of total cholesterol, unesterified cholesterol and phospholipids were significantly reduced overall by probucol, while triglycerides were not affected. 4. No statistically significant differences were observed in the presence of oxidized products in low density lipoproteins (LDL) isolated from plasma of controls compared to probucol-treated rabbits. However, LDL from probucol-treated animals was resistant to oxidation in the presence of Cu2+ (3 microM). 5. Group I had aortic atherosclerosis covering 70 +/- 5% of intimal area of thoracic aortae, that increased to 91 +/- 3% in Group II. This was associated with cholesterol contents of aortae increasing from 1.4 +/- 0.2 microgram mg-1 in Group I to 2.7 +/- 0.3 microgram mg-1 in Group II. Probucol administration did not produce a statistically significant reduction of atherosclerotic lesion area (78 +/- 7%). However, probucol treatment reduced cholesterol content to 1.9 + 0.3,ugmg-' (P < 0.01). Collagen content of aortae was not affected by probucol treatment. 6. Thus, while probucol did not promote regression, the drug did retard the continued deposition of cholesterol esters into atherosclerotic lesions of mature WHHL rabbits.

Selective regulation of cellular cyclooxygenase by dexamethasone and endotoxin in mice.

We have studied the effect of glucocorticoids administered in vivo on the activity and synthesis of the cyclooxygenase enzyme (COX) in mice treated with or without concurrent intravenous administration of LPS. Mouse peritoneal macrophages from LPS-treated animals showed a two to three fold increase in COX activity determined by the production of PGE2 and PGI2 after stimulation of the cells with exogenous arachidonate. Dexamethasone injected simultaneously with LPS, 12 h before killing of the animal and removal of the macrophages, completely blocked the LPS-induced increase COX activity in peritoneal macrophages. The regulation observed in COX activity by LPS and dexamethasone are due primarily to changes in COX mass as determined by immunoprecipitation of [35S]methionine endogenously labeled enzyme. In contrast, the COX present in the nonadherent cells and in renal medullary microsomes obtained from the same animals, showed no significant changes between treatments. These results indicate that LPS in vivo stimulates COX synthesis in the peritoneal macrophages but not in the kidney. The effect of dexamethasone to inhibit COX synthesis is selective to the LPS-induced enzyme.

Probucol attenuates the development of aortic atherosclerosis in cholesterol-fed rabbits.

1. Probucol was administered to rabbits fed a cholesterol-enriched (2% wt/wt) diet to determine potential anti-atherogenic effects in a preparation in which the disease process is due to elevated plasma concentrations of cholesterol ester-rich very low density lipoproteins (CER-VLDL). 2. Probucol was supplemented to the diet at 1% wt/wt which resulted in plasma concentrations rising steadily to 53 +/- 8 micrograms ml-1 after 14 days, with no significant changes during continued administration. Dietary consumption and body weight gains were comparable in the drug-treated and control groups during the observation period. 3. Probucol treatment did not significantly affect plasma concentrations of total cholesterol, unesterified cholesterol, triglycerides or phospholipids. 4. The concentration of CER-VLDL in plasma and its physicochemical characteristics were not significantly changed during administration of probucol. CER-VLDL from both control and probucol-treated animals was a potent stimulant of the augmentation of the intracellular incorporation of [3H]-oleate into cholesteryl-[3H]-oleate in cultured macrophages. 5. Despite the lack of effect of probucol on concentrations of plasma lipids and the cell interaction characteristics of CER-VLDL, administration of the drug markedly decreased the extent of intimal aortic surface area covered by grossly discernible atherosclerotic lesions from 55.6 +/- 11.8% to 11.6 +/- 1.9% in thoracic sections, and from 49.1 +/- 10.2% to 7.2 +/- 0.4% in abdominal sections. Furthermore, probucol treatment significantly reduced the deposition of total cholesterol in vascular tissue. 6. Probucol reduced the extent of aortic atherosclerosis produced by diet-induced hypercholesterolemia in rabbits. This reduction occurred in the absence of any significant change in the characteristics of plasma lipoproteins that were determined. These results indicate that either there is a role of oxidation in the disease process of this animal model of atherosclerosis or that probucol is acting via a presently undefined mechanism.

Isolation of low density lipoprotein from atherosclerotic vascular tissue of Watanabe heritable hyperlipidemic rabbits.

Atherogenic properties of low density lipoproteins (LDL) in vivo may reflect modification of lipoproteins associated with endothelial translocation and exposure to extracellular matrix and interstitial fluid. To examine whether modifications of LDL occur in vivo, lipoproteins were isolated from plasma and vascular tissue of Watanabe heritable hyperlipidemic (WHHL) rabbits. LDL was extracted from vascular tissue (LDL-V) by homogenization in the presence of a sodium carbonate buffer. Control experiments demonstrated that modifications did not occur under the preparative conditions used to release LDL from tissue. LDL-V contained less esterified cholesterol, but more cholesterol esters, than did LDL from plasma (LDL-P). The diameters of both LDL-V and LDL-P followed gaussian distributions, but LDL-V particles were smaller (20.3 +/- 0.1 and 26.3 +/- 0.1 nm). Mild lipid peroxidation was evident in LDL-V. The sphingomyelin content was increased in LDL-V, with less phosphatidylcholine than in LDL-P. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) indicated that apolipoprotein B was depleted in LDL-V, but Western blot analyses identified lower molecular weight proteins antigenically related to apolipoprotein B. LDL-V markedly stimulated cholesterol esterification in mouse peritoneal macrophages and also in rabbit alveolar macrophages, a cell type that did not respond to acetylated LDL. LDL-V was not recognized by cultured rabbit skin fibroblasts. Thus, LDL isolated from atherosclerotic vascular tissue in vivo was modified in a fashion that could confer atherogenic properties reflected by augmentation of cholesterol esterification in macrophages in vitro.