Amiram Raz

Perioperative Use of β-blockers and COX-2 Inhibitors May Improve Immune Competence and Reduce the Risk of Tumor Metastasis

BackgroundCOX inhibitors and β-blockers were recently suggested to reduce cancer progression through inhibition of tumor proliferation and growth factor secretion, induction of tumor apoptosis, and prevention of cellular immune suppression during the critical perioperative period. Here we evaluated the perioperative impact of clinically applicable drugs from these categories in the context of surgery, studying natural killer (NK) cell activity and resistance to experimental metastases.MethodsF344 rats were treated with COX-1 inhibitors (SC560), COX-2 inhibitors (indomethacin, etodolac, or celecoxib), a β-blocker (propranolol), or a combination of a COX-2 inhibitor and a β-blocker (etodolac and propranolol). Rats underwent laparotomy, and were inoculated intravenously with syngeneic MADB106 tumor cells for the assessment of lung tumor retention (LTR). Additionally, the impact of these drug regimens on postoperative levels of NK cytotoxicity was studied in peripheral blood and marginating-pulmonary leukocytes.ResultsSurgery increased MADB106 LTR. COX-2 inhibition, but not COX-1 inhibition, reduced postoperative LTR. Etodolac and propranolol both attenuated the deleterious impact of surgery, and their combined use abolished it. Surgery decreased NK cytotoxicity per NK cell in both immune compartments, and only the combination of etodolac and propranolol significantly attenuated these effects. Lastly, the initiation of drug treatment three days prior to surgery yielded the same beneficial effects as a single pre-operative administration, but, as discussed, prolonged treatment may be more advantageous clinically.ConclusionsExcess prostaglandin and catecholamine release contributes to postoperative immune-suppression. Treatment combining perioperative COX-2 inhibition and β-blockade is practical in operated cancer patients, and our study suggests potential immunological and clinical benefits.

Platelet and blood vessel arachidonate metabolism and interactions.

Exogenous arachidonate addition to intact platelets, in the absence or the presence of blood vessel microsomes, results in the production of thromboxane B(2) (the stable degradation product of thromboxane A(2)) only. Prostaglandin (PG) endoperoxides are released from intact platelets only when thromboxane synthetase is inhibited. Thus, addition of exogenous arachidonate to imidazole-pretreated platelets in the presence of bovine aorta microsomes (source of prostacyclin synthetase) results predominantly in the synthesis of 6-keto-PGF(1alpha) (the stable degradation product of prostacyclin). Strips of intact aorta were removed from aspirin-treated rabbits, thus the isolated blood vessels were unable to convert endogenous or exogenous arachidonate to prostacyclin. Human platelets, with [(14)C]arachidonate-labeled phospholipids, adhered to the blood vessel segments and released some thromboxane B(2). The subsequent addition of thrombin facilitated the release of endogenous arachidonate and thromboxane, but no labeled 6-keto-PGF(1alpha) was detectable. There is therefore no direct chemical evidence of PG-endoperoxide release from human platelets during either aggregation or adhesion, which therefore precludes the possibility that blood vessels use platelet PG-endoperoxide for prostacyclin synthesis. Imidazole inhibited the thromboxane synthetase in the labeled platelets, and thereafter thrombin stimulation resulted in the release of platelet-derived, labeled PG-endoperoxides that were converted to labeled prostacyclin by the vascular prostacyclin synthetase. The latter result suggests a potential antithrombotic therapeutic benefit might be achieved using an effective thromboxane synthetase inhibitor.

Is inhibition of cyclooxygenase required for the anti-tumorigenic effects of nonsteroidal, anti-inflammatory drugs (NSAIDS)? In vitro versus in vivo results and the relevance for the prevention and treatment of cancer

Active research is being conducted to unravel the cellular mechanisms mediating the anti-tumorigenic effects of nonsteroidal anti-inflammatory drugs (NSAIDs) and their association with cyclooxygenase (COX) inhibition. The majority of NSAIDs inhibit either COX-1, COX-2, or both and exert their anti-COX, anti-inflammatory, and anti-tumorigenic effects in vivo in a parallel dose-dependent manner. The effects are seen at NSAID blood plasma concentrations of 0.1-5 microM. Significantly, the same compounds tested at the same concentrations in incubations with cultured tumor cells in vitro similarly inhibit COX activities but are devoid of anti-proliferative activity. Yet, at much higher concentrations (100-20,000 microM), these same NSAIDs do exert anti-proliferative effects in vitro due to apparent non-specific toxic effects, as evidenced by disruption of ion transport and mitochondrial oxidation in some cells. A small group of NSAIDs (e.g. sulindac) do not inhibit COX enzymes significantly but can reduce the synthesis of prostanoids by alternate mechanisms. One such mechanism is inhibition of agonist-stimulated phospholipase-mediated release of arachidonic acid from phospholipids leading to depressed synthesis of prostanoids, especially prostaglandin E(2) (PGE(2)). Another group of non-COX inhibitors are the R-isomers of NSAIDs, based on the structure of 2-arylpropionic acid. These compounds exert anti-proliferative effects in vivo, acting by an as yet undetermined mechanism. A possible caveat in these data is an R to S chiral transformation in vivo that would render the R-isomer effect as being due to the S-isomer generated in vivo from it. Demonstration of minimal or no R to S inversion under the experimental in vivo conditions employed is, therefore, a necessary control in these studies. The overall body of data supports the conclusion that, for COX-inhibiting NSAIDs, their anti-tumorigenic effect in vivo is due to, and depends upon, inhibition of tumor COX enzymes, primarily COX-2. The cellular effects seen when adding high concentrations of NSAIDs to tumor cells cultured in vitro and the mechanisms proposed to mediate these effects may not have substantial relevance to the mechanisms that mediate the effects of NSAIDs in vivo.

Differential metabolism of dihomo-γ-linolenic acid and arachidonic acid by cyclo-oxygenase-1 and cyclo-oxygenase-2: implications for cellular synthesis of prostaglandin E1 and prostaglandin E2

Prostaglandin (PG) E(1) has been shown to possess anti-inflammatory properties and to modulate vascular reactivity. These activities are sometimes distinct from those of PGE(2), suggesting that endogenously produced PGE(1) may have some beneficial therapeutic effects compared with PGE(2). Increasing the endogenous formation of PGE(1) requires optimization of two separate processes, namely, enrichment of cellular lipids with dihomo-gamma-linolenic acid (20:3 n-6; DGLA) and effective cyclo-oxygenase-dependent oxygenation of substrate DGLA relative to arachidonic acid (AA; 20:4 n-6). DGLA and AA had similar affinities (K(m) values) and maximal reaction rates (V(max)) for cyclo-oxygenase-2 (COX-2), whereas AA was metabolized preferentially by cyclo-oxygenase-1 (COX-1). To overcome the kinetic preference of COX-1 for AA, CP-24879, a mixed Delta(5)/Delta(6) desaturase inhibitor, was used to enhance preferential accumulation of DGLA over AA in cells cultured in the presence of precursor gamma-linolenic acid (18:3 n-6). This protocol was tested in two cell lines and both yielded a DGLA/AA ratio of approx. 2.8 in the total cellular lipids. From the enzyme kinetic data, it was calculated that this ratio should offset the preference of COX-1 for AA over DGLA. PGE(1) synthesis in the DGLA-enriched cells was increased concurrent with a decline in PGE(2) formation. Nevertheless, PGE(1) synthesis was still substantially lower than that of PGE(2). It appears that employing a dietary or a combined dietary/pharmacological paradigm to augment the cellular ratio of DGLA/AA is not an effective route to enhance endogenous synthesis of PGE(1) over PGE(2), at least in cells/tissues where COX-1 predominates over COX-2.

Comparative effects of indomethacin on cell proliferation and cell cycle progression in tumor cells grown in vitro and in vivo.

Considerable research effort is currently being directed towards understanding the mechanisms mediating the antiproliferative effects of non-steroidal anti-inflammatory drugs (NSAIDs) and, more recently, of cyclooxygenase (COX)-2 inhibitors as well. A key question is whether NSAIDs (excluding sulindac) exert their anticarcinogenic effects in vivo by a mechanism that is dependent on their capacity to inhibit COX activity. Some studies with cultured tumor cells in vitro have argued against such a linkage, showing that NSAIDs inhibit cell replication and/or augment apoptosis only at concentrations that exceed those required to inhibit COX activities 10- to 100-fold. The significance of these results for the observed anticarcinogenic effects of NSAIDs in vivo has not yet been evaluated. We addressed this question by comparing, for the same tumor cells, the effects of the NSAID indomethacin on cell growth parameters when the cells were grown in culture to the effects seen in the in vivo growing tumor in the mouse. Indomethacin added to cultured Lewis lung carcinoma cells exerted a potent antiproliferative effect ((3)H thymidine assay) and reduced cell viability (MTT[3-(4,5-dimethyl(thiazol-2-yl)-2,5 diphenyl tetrazolium bromide] assay) at low doses (10-20 microM) in parallel with its inhibitory effect on cellular cyclooxygenase. These effects of indomethacin appeared to arise from a clear antiproliferative shift in the profile of the cell cycle parameters towards a reduced percentage of cells at the S and G(2)/M phases, together with an increased percentage of cells at the G(1) phase. Significantly, similar results were seen when indomethacin was given in vivo at the low dose of 2 mg per kg/day, which blocked blood platelet COX activity and at the same time produced a delay in tumor growth initiation and attenuation of apparent primary tumor growth as well as growth of lung metastases. These results thus provide strong support for the notion that COX inhibition is a major determinant in the antitumorigenic effect of indomethacin in vivo.

Interaction of prostaglandins with blood plasma proteins: I. Binding of prostaglandin E2 to human plasma proteins and its effect on the physiological activity of prostaglandin E2 in vitro and in vivo☆

The binding of (PGE2) prostaglandin E2 to human plasma proteins was investigated by DEAE-Sephadex column chromatography and acrylamide gel electrophoresis, and quantitatively assessed by equilibrium dialysis. PGE2 added to human plasma in vitro was found to become mainly bound to plasma albumin. This binding was also demonstrated by adding PGE2 to human serum albumin solutions. The binding of PGE2 to human serum albumin inhibits the contraction-producing effect of PGE2 on the isolated gerbil colon in vitro. The depressor effect of PGE2 on the rat blood pressure was used to assess the in vivo effect of PGE2 albumin interaction. The blood pressure lowering activities of free and albumin-bound PGE2 were found to be the same when administered either intravenously or intraarterially. The significance of these observations with regard to estimation of PG concentration in whole blood or plasma, and their possible effects on PG metabolism is discussed.

Identification and Characterization of a Novel Δ6/Δ5 Fatty Acid Desaturase Inhibitor As a Potential Anti-Inflammatory Agent

Abstract The anti-inflammatory properties of essential fatty acid deficiency or n-3 polyunsaturated fatty acid supplementation have been attributed to a reduced content of arachidonic acid (AA; 20:4 n-6). An alternative, logical approach to depleting AA would be to decrease endogenous synthesis of AA by selectively inhibiting the Δ5 and/or the Δ6 fatty acid desaturase. High-throughput radioassays were developed for quantifying Δ5, Δ6, and Δ9 desaturase activities in vitro and in vivo . CP-24879 ( p -isopentoxyaniline), an aniline derivative, was identified as a mixed Δ5/Δ6 desaturase inhibitor during the screening of chemical and natural product libraries. In mouse mastocytoma ABMC-7 cells cultured chronically with CP-24879, there was a concentration-dependent inhibition of desaturase activity that correlated with the degree of depletion of AA and decreased production of leukotriene C 4 (LTC 4 ). Production of LTC 4 was restored by stimulating the cells in the presence of exogenous AA, indicating that endogenous AA was limiting as substrate. In the livers of mice treated chronically with the maximally tolerated dose of CP-24879 (3 mg/kg, t.i.d.), combined Δ5/Δ6 desaturase activities were inhibited approximately 80% and AA was depleted nearly 50%. These results suggest that Δ5 and/or Δ6 desaturase inhibitors have the potential to manifest an anti-inflammatory response by decreasing the level of AA and the ensuing production of eicosanoids.

Fish oil inhibits Δ6 desaturase activity in vivo: Utility in a dietary paradigm to obtain mice depleted of arachidonic acid

Abstract In mice that were alternately fasted and then refed an essential fatty acid-deficient (EFAD) diet, there was a rapid and substantial decline in tissue n-3 and n-6 polyunsaturated fatty acids (PUFAs) and a corresponding increase in n-9 fatty acids. Combined in vivo activities of Δ6 + Δ5 desaturases were quantified directly by measuring the conversion of 14C-linoleic acid (intraperitoneal injection) to 14C-arachidonic acid in liver lipids. Δ5 desaturase activity was quantified by measuring the conversion of 14C-dihomo-γ-linolenic acid (intraperitoneal injection) to 14C-arachidonic acid in liver lipids. The combined Δ6 + Δ5 desaturase activities in EFAD mice was very similar to that in chow-fed control mice (35% vs. 33% conversion of 14C-linoleic acid to 14C-arachidonic acid, respectively). Subsequent refeeding of EFAD mice with an EFAD diet supplemented with corn oil restored tissue n-6 PUFA levels, but did not alter Δ6 + Δ5 desaturase activities (33%). In contrast, subsequent refeeding of EFAD mice with a fish oil-supplemented diet markedly inhibited Δ6 + Δ5 desaturase activities (7%). Fatty acid analysis of the livers from the fish oil-fed mice showed that there was a depletion of the n-6 PUFAs, linoleic acid, and arachidonic acid, and an increase in the n-3 PUFAs, eicosapentaenoic acid (20:5 n-3) and docosahexaenoic acid (22:6 n-3). The inhibition of Δ6 + Δ5 desaturase activities was also maintained in EFAD mice fed a 1:1 mixture of fish oil:corn oil. As a consequence, a unique fatty acid composition in liver and plasma was obtained in which arachidonic acid was selectively depleted, whereas linoleic acid and n-3 PUFAs were increased. Δ5 desaturase activity was not affected by any of the fasting/refeeding paradigms. The data demonstrate that dietary n-3 PUFAs negatively regulate the in vivo synthesis of n-6 PUFAs at the level of the Δ6 desaturase. The inhibition of Δ6 desaturase activity by n-3 PUFAs provides a basis for a unique dietary route to selectively reduce tissue arachidonic acid, while providing sufficient linoleic acid, an essential fatty acid, to support normal cellular metabolism. This dietary paradigm may be effective in attenuating diseases characterized by excessive production of arachidonic acid-derived eicosanoids.

Indomethacin and aspirin inhibition of prostaglandin E2 synthesis by sheep seminal vesicles microsome powder

Abstract Sheep seminal vesicles microsome powder was used as a source of prostaglandin synthetase in studies on the nature of inhibition of prostaglandin synthesis by indomethacin and aspirin. Irdomethacin inhibition was found to be highly irreversible, although partial recovery of synthetase activity was obtained after extensive dialysis. A major difference was observed between the effects of aspirin and indomethacin on prostaglandin synthetase activity in seminal vesicles slices. Enzyme activity in microsomes prepared from slices incubated with aspirin was markedly inhibited while the activity in microsomes prepared after incubation with indomethacin was not affected. These results suggest that indomethacin may penetrate intracellularly very slowly, or not at all, and raise a question as to whether the inhibition by indomethacin in vivo is mediated via direct inhibition of prostaglandin synthesis.

Prostaglandin generation in rabbit kidney. Hormone-activated selective lipolysis coupled to prostaglandin biosynthesis.

The endogenous release of prostaglandins and free fatty acids from the isolated perfused rabbit kidney in the absence or presence of stimulation by bradykinin or angiotensin-II was investigated. Basal (nonstimulated) release of prostaglandin-precursor arachidonic acid was 15-20-fold higher than that of prostaglandin E2 indicating a low conversion of released arachidonate to prostaglandins. Addition of bovine serum albumin to the perfusion medium caused a substantial (50-250%) increase in the release of all fatty acids except myristic and arachidonic acids, and no significant change in prostaglandin E2 generation. In contrast, administration of bradykinin (0.5 microgram) or angiotensin-II (1 microgram) caused a 10-15-fold increase in prostaglandin E2 release, and with albumin present, also a 2-3-fold selective increase in arachidonic acid release. Thus, unlike what was observed under basal conditions, arachidonic acid released following hormone stimulation is efficiently converted to prostaglandin E2. We conclude that administration of bradykinin or angiotensin-II into the perfused kidney activates a lipase which selectively releases arachidonic acid, probably from a unique lipid entity. This lipase reaction is tightly coupled to a prostaglandin generating system so that the released arachidonate is first made available to the prostaglandin cyclooxygenase, resulting in its substantial conversion to prostaglandins.