Anticyclooxygenase Antilipoxygenase Activities and Regulation of Cytokine Secretion Immunomodulatory Effect

Among the various beneficial effects of the extract prepared from the roots of P. capsidatum (the plant from which RSV was first isolated) were its magical effects on allergic and inflammatory diseases. Invariably, these pathological states are a direct or indirect outcome of a hyperactive immune system as a result of an increase in activity of leukocytes that churn out excess of biological response modifiers. The two enzyme systems involved in the synthesis of proinflammatory mediators such as 5-HETE (5-hydroxy-6,8,11,14-eicosatetraenoic acid), thromboxane A2, prostaglandins (PG), and HTT (12-hydroxy-5,8,10-heptadecatrienoic acid) are the cycloxygenase (COX) and the lipoxygenase pathways (Cuendet and Pezzuto, 2000). COX-1 (also referred to as PGH synthase) is constitutively expressed, whereas an inducible COX-2 is also expressed constitutively in certain regions of the brain, kidneys, and cancerous tissues. COX-2 activity, usually undetected in normal tissues, generates proinflammatory substances by the oxygenation of arachidonic acid to PGs (PGD2 and PGE2) (Cuendet and Pezzuto, 2000). In addition, COX-2 can catalyze the formation of chemotactic substances such as HHT and thromboxone A2 from PGH2 via the thromboxone synthetase (Gierse et al., 1995). This chemotactic activity can in turn lead to platelet aggregation. The leukocyte lipoxygenase is known to catalyze the initial reaction that leads to the formation of 5-HETE, a stable derivative of the peroxy form 5-HPETE (Kimura et al., 1995). These substances have high chemotactic activity and are potent inducers of histamine release from basophil.

The PGs have been implicated in promoting cell proliferation, suppressing immune surveillance, and stimulating tumorigenesis (Gusman et al., 2001). Owing to these deleterious effects of PGs and other inflammatory substances generated by COX and lipoxygenase pathways, identification of molecules with the potential to inhibit these pathways has been a major focus of biomedical research. In this regard, the effects of stilbenes, in particular RSV, on COX and leukocyte lipoxygenase pathways have produced interesting results. RSV was found to inhibit the 5-lipoxegenase product 5-HETE and the COX products HHT and thromboxane B2 (Kimura et al., 1985). Based on these results, the researchers further established that this inhibitory activity was directly responsible for the antiplatelet aggregation induced by RSV.

The COX and lipoxygenase inhibitory activities of RSV have also been reported to account for its protective effect against oxidative-stress-induced death of human erythroleukemia K562 cells (MacCarrone et al., 1999). The mechanism of this death-inhibitory activity of RSV involved inhibition of H2O2-induced increases in PGE2 (product of COX activity) and leukotriene B4 (product of lipoxygenase activity) concentrations. This inhibitory effect on COX and lipoxygenase activities has been proposed as a possible mechanism for the antitumor activity of RSV; however, our studies (in HL60 leukemia and T47D breast carcinoma cells) and recent data from other groups provide evidence that the antitumor activity may, in part, be due to its ability to trigger apoptotic death in tumor cells (Ahmad et al., 2001; Clement et al., 1998a; Ding and Adrian, 2002; Ferry-Dumazet et al., 2002; Huang et al., 1999; Morris et al., 2002). Nevetheless, given the role that inflammatory mediators play in the induction and promotion of carcinogenesis, it is plausible that both these observed activities may play a role in cancer chemoprevention.

The anti-inflammatory activity of RSV has also been demonstrated in a rat model of carrageenan-induced paw edema (Gentilli et al., 2001). RSV inhibited both acute and chronic phases of this inflammatory process, with an activity greater than that of indomethacin or phenylbutazone. This effect was also attributed to the impairment of PG synthesis via selective inhibition of COX-1. Similarly, preincubation with RSV decreased arachidonic acid release and COX-2 induction in mouse peritoneal macrophages stimulated with tumor promoter PMA, ROI, or lipopolysaccharides (LPS) (Tsai et al., 1999). Gene transfer experiments using a reporter construct containing COX-2-luciferase confirmed that RSV-mediated decrease in COX-2 activity was indeed due to its inhibitory effect on protein kinase C (PKC)-driven activation of COX-2 transcription (Subbaramaiah et al., 1998). A more detailed account of the effect of RSV on gene transcription and transcriptional factors will be presented in a later section.

The effect of RSV on macrophages and polymorphonuclear cells (PMN) has also been evaluated. These cells are the major players in the body's response to immunogenic challenges, and biological response modifier secreted from these cells can contribute to the development of disease states such as allergy and inflammation (Harlan, 1987). One classic model of macrophage activation is the bacterial LPS. Under normal physiological settings this activation leads to a moderate increase in iNOS activity resulting in NO production that has bactericidal effects. However, abnormally high concentrations of NO and its derivatives peroxynitrite and nitrogen dioxide give rise to inflammation and have been shown to contribute to the process of carcinogenesis (Halliwell, 1994). In this regard, exposure of RAW 264.7 macrophage cells to LPS resulted in the induction of iNOS and the resultant release of nitrite into the culture medium (Tsai et al., 1999; Wadsworth and

Koop, 1999). Preincubation of cells with RSV resulted in a dose-dependent inhibition of iNOS induction, and decreases in the steady-state levels of iNOS mRNA and protein. In contrast, RSV has also been shown to activate NO production through the induction of NOS activity in cultured bovine pulmonary artery endothelial cells (Hsieh et al., 1999b). In this model the vaso-dilatory activity of NO has been proposed as a possible mechanism for the prevention of initiation of atherosclerosis.

The effect of RSV on PMN-induced proinflammatory signals has also been investigated. In these series of experiments, PMN were stimulated by exposure to formyl methionyl leucyl phenylalanine (fMLP), the complement fragment C5a, the Ca2+ ionophore A23187, or amonoclonal antibody to the h2-integrin Mac-1, and the effects on ROI production, neutrophil degranulation, and the cell surface expression of Mac-1 were assessed (Rotondo et al., 1998). Enhanced ROI generation by PMN can result in membrane lipid peroxidation, endothelial damage, and increase in vascular permeability. Degranulation of neutrophils can result in the release of enzymes such as elastase and h-glucoronidase, which have also been linked to endothelial damage and subendothelial smooth muscle proliferation (Harlan, 1987; Totani et al., 1994). A third major contributor to endothelial injury is the increase in the cell surface expression of adhesion molecules of the h-2 integrin family such as CD11a/CD18 (LFA1), CD11b/CD18 (Mac-1), and CD11c/ CD18 (p150/95) (Arnaout, 1990). RSV remarkably inhibited ROI production, release of elastase and h-glucoronidase from neutrophil granules, and the cell surface expression of the h2 integrin MAC-1, upon PMN stimulation. These results strongly indicate that RSV elicits inhibitory effect at all physiological phases of the inflammatory response, i.e., from the initial recruitment of PMNs to their activation and the subsequent release of inflammatory mediators. As the inflammatory response is a critical common denominator in the development of many systemic disorders, such as atherosclerosis and carcinogenesis, the strong anti-inflammatory activity of RSV could have tremendous clinical implications.

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