Hasan Safayhi

Acetyl-11-keto-β-boswellic acid (AKBA): structure requirements for binding and 5-lipoxygenase inhibitory activity

1 5‐Lipoxygenase (5‐LOX) products from endogenous arachidonic acid in ionophore‐stimulated peritoneal polymorphonuclear leukocytes (PMNL) and from exogenous substrate (20 μm) in 105,000 g supernatants were measured. 2 The effects of natural pentacyclic triterpenes and their derivatives on 5‐LOX activity were compared with the inhibitory action of acetyl‐11‐keto‐β‐boswellic acid (AKBA), which has been previously shown to inhibit the 5‐LOX by a selective, enzyme‐directed, non‐redox and non‐competitive mechanism. 3 The 5‐LOX inhibitory potency of AKBA was only slightly diminished by deacetylation of the acetoxy group or reduction of the carboxyl function to alcohol in intact cells (IC50=1.5 vs. 3 and 4.5 μm, respectively) and in the cell‐free system (8 vs. 20 and 45 μm). 4 β‐Boswellic acid (β‐BA), lacking the 11‐keto function, inhibited 5‐LOX only partially and incompletely, whereas the corresponding alcohol from β‐BA, as well as amyrin, acetyl‐11‐keto‐amyrin, 11‐keto‐β‐boswellic acid methyl ester had no 5‐LOX inhibitory activity up to 50 μm in either system. 5 β‐BA only partially prevented the AKBA‐induced 5‐LOX inhibition, whereas the non‐inhibitory compounds, amyrin and acetyl‐11‐keto‐amyrin, almost totally antagonized the AKBA effect and shifted the concentration‐inhibition curve for the incomplete inhibitor β‐BA to the right. In contrast, the non‐inhibitory 11‐keto‐β‐BA methyl ester exerted no antagonizing effect. 6 The results demonstrate that the pentacyclic triterpene ring system is crucial for binding to the highly selective effector site, whereas functional groups (especially the 11‐keto function in addition to a hydrophilic group on C4 of ring A) are essential for 5‐LOX inhibitory activity.

A test system for leukotriene synthesis inhibitors based on the in-vitro differentiation of the human leukemic cell lines HL-60 and Mono Mac 6

Differentiation of HL-60 cells along the granulocytic lineage by DMSO in the presence of transforming growth factor-β and low concentrations of 1,25-dihydroxyvitamin D3 leads to the upregulation of 5-lipoxygenase activity in 100,000 g supernatants and intact cells to levels which are comparable to normal granulocytes. Similarly, differentiation of the human monocytic cell line Mono Mac 6 by 1,25-dihydroxyvitamin D3 and transforming growth factor-β strongly upregulates the 5-lipoxygenase pathway. Here, we describe an assay system for leukotriene biosynthesis inhibitors which is based on the in-vitro differentiation of HL-60 and Mono Mac 6 cells. Different leukotriene biosynthesis inhibitors like the nonredox type inhibitor ZM 230487, the redox type inhibitor BW A4C and the FLAP inhibitor MK886 were tested and the results were compared with an assay system based on normal human granulocytes. ZM 230487, BWA4C and MK886 showed similar potencies in these cell lines as compared to normal leukocytes. Thus, the in-vitro differentiation of HL-60 and Mono Mac 6 cells provides an excellent model for the screening of drugs affecting the 5-lipoxygenase pathway.

MEK-1/2 inhibition prevents 5-lipoxygenase translocation in N-formylpeptide-challenged human neutrophils

In order to elucidate the role of mitogen-activated protein kinase kinase (MEK-1/2) in 5-lipoxygenase (5-LO) activation we studied the N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced 5-LO translocation in human blood neutrophils (PMNs). In non-primed, Ca(2+)-repleted PMNs, fMLP consistently stimulated MEK-1/2 phosphorylation, but induced 5-LO translocation and product formation (430+/-128 pmol; SEM, n=13) only in 13 of 18 PMN preparations from different healthy donors. In fMLP-responsive cells, the MEK-1/2 inhibitor PD098059 (50 microM) attenuated MEK phosphorylation and abolished 5-LO activation at the translocation step. The fMLP-mediated 5-LO product formation was also sensitive to MEK inhibition by U0126 and to p38 inhibition by SB203580. But in contrast to PD098059, U0126 at 10 microM and SB203580 at 20-50 microM impaired 5-LO activity in the cell-free assay setting, suggesting direct actions of higher concentrations of U0126 and SB203580 on 5-LO apart from MEK and p38 inhibition, respectively. These data show that fMLP initiates 5-LO product formation in non-primed, Ca(2+)-repleted human blood PMNs from healthy donors, and that MEK signaling is pivotal, but not sufficient for 5-LO activation in response to the receptor agonist fMLP.

5-Lipoxygenase inhibition by acetyl-11-keto-β-boswellic acid (AKBA) by a novel mechanism.

Acetyl-11-keto-β-boswellic acid (AKAB) from Boswellia serrata and B. carterii acts directly on purified 5-lipoxygenase of human blood leukocytes at a selective site for pentacyclic triterpenes that is different from the arachidonate substrate binding site. The pentacyclic triterpene ring is crucial for binding to the enzyme, whereas functional groups (11-keto function in addition to a hydrophilic group on C 4 of ring A) are essential for the 5-lipoxygenase activity.

KCl-induced insulin secretion from RINm5F cells is mediated through Ca2+ influx along L-type Ca2+ channels

SummaryIn order to characterize the voltage-dependent Ca2+ channels of insulin secretory RINm 5F cells, we have studied the binding of the dihydropyridine (DHP) type Ca2+ antagonist PN 200-110 and its effect on insulin release. In the membrane preparation from RINm 5F cells [3H]-(+)-PN 200-110 bound to a high affinity binding site in a stereoselective manner (KD: 7.0 nM, Bmax: 858 fmol/mg protein). The benzothiazepine type Ca2+ antagonist D-cis-diltiazem increased the binding of [3H]-(+)-PN 200-110 in a temperature-dependent manner. The phenylalkylamine-type Ca2+ antagonist verapamil decreased PN binding with an IC50 of 100 μM. (+)-PN 200-110 inhibited KCl-(25 mM)-induced insulin release (IC50 = 10 nM), Effects on binding and hormone release occurred over comparable concentration ranges: 1 μM PN 200-110 produced 100% displacement and totally abolished depolarization-mediated insulin release. The N-type Ca2+-antagonist ω-conotoxin showed no effect on KCl-induced insulin release. The data suggest that in RINm 5 F cells only l-type Ca2+ channels are involved in the mechanism of depolarization-mediated insulin release.

CGS 9343B and W7 (calmodulin antagonists) inhibit KCl-induced increase in cytosolic free calcium and insulin secretion of RINm5F cells

SummaryCGS 9343B:1,3-Dihydro-l-[1-((4-methyl-4H,6H-pyrrolo[1,2-a]-[4,1]benzoxazepin-4-yl)methyl)-4-piperidinyl]-2 H-benzimidazol-2-one maleate and W7: N-(6-aminohexyl)-5-chloro-l-naphthalenesulfonamide) are calmodulin antagonists with different specificities. The effects of CGS 9343B and W7 on cytosolic free calcium concentration ([Ca2+]i) and insulin release were investigated in rat insulinoma cells (RINm5F). As measured with the Quin-2 technique, preincubation with CGS 9343B (0.3–10 μM) and W7 (5–50 μM) concentration dependently decreased KCl (25 mM)-mediated accumulation of cytosolic calcium. Both, CGS 9343B (10 μM) and W7 (50–100 μM) almost abolished the alanine- and KCl-induced increase in [Ca2+]i and significantly inhibited KCl (25 mM)- and alanine (10 mM)-mediated insulin release. W5 (100 μLM), the chlorine-deficient analogue of W7 with decreased affinity for calmodulin, did not inhibit the KCl-induced increase in [Ca2+]i and enhanced basal and KCl-mediated insulin release by 56% and 189%, respectively. Our data suggest that CGS 9343B and W7 inhibit the depolarization-induced calcium uptake and subsequent increase in [Ca2+]i.

Boswellic acids and protease activities

The involvement of human leukocyte elastase (HLE) in several inflammatory processes and the reported inhibitory effect of ursolic acid on HLE has prompted the authors to start investigation on the effects of acetyl-11-keto-β-boswellic acid (AKBA) on serine proteinases including HLE.

Effect of W-7 on ionic fluxes and electrical activity of mouse pancreatic islets

W-7 (N-(6-amino-hexyl)-5-chloro-1-naphthalenesulfonamide) (0.1 mM), a calmodulin inhibiting compound, suppressed the reincrease of 86Rb+ efflux from pancreatic islets normally seen in response to lowering the glucose concentration from stimulated to basal value. Ionophore (A23187)-induced increase was completely abolished. W-7 inhibited 45Ca2+ uptake and stimulation of 45Ca2+ efflux in response to glucose (11.1 mM) but did not affect K+ (20 mM)-induced 45Ca2+ uptake. Electrical activity of B-cells at 11.1 mM glucose showed a prolongation in burst length in the presence of 0.1 mM W-7. The data suggest that W-7 affects the opening properties of K+ channels resulting in a delayed repolarisation of the cells possibly through its inhibitory action on Ca2(+)-activated calmodulin.

Insulin secretion without the participation of arachidonic acid

In order to study the role of arachidonic acid (AA) in depolarization-induced insulin secretion rat insulinoma cells (RINm5F) were depleted of AA by cultivation in essential fatty acid-free medium. Within 2 weeks AA content of these cells was decreased to a non-detectable level as assessed by gas chromatography (GC). Different cell lines were obtained by supplementation of the defatted medium with oleic acid or the AA precursor linoleic acid (7 and 70 microM, each). The AA content varied in dependence from the precursor availability from 0 to about 14% of long chain fatty acids. Variation in AA content or the depletion of AA to a non-detectable level did not modulate insulin synthesis, basal and potassium-induced insulin release, cell growth (cell number and protein), membrane depolarization and increases in cytosolic Ca2+. In AA containing cells no eicosanoids was produced in the course of stimulated hormone release. The data suggest that in RINm5F cells release of AA and/or formation of oxidized metabolites from AA are not essential for functional integrity.

Acetyl-11-keto-β-boswellic acid (AKBA) is cytotoxic for meningioma cells and inhibits phosphorylation of the extracellular-signal regulated kinase 1 and 2

Acetyl-11-keto-beta-boswellic acid (AKBA) is a naturally occurring pentacyclic triterpene isolated from the gum resin exudate from the stem of the tree Boswellia serrata (frankincense). AKBA has been recently identified as a novel, orally active, non-redox and non-competitive 5-lipoxygenase inhibitor that also inhibits topisomerase I and II in vitro. Because natural pentacyclic triterpenes have an antiproliferative effect against different tumor types, we investigated the effects of AKBA on the proliferation of 11 primary cell cultures established from human surgical specimens of meningiomas, common central nervous system tumors. Treatment of meningioma cells by AKBA revealed a potent cytotoxic activity with half-maximal inhibitory concentrations in the range of 2-8 microM. At similar, physiologically achievable concentrations, AKBA rapidly (within minutes) and potently inhibited the phosphorylation of extracellular signal-regulated kinase 1 and 2 (Erk-1 and Erk-2) in meningioma cells stimulated with platelet-derived growth factor BB. High expression level of 5-LO was detected in primary meningioma cells and surgical specimens by immunoblotting analysis, suggesting the possible role of 5-LO in meningioma tumorigenesis. Considering the critical importance of the Erk-1/2 signal transduction pathway not only in meningiomas but in other human neoplasms, the interruption of signaling through this evolutionarily conserved pathway might be one of the mechanisms by which AKBA induces suppression of proliferation and apoptosis of different tumor types.