Isozymes inhibited by active site blocking: versatility of calcium indifferent hesperidin binding to phospholipase A2 and its significance.

sPLA2 is released under inflammatory conditions from neutrophils, basophils and T-cells. They cleave the cellular phospholipids leading to the release of arachidonic acid and there by provide intermediates for biosynthesis of inflammatory mediators. The focus of this study is on the interaction of hesperidin, a natural flavonoid with Group IB, IIA, and V and X isozymes of sPLA2. Affinity of hesperidin towards PLA2 isozymes was analyzed through enzymatic studies and molecular modeling. The experiments showed that hesperidin competitively inhibited PLA2 with IC50 of 5.1 µM. Molecular modeling studies revealed the association of hesperidin with the docking scores -6.90, -9.53, -5.63 and -8.29 kcal for isozymes Group IB, IIA, V and X of PLA2 respectively. Their binding energy values were calculated as -20.25, -21.63, -21.66 and -33.43 kcal for the Group IB, IIA, V and X respectively. Structural model for Group V was made by homology modeling since no structural coordinates were available. Molecular dynamics studies were carried out to evaluate the structural stability of protein ligand complex. The analyses showed that hesperidin blocked the entry of the substrate to the active site of PLA2 and it was indifferent to the differences of the isozymes. Hence, hesperidin might serve as lead for designing highly specific anti-inflammatory drugs directed to the PLA2 isozyme specific to various diseases, with IC50 value of therapeutic significance.

Group X secreted phospholipase A(2) induces lipid droplet formation and prolongs breast cancer cell survival.

BACKGROUND: Alterations in lipid metabolism are inherent to the metabolic transformations that support tumorigenesis. The relationship between the synthesis, storage and use of lipids and their importance in cancer is poorly understood. The human group X secreted phospholipase A2 (hGX sPLA2) releases fatty acids (FAs) from cell membranes and lipoproteins, but its involvement in the regulation of cellular FA metabolism and cancer is not known. RESULTS: Here we demonstrate that hGX sPLA2 induces lipid droplet (LD) formation in invasive breast cancer cells, stimulates their proliferation and prevents their death on serum deprivation. The effects of hGX sPLA2 are shown to be dependent on its enzymatic activity, are mimicked by oleic acid and include activation of protein kinase B/Akt, a cell survival signaling kinase. The hGX sPLA2-stimulated LD biogenesis is accompanied by AMP-activated protein kinase (AMPK) activation, up-regulation of FA oxidation enzymes and the LD-coating protein perilipin 2, and suppression of lipogenic gene expression. Prolonged activation of AMPK inhibited hGX sPLA2-induced LD formation, while etomoxir, an inhibitor of FA oxidation, abrogated both LD formation and cell survival. The hGX sPLA2-induced changes in lipid metabolism provide a minimal immediate proliferative advantage during growth under optimal conditions, but they confer to the breast cancer cells a sustained ability to resist apoptosis during nutrient and growth factor limitation. CONCLUSION: Our results identify hGX sPLA2 as a novel modulator of lipid metabolism that promotes breast cancer cell growth and survival by stimulating LD formation and FA oxidation.

Blockade of human group X secreted phospholipase A2 (GX-sPLA2)-induced airway inflammation and hyperresponsiveness in a mouse asthma model by a selective GX-sPLA2 inhibitor.

Group X (GX) phospholipase A(2), a member of a large group of secreted phospholipases A(2) (sPLA(2)s), has recently been demonstrated to play an important in vivo role in the release of arachidonic acid and subsequent formation of eicosanoids. In a Th2 cytokine-driven mouse asthma model, deficiency of mouse GX (mGX)-sPLA(2) significantly impairs development of the asthma phenotype. In this study, we generated mGX-sPLA(2)(-/-) mice with knock-in of human GX (hGX)-sPLA(2) (i.e. hGX-sPLA(2)(+/+) knock-in mice) to understand more fully the role of GX-sPLA(2) in these allergic pulmonary responses and to assess the effect of pharmacological blockade of the GX-sPLA(2)-mediated responses. Knock-in of hGX-sPLA(2) in mGX-sPLA(2)(-/-) mice restored the allergen-induced airway infiltration by inflammatory cells, including eosinophils, goblet cell metaplasia, and hyperresponsiveness to methacholine in the mGX-sPLA(2)-deficient mice. This knock-in mouse model enabled the use of a highly potent indole-based inhibitor of hGX-sPLA(2), RO061606 (which is ineffective against mGX-sPLA(2)), to assess the potential utility of GX-sPLA(2) blockade as a therapeutic intervention in asthma. Delivery of RO061606 via mini-osmotic pumps enabled the maintenance in vivo in the mouse asthma model of plasma inhibitor concentrations near 10 µm, markedly higher than the IC(50) for inhibition of hGX-sPLA(2) in vitro. RO061606 significantly decreased allergen-induced airway inflammation, mucus hypersecretion, and hyperresponsiveness in the hGX-sPLA(2)(+/+) knock-in mouse. Thus, development of specific hGX-sPLA(2) inhibitors may provide a new pharmacological opportunity for the treatment of patients with asthma.

Phase composition of lipoprotein SM/cholesterol/PtdCho affects FA specificity of sPLA2s.

We have previously reported preferential release of polyunsaturated FAs during hydrolysis of lipoprotein phosphatidylcholine (PtdCho) by group X secretory phospholipase A2 (sPLA2) and preferential release of oligounsaturated FAs during hydrolysis of lipoprotein PtdCho by group V sPLA2, but the mechanism of this selectivity has remained unknown. We now show that the rate and specificity of hydrolysis are affected by relative increases in endogenous SM and free cholesterol (FC) during the lipase digestion. The highest preference for arachidonate release from LDL and HDL by group X sPLA2 was observed for residual SM/PtdCho molar ratio of 1.2 and 0.4, compared with the respective starting ratios of 0.4 and 0.2, as measured by liquid chromatography/electrospray ionization-mass spectrometry. Group V sPLA2 showed preferential release of linoleate from LDL and HDL at SM/PtdCho ratio 1.5 and 0.6, respectively. We have attributed the change in FA specificity to segregation of molecular species of PtdCho and of sPLA2s between disordered and ordered SM/FC/PtdCho lipid phases. The increases in SM and FC during digestion with group IIA sPLA2 were more limited, and a preferential hydrolysis of any FAs was not observed. The significance of SM and FC SM and FC accumulation during sPLA2 hydrolysis of lipoprotein PtdCho has been previously overlooked.

Reduction in myocardial ischemia/reperfusion injury in group X secretory phospholipase A2-deficient mice.

BACKGROUND: Group X secretory phospholipase A(2) (sPLA(2)-X) has the most potent hydrolyzing activity toward phosphatidylcholine and elicits a marked release of arachidonic acid among several types of sPLA(2). sPLA(2)-X is expressed in neutrophils, but its pathogenic role remains unclear. METHODS AND RESULTS: We generated mice that lack sPLA(2)-X and studied their response to myocardial ischemia/reperfusion. The sPLA(2)-X(-/-) mice had a significant reduction in myocardial infarct size and a decrease in myocardial myeloperoxidase activity compared with sPLA(2)-X(+/+) mice. Myocardial infarct size was also significantly reduced in lethally irradiated sPLA(2)-X(+/+) mice reconstituted with sPLA(2)-X(-/-) bone marrow compared with sPLA(2)-X(+/+) bone marrow. The extent of myocardial ischemia/reperfusion injury was comparable between sPLA(2)-X(-/-) and sPLA(2)-X(+/+) mice in Langendorff experiments using isolated hearts and blood-free perfusion buffer, supporting a potential role of sPLA(2)-X in blood in myocardial ischemia/reperfusion injury. In the infarcted myocardium of sPLA(2)-X(+/+) mice, sPLA(2)-X was released from neutrophils but not myocardial tissues and platelets and was undetectable in the peripheral serum. The sPLA(2)-X(-/-) mice had lower accumulation of neutrophils in ischemic myocardium, and the isolated sPLA(2)-X(-/-) neutrophils had lower release of arachidonic acid and attenuated cytotoxic activities including respiratory burst compared with sPLA(2)-X(+/+) neutrophils. The attenuated functions of sPLA(2)-X(-/-) neutrophils were reversible by the exogenous addition of sPLA(2)-X protein. Furthermore, administration of a sPLA(2) inhibitor reduced myocardial infarct size and suppressed the cytotoxic activity of sPLA(2)-X(+/+) neutrophils. CONCLUSIONS: Myocardial ischemia/reperfusion injury was attenuated in sPLA(2)-X(-/-) mice partly through the suppression of neutrophil cytotoxic activities.