Presentation_1_Modulations of bioactive lipids and their receptors in postmortem Alzheimer’s disease brains.PDF

BackgroundAnalyses of brain samples from Alzheimer’s disease (AD) patients may be expected to help us improve our understanding of the pathogenesis of AD. Bioactive lipids, including sphingolipids, glycerophospholipids, and eicosanoids/related mediators have been demonstrated to exert potent physiological actions and to be involved in the pathogenesis of various human diseases. In this cross-sectional study, we attempted to elucidate the associations of these bioactive lipids with the pathogenesis/pathology of AD through postmortem studies of human brains.MethodsWe measured the levels of glycerophospholipids, sphingolipids, and eicosanoids/related mediators in the brains of patients with AD (AD brains), patients with Cerad score B (Cerad-b brains), and control subjects (control brains), using a liquid chromatography-mass spectrometry method; we also measured the mRNA levels of specific receptors for these bioactive lipids in the same brain specimens.ResultsThe levels of several species of sphingomyelins and ceramides were higher in the Cerad-b and AD brains. Levels of several species of lysophosphatidic acids (LPAs), lysophosphatidylcholine, lysophosphatidylserine, lysophosphatidylethanolamine (LPE), lysophosphatidylinositol, phosphatidylcholine, phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylinositol, and phosphatidylglycerol were especially high in the Cerad-b brains, while those of lysophosphatidylglycerol (LPG) were especially high in the AD brains. Several eicosanoids, including metabolites of prostaglandin E2, oxylipins, metabolites of epoxide, and metabolites of DHA and EPA, such as resolvins, were also modulated in the AD brains. Among the lipid mediators, the levels of S1P2, S1P5, LPA1, LPA2, LPA6, P2Y10, GPR174, EP1, DP1, DP2, IP, FP, and TXA2r were lower in the AD and/or Cerad-b brains. The brain levels of ceramides, LPC, LPI, PE, and PS showed strong positive correlations with the Aβ contents, while those of LPG showed rather strong positive correlations with the presence of senile plaques and neurofibrillary tangles. A discriminant analysis revealed that LPG is especially important for AD and the LPE/PE axis is important for Cerad-b.ConclusionsComprehensive lipidomics, together with the measurement of lipid receptor expression levels provided novel evidence for the associations of bioactive lipids with AD, which is expected to facilitate future translational research and reverse translational research.</p

Increased mRNA Levels of Sphingosine Kinases and S1P Lyase and Reduced Levels of S1P Were Observed in Hepatocellular Carcinoma in Association with Poorer Differentiation and Earlier Recurrence

<div><p>Although sphingosine 1-phosphate (S1P) has been reported to play an important role in cancer pathophysiology, little is known about S1P and hepatocellular carcinoma (HCC). To clarify the relationship between S1P and HCC, 77 patients with HCC who underwent surgical treatment were consecutively enrolled in this study. In addition, S1P and its metabolites were quantitated by LC-MS/MS. The mRNA levels of sphingosine kinases (SKs), which phosphorylate sphingosine to generate S1P, were increased in HCC tissues compared with adjacent non-HCC tissues. Higher mRNA levels of SKs in HCC were associated with poorer differentiation and microvascular invasion, whereas a higher level of SK2 mRNA was a risk factor for intra- and extra-hepatic recurrence. S1P levels, however, were unexpectedly reduced in HCC compared with non-HCC tissues, and increased mRNA levels of S1P lyase (SPL), which degrades S1P, were observed in HCC compared with non-HCC tissues. Higher SPL mRNA levels in HCC were associated with poorer differentiation. Finally, in HCC cell lines, inhibition of the expression of SKs or SPL by siRNA led to reduced proliferation, invasion and migration, whereas overexpression of SKs or SPL enhanced proliferation. In conclusion, increased SK and SPL mRNA expression along with reduced S1P levels were more commonly observed in HCC tissues compared with adjacent non-HCC tissues and were associated with poor differentiation and early recurrence. SPL as well as SKs may be therapeutic targets for HCC treatment.</p></div

Correlation of the mRNA levels of SKs and SPL with microvascular invasion in HCC tissues (n = 77).

<p>Correlation of the mRNA levels of SKs and SPL with microvascular invasion in HCC tissues (n = 77).</p

Concentration-dependent inhibition of U46619- and collagen-induced platelet aggregation by licochalcones.

<p><b>(A)</b> Licochalcone A (Lico A, 100 μM), licochalcone C (Lico C, 100 μM), licochalcone D (Lico D, 100 μM) or DMSO (-) was preincubated for 5 min before addition of U46619 (3 μM), collagen (3 μg/ml) or thrombin (0.03 U/ml) in the presence of 1 mM CaCl₂. Results are shown as mean±S.E.M. (*<i>P</i><0.05 compared with control, n = 3, Tukey–Kramer’s method). <b>(B)</b> Licochalcones (2–100 μM) or DMSO (control) were preincubated for 5 min before addition of U46619 (3 μM) or collagen (3 μg/ml) in the presence of 1 mM CaCl₂. Results are shown as mean±S.E.M. (*<i>P</i><0.05 compared with control, n = 3–8, Dunnett’s method). <b>(C)</b> Licochalcone A (10 or 100 μM) or DMSO (control) were preincubated for 5 min before addition of collagen (3 μg/ml) in the presence of 1 mM CaCl₂. Representative traces of the collagen-induced platelet aggregation with or without licochalcone A are shown.</p

Levels of S1P and its metabolites in HCC tissues and the correlation between S1P levels and SKs.

<p>S1P levels (A) were reduced in HCC tissues compared with non-tumorous tissues in 84.5% of the patients; the mean S1P level was lower in HCC tissues than in non-tumorous tissues (<i>P</i> = 0.009, n = 58). The levels of sphingosine (Sph) and ceramide (Cer; 16:0, 18:0, 20:0, 22:0 and 24:0) (B) did not differ between HCC tissues and non-tumorous tissues. The asterisk indicates a significant difference. A correlation between S1P levels and SK1 or SK2 (C, D) mRNA levels was not observed in HCC tissues but was observed in non-tumorous tissues (<i>P</i> = 0.0068 and 0.0033, n = 58).</p

Patient characteristics.

<p>Patient characteristics.</p

Effects of overexpression and inhibition of SKs or SPL expression.

<p>(A, B) PLC/PRF/5 cells transfected with SK1 siRNA and SPL siRNA and HuH7 cells transfected with SK2 siRNA had reduced SK1, SK2 and SPL mRNA levels, respectively, compared with the cells that were transfected with the negative control siRNA (NC; <i>P</i> <0.0001, <i>P</i> <0.0001and <i>P</i> <0.0001, n = 3). Reduced proliferation, invasion and migration were observed as follows: SK1 siRNA <i>P</i> <0.001, <i>P</i> = 0.007 and 0.03; SK2 siRNA <i>P</i> <0.0001, <i>P</i> = 0.003 and <i>P</i> <0.0001; SPL siRNA <i>P</i> = 0.0002, <i>P</i> = 0.03 and <i>P</i> = 0.09, n = 3. (C, D) The FLAG-tagged mammalian expression vector p3FLAG-CMV10 enabled us to obtain stably transfected cells expressing SK1 (HuH7 cells), SK2 (PLC/PRF/5 cells) and SPL (HuH7 cells) (<i>P</i> <0.0001, 0.0001 and 0.0001, n = 3). In these cells, proliferation was enhanced compared with the non-transfected cells (<i>P</i> <0.0001, <i>P</i> = 0.0002, and <i>P</i> <0.0001, n = 3). The asterisk indicates a significant difference.</p

Enhanced SK1 and SK2 mRNA expression in HCC tissues and its association with poorer differentiation.

<p>(A) The metabolic pathways involved in the formation and degradation of S1P are depicted. SK1 (B) and SK2 (C) mRNA levels were increased in HCC compared with adjacent non-tumorous tissues in 54.5% and 93.5% of the patients, respectively; the mean mRNA expression level of SK1 and SK2 in HCC tissues was 3.8-fold and 3.0-fold higher, respectively, than that in non-tumorous tissues (<i>P</i> = 0.02 and <i>P</i> <0.0001, n = 77). (D) The mRNA expression levels of both SKs in HCC tissues compared with those in non-tumorous tissues correlated with the degree of tumor differentiation.</p

Licochalcones extracted from <i>Glycyrrhiza inflata</i> inhibit platelet aggregation accompanied by inhibition of COX-1 activity

<div><p>Licochalcones extracted from <i>Glycyrrhiza inflata</i> are known to have a variety of biological properties such as anti-inflammatory, anti-bacterial, and anti-tumor activities, but their action on platelet aggregation has not yet been reported. Therefore, in this study we investigated the effects of licochalcones on platelet aggregation. Collagen and U46619, a thromboxane A₂ receptor agonist, caused rabbit platelet aggregation, which was reversed by pretreatment with licochalcones A, C and D in concentration-dependent manners. Among these compounds, licochalcone A caused the most potent inhibitory effect on collagen-induced platelet aggregation. However, the licochalcones showed marginal inhibitory effects on thrombin or ADP-induced platelet aggregation. In addition to rabbit platelets, licochalcone A attenuated collagen-induced aggregation in human platelets. Because licochalcone A also inhibited arachidonic acid-induced platelet aggregation and production of thromboxane A₂ induced by collagen in intact platelets, we further examined the direct interaction of licochalcone A with cyclooxygenase (COX)-1. As expected, licochalcone A caused an inhibitory effect on both COX-1 and COX-2 <i>in vitro</i>. Regarding the effect of licochalcone A on COX-1 enzyme reaction kinetics, although licochalcone A showed a stronger inhibition of prostaglandin E₂ synthesis induced by lower concentrations of arachidonic acid, Vmax values in the presence or absence of licochalcone A were comparable, suggesting that it competes with arachidonic acid at the same binding site on COX-1. These results suggest that licochalcones inhibit collagen-induced platelet aggregation accompanied by inhibition of COX-1 activity.</p></div

Licochalcones extracted from <i>Glycyrrhiza inflata</i> inhibit platelet aggregation accompanied by inhibition of COX-1 activity - Fig 1

<p>Chemical structures of licochalcones A, C and D.</p