Takashi Miida

MDM2 Antagonist Nutlin-3 Displays Antiproliferative and Proapoptotic Activity in Mantle Cell Lymphoma

Purpose: Mantle cell lymphoma (MCL) has one of the poorest prognoses of the non-Hodgkins lymphomas, and novel therapeutic approaches are needed. We wished to determine whether Nutlin-3, a novel small-molecule murine double minute 2 (MDM2) antagonist that efficiently activates TP53, might be effective in inducing cell death in MCL. Experimental Design: MCL cell lines with known TP53 status were treated with Nutlin-3, and biological and biochemical consequences were studied. Synergies with the prototypic genotoxic agent doxorubicin and the novel proteasome inhibitor bortezomib were assessed. Results: Nutlin-3 resulted in a reduction in cell proliferation/viability (IC50 < 10 μmol/L), an increase in the apoptotic fraction, and cell cycle arrest in wild-type (wt) TP53 Z-138 and Granta 519 cells. These effects were accompanied by TP53 accumulation and induction of TP53-dependent proteins p21, MDM2, Puma, and Noxa. Cell cycle arrest was characterized by suppression of S phase and an increase in the G0-G1 and G2-M fractions and accompanied by suppression of total and phosphorylated retinoblastoma protein and a decrease in G2-M-associated proteins cyclin B and CDC2. The combination of Nutlin-3 with doxorubicin or bortezomib was synergistic in wt-TP53 MCL cells. Nutlin-3 also induced cell cycle arrest and reduced cell viability in the mutant TP53 MINO cells but at a significantly higher IC50 (22.5 μmol/L). These effects were associated with induction of the TP53 homologue p73, slight increases in p21 and Noxa, and caspase activation. Nutlin-3 and bortezomib synergistically inhibited cell growth of MINO. Conclusion: These findings suggest that the MDM2 antagonist Nutlin-3 may be an effective agent in the treatment of MCL with or without wt-TP53.

Small dense LDL: An emerging risk factor for cardiovascular disease.

Although low-density lipoprotein cholesterol (LDL-C) is a strong risk factor for coronary artery disease (CAD), LDL-C levels are not always elevated in CAD patients. LDL consists of several subclasses with distinct sizes, densities, and physicochemical compositions. Thus, LDL subclasses can be separated by various laboratory procedures. Among them, ultracentrifugation and electrophoresis have been used most frequently for determining LDL subclasses. Accumulating evidence has shown that a predominance of small dense LDL (sd-LDL) is closely associated with CAD. Moreover, sd-LDL-cholesterol (sd-LDL-C) concentrations are elevated in groups at a high risk for CAD, such as patients with type 2 diabetes and metabolic syndrome. Therefore, sd-LDL concentration is recognized as a surrogate marker for CAD. However, some studies failed to show therapeutic modulation of sd-LDL, likely because separating methods and sd-LDL particle definitions have not yet been standardized. Recently, a detergent-based homogenous assay for sd-LDL-C has been developed. This method does not require any pretreatment, and the measured values are highly reproducible with an automated analyzer. These features are suitable for large-scale clinical studies. This homogeneous assay is a useful tool for clarifying whether sd-LDL-C is a superior marker to LDL-C, and whether sd-LDL-C lipid-lowering therapies decrease the incidence of CAD.

Probucol markedly reduces HDL phospholipids and elevated preβ1-HDL without delayed conversion into α-migrating HDL: Putative role of angiopoietin-like protein 3 in probucol-induced HDL remodeling

Probucol is a unique hypolipidemic agent that increases cholesteryl ester transfer protein (CETP) activity. Enhanced CETP-mediated conversion of high-density lipoprotein (HDL) partly explains the probucol-induced decrease in HDL cholesterol and increase in plasma prebeta1-HDL (native lipid-poor HDL) concentrations. However, HDL cholesterol is reduced in patients that are completely deficient in CETP. Angiopoietin-like protein 3 (ANGPTL3) is an endogenous suppressor of endothelial lipase that promotes the hydrolysis of HDL phospholipids and may generate prebeta1-HDL. To determine whether probucol decreases ANGPTL3 and HDL phospholipids while increasing prebeta1-HDL, we measured these parameters before and after a 4-week probucol treatment in 39 hypercholesterolemic patients and age- and sex-matched controls. The median ANGPTL3 had decreased from 143 to 113 microg/L by week 4 (p<0.05). High-performance liquid chromatography revealed that probucol decreased the phospholipid content of very large (13.5-15 nm) and large (12.1 nm) HDL particles predominantly by 65% (p<0.01) and 53% (p<0.001), respectively. The change in ANGPTL3, but not CETP mass, was positively correlated with that in large HDL phospholipids (r=0.455, p<0.05). The absolute and relative concentrations of prebeta1-HDL increased by 14% (p<0.01) and 60% (p<0.001), respectively. The conversion rate of prebeta1-HDL into alpha-migrating HDL by lecithin-cholesterol acyltransferase did not change significantly. In conclusion, probucol decreases plasma ANGPTL3 and HDL phospholipids while increasing prebeta1-HDL. We speculate that probucol induces HDL remodeling via an endothelial lipase-mediated pathway.

Experimental evidence that phenylalanine is strongly associated to oxidative stress in adolescents and adults with phenylketonuria

Few studies have looked at optimal or acceptable serum phenylalanine levels in later life in patients with phenylketonuria (PKU). This study examined the oxidative stress status of adolescents and adults with PKU. Forty PKU patients aged over fifteen years were enrolled, and were compared with thirty age-matched controls. Oxidative stress markers, anti-oxidant enzyme activities in erythrocytes, and blood anti-oxidant levels were examined. Nitric oxide (NO) production was also examined as a measure of oxidative stress. Plasma thiobarbituric acid reactive species and serum malondialdehyde-modified LDL levels were significantly higher in PKU patients than control subjects, and correlated significantly with serum phenylalanine level (P<0.01). Plasma total anti-oxidant reactivity levels were significantly lower in the patient group, and correlated negatively with phenylalanine level (P<0.001). Erythrocyte superoxide dismutase and catalase activities were higher and correlated significantly with phenylalanine level (P<0.01). Glutathione peroxidase activity was lower and correlated negatively with phenylalanine level (P<0.001). The oxidative stress score calculated from these six parameters was significantly higher in patients with serum phenylalanine of 700-800 μmol/l. Plasma anti-oxidant substances, beta-carotene, and coenzyme Q(10) were also lower (P<0.001), although the decreases did not correlate significantly with the phenylalanine level. Serum nitrite/nitrate levels, as stable NO products, were higher together with low serum asymmetric dimethylarginine, as an endogenous NO inhibitor. Oxidative stress status is closely linked with serum phenylalanine levels. Phenylalanine level in should be maintained PKU below 700-800 μmol/l even in adult patients.

High preβ1-HDL levels in hypercholesterolemia are maintained by probucol but reduced by a low-cholesterol diet

Previous study has shown that prebeta1-HDL levels increase in hypercholesterolemia, or high cholesteryl ester transfer protein (CETP) activity. To determine how prebeta1-HDL levels change after treatment with probucol or by following a low-cholesterol diet, we randomly assigned 24 hypercholesterolemic patients to either the probucol (P), or low-cholesterol diet group (D), and measured prebeta1-HDL levels before and after treatments using native two-dimensional gel electrophoresis. We also examined 12 subjects with normolipidemia (N). At baseline, prebeta1-HDL levels were higher in P (P < 0.05) and D (P < 0.05) than in N (9.2 +/- 4.3, 10.4 +/- 5.5, and 5.9 +/- 2.3 mg/dl apo A-I). After a 4-week treatment, prebeta1-HDL levels were still high in P (10.5 +/- 4.2 mg/dl apo A-I, N.S.), but reduced in D (7.7 +/- 3.0 mg/dl apo A-I, P < 0.001). Delta prebeta1-HDL (Y) was positively correlated with deltaCETP mass (X) in P (y = 7.83x - 1.93; r = 0.584, P < 0.05). In summary, high prebeta1-HDL levels in hypercholesterolemia are maintained by probucol but reduced by a low-cholesterol diet. These findings suggest that prebeta1-HDL levels may be regulated by cholesterol and CETP levels.

Analytical performance of a sandwich enzyme immunoassay for preβ1-HDL in stabilized plasma

We have established an immunoassay for preβ1-HDL (the initial acceptor of cellular cholesterol) using a monoclonal antibody, MAb55201. Because preβ1-HDL is unstable during storage, fresh plasma must be used for preβ1-HDL measurements. In this study, we describe a method of stabilizing preβ1-HDL, and evaluate the analytical performance of the immunoassay for preβ1-HDL. Fresh plasma was stored under various conditions with or without a pretreatment consisting of a 21-fold dilution into 50% (v/v) sucrose. Preβ1-HDL concentration was measured by immunoassay. In nonpretreated samples, preβ1-HDL decreased significantly from the baseline after 6 h at room temperature. Although preβ1-HDL was more stable at 0°C than at room temperature, it increased from 30.2 ± 8.5 (SE) to 56.5 ± 5.5 mg/l apolipoprotein A-I (apoA-I) (P < 0.001) in hyperlipidemics, and from 18.4 ± 1.2 to 37.9 ± 3.3 mg/l apoA-I (P < 0.001) in normolipidemics after 5-day storage. After 30-day storage at −80°C, preβ1-HDL increased from 29.0 ± 4.0 to 38.0 ± 5.7 mg/l apoA-I (P < 0.001) in hyperlipidemics, whereas it did not change in normolipidemics. In pretreated samples, preβ1-HDL concentration did not change significantly under any of the above conditions. Moreover, preβ1-HDL concentrations determined by immunoassay correlated with those determined by native two-dimensional gel electrophoresis (n = 24, r = 0.833, P < 0.05). An immunoassay using MAb55201 with pretreated plasma is useful for clinical measurement of preβ1-HDL.

Role of stromal microenvironment in nonpharmacological resistance of CML to imatinib through Lyn/CXCR4 interactions in lipid rafts

We and others have previously demonstrated that p210 Bcr-Abl tyrosine kinase inhibits stromal cell-derived factor-1α/CXCR4 chemokine receptor signaling, contributing to the deficient adhesion of chronic myeloid leukemia (CML) cells to bone marrow stroma. Conversely, exposure of CML cells to a tyrosine kinase inhibitor (TKI) enhances migration of CML cells towards stromal cell layers and promotes non-pharmacological resistance to imatinib. Src-related kinase Lyn is known to interact with CXCL12/CXCR4 signaling and is directly activated by p210 Bcr-Abl. In this study, we demonstrate that TKI treatment promoted CXCR4 redistribution into the lipid raft fraction, in which it co-localized with active phosphorylated form of Lyn (LynTyr396) in CML cells. Lyn inhibition or cholesterol depletion abrogated imatinib-induced migration, and dual Src/Abl kinase inhibitor dasatinib induced fewer CML cells to migrate to the stroma. These findings demonstrate the novel mechanism of microenvironment-mediated resistance through lipid raft modulation, which involves compartmental changes of the multivalent CXCR4 and Lyn complex. We propose that pharmacological targeting of lipid rafts may eliminate bone marrow-resident CML cells through interference with microenvironment-mediated resistance.

Impacts of angiopoietin-like proteins on lipoprotein metabolism and cardiovascular events.

Purpose of review Angiopoietin-like proteins (Angptls) comprise a family of secreted glycoproteins with high homology to angiopoietins which are important regulators of angiogenesis. Angptl3 and Angptl4 inhibit lipoprotein lipase in mice. This article reviews recent human studies on Angptls and their effects on lipoprotein profiles and cardiovascular disease. Recent findings Population-based studies have indicated that loss-of-function mutations of Angptl3, Angptl4, and Angptl5 are associated with a low triglyceride concentration. Angptl3 concentration is positively correlated with HDL-cholesterol, but not with triglyceride, suggesting that Angptl3 regulates HDL metabolism by inhibiting endothelial lipase. Angptl4 concentration is correlated positively with triglycerides in patients with metabolic syndrome, although most studies have failed to find such a correlation. Angptl6 has no effects on lipoprotein profiles. Angptl3 is associated with atherosclerosis in coronary, carotid, and femoral arteries. Conflicting results have been obtained regarding whether the E40K variant (a loss-of-function mutation of Angptl4) is associated with an increased risk for cardiovascular disease, which may occur due to the lipid-independent actions of Angptl4. Summary Angptl3, Angptl4, and possibly Angptl5 are regulators of lipoprotein metabolism in humans. Whether inhibition of these Angptls will be useful for dyslipidemia to prevent cardiovascular disease remains to be elucidated in future studies.

Single Administration of α-Glucosidase Inhibitors on Endothelial Function and Incretin Secretion in Diabetic Patients With Coronary Artery Disease - Juntendo University Trial: Effects of Miglitol on Endothelial Vascular Reactivity in Type 2 Diabetic Patients With Coronary Heart Disease (J-MACH) -

Background: Post-prandial hyperglycemia, hyperlipidemia, and endothelial dysfunction play an important role in the pathogenesis of atherosclerosis. Improvement in post-prandial hyperglycemia on α-glucosidase inhibitors (α-GIs) is associated with a risk reduction of cardiovascular diseases, but the post-prandial effects of α-GIs on endothelial function and incretin secretion in type 2 diabetic patients with coronary artery disease (CAD) remain unclear. Methods and Results: The post-prandial effects of a single administration of miglitol and voglibose on endothelial function and changing levels of glucose, insulin, lipids, glucagon-like peptide (GLP)-1, and gastric inhibitory polypeptide (GIP) were compared after a standard meal loading in 11 diabetic patients with CAD, using a placebo-controlled cross-over design. The changing levels of glucose, insulin and triglycerides at 60 min were significantly lower in the miglitol group than in the voglibose and placebo groups (all P<0.01). GLP-1 levels were significantly higher at 120 min (P<0.05) and GIP levels were significantly lower at 30 min and 60 min (P<0.05) in the miglitol group compared to other treatments. The reactive hyperemia duration at 120 min was significantly maintained in the miglitol group compared to the other groups. Conclusions: A single administration of miglitol significantly improved post-prandial glucose/lipid metabolism, incretin secretion, and endothelial dysfunction in diabetic patients with CAD, suggesting that miglitol may be a useful anti-atherogenic agent (UMIN000002264).  (Circ J 2010; 74: 1471 - 1478)

TGF-β-Neutralizing Antibody 1D11 Enhances Cytarabine-Induced Apoptosis in AML Cells in the Bone Marrow Microenvironment.

Hypoxia and interactions with bone marrow (BM) stromal cells have emerged as essential components of the leukemic BM microenvironment in promoting leukemia cell survival and chemoresistance. High levels of transforming growth factor beta 1 (TGFβ1) produced by BM stromal cells in the BM niche regulate cell proliferation, survival, and apoptosis, depending on the cellular context. Exogenous TGFβ1 induced accumulation of acute myeloid leukemia (AML) cells in a quiescent G0 state, which was further facilitated by the co-culture with BM-derived mesenchymal stem cells (MSCs). In turn, TGFβ-neutralizing antibody 1D11 abrogated rhTGFβ1 induced cell cycle arrest. Blocking TGFβ with 1D11 further enhanced cytarabine (Ara-C)–induced apoptosis of AML cells in hypoxic and in normoxic conditions. Additional constituents of BM niche, the stroma-secreted chemokine CXCL12 and its receptor CXCR4 play crucial roles in cell migration and stroma/leukemia cell interactions. Treatment with 1D11 combined with CXCR4 antagonist plerixafor and Ara-C decreased leukemia burden and prolonged survival in an in vivo leukemia model. These results indicate that blockade of TGFβ by 1D11 and abrogation of CXCL12/CXCR4 signaling may enhance the efficacy of chemotherapy against AML cells in the hypoxic BM microenvironment.