Simvastatin-romidepsin combination kills bladder cancer cells synergistically

The HMG-CoA reductase inhibitor simvastatin activates AMP-activated protein kinase (AMPK) and thereby induces histone acetylation. We postulated that combining simvastatin with the histone deacetylase (HDAC) inhibitor romidepsin would kill bladder cancer cells by inducing histone acetylation cooperatively. The combination of romidepsin and simvastatin induced robust apoptosis and killed bladder cancer cells synergistically. In murine subcutaneous tumor models using MBT-2 cells, a 15-day treatment with 0.5 mg/kg romidepsin and 15 mg/kg simvastatin was well tolerated and inhibited tumor growth significantly. Mechanistically, the combination induced histone acetylation by activating AMPK. The combination also decreased the expression of HDACs, thus further promoting histone acetylation. This AMPK activation was essential for the combination's action because compound C, an AMPK inhibitor, suppressed the combination-induced histone acetylation and the combination's ability to induce apoptosis. We also found that the combination increased the expression of peroxisome proliferator-activated receptor (PPAR) γ, leading to reactive oxygen species production. Furthermore, the combination induced endoplasmic reticulum (ER) stress and this ER stress was shown to be associated with increased AMPK expression and histone acetylation, thus playing an important role in the combination's action. Our study also suggests there is a positive feedback cycle between ER stress induction and PPARγ expression.     

Accuracy of specimen-specific nonlinear finite element analysis for evaluation of radial diaphysis strength in cadaver material

The feasibility of a user-specific finite element model for predicting the in situ strength of the radius after implantation of bone plates for open fracture reduction was established. The effect of metal artifact in CT imaging was characterized. The results were verified against biomechanical test data. Fourteen cadaveric radii were divided into two groups: (1) intact radii for evaluating the accuracy of radial diaphysis strength predictions with finite element analysis and (2) radii with a locking plate affixed for evaluating metal artifact. All bones were imaged with CT. In the plated group, radii were first imaged with the plates affixed (for simulating digital plate removal). They were then subsequently imaged with the locking plates and screws removed (actual plate removal). Fracture strength of the radius diaphysis under axial compression was predicted with a three-dimensional, specimen-specific, nonlinear finite element analysis for both the intact and plated bones (bones with and without the plate captured in the scan). Specimens were then loaded to failure using a universal testing machine to verify the actual fracture load. In the intact group, the physical and predicted fracture loads were strongly correlated. For radii with plates affixed, the physical and predicted (simulated plate removal and actual plate removal) fracture loads were strongly correlated. This study demonstrates that our specimen-specific finite element analysis can accurately predict the strength of the radial diaphysis. The metal artifact from CT imaging was shown to produce an overestimate of strength.     

Experimental Verification of the Effects on Normal Domestic Cats by Feeding Prescription Diet for Decreasing Stress

The objective of this study was to evaluate the effects of diet on the feline stress response by measuring plasma and urinary cortisol. A study diet was developed with a unique combination of nutrients that supports the management of stressful situations. The specific formulation of the diet included alpha-casozepine, which is believed to have an anxiolytic effect, and tryptophan supplementation. Tryptophan is the precursor for the synthesis of the neurotransmitter serotonin. Twenty-one indoor cats were fed with the study diet (n = 10) or a control diet (n = 11) for 8 weeks, after which physiological responses were evaluated. The study diet significantly increased the ratio of plasma tryptophan to large neutral amino acids and decreased urinary cortisol concentrations after being consumed daily for 8 weeks, but there was no effect on plasma cortisol levels following a stressful event (veterinary examination and blood draw). Further studies, such as behavioral analyses, are needed to clarify the effects of the study diet.     

Global overexpression of divalent metal transporter 1 delays crocidolite-induced mesothelial carcinogenesis in male mice

Abstract

Exposure to asbestos fiber is central to mesothelial carcinogenesis, for which iron overload in or near mesothelial cells is a key pathogenic mechanism. Alternatively, iron chelation therapy with deferasirox or regular phlebotomy was significantly preventive against crocidolite-induced mesothelial carcinogenesis in rats. However, the role of iron transporters during asbestos-induced carcinogenesis remains elusive. Here, we studied the role of divalent metal transporter 1 (DMT1; Slc11a2), which is a Fe(II) transporter, that is present not only on the apical plasma membrane of duodenal cells but also on the lysosomal membrane of every cell, in crocidolite-induced mesothelial carcinogenesis using DMT1 transgenic (DMT1Tg) mice. DMT1Tg mice show mucosal block of iron absorption without cancer susceptibility under normal diet. We unexpectedly found that superoxide production was significantly decreased upon stimulation with crocidolite both in neutrophils and macrophages of DMT1Tg mice, and the macrophage surface revealed higher iron content 1?h after contact with crocidolite. Intraperitoneal injection of 3?mg crocidolite ultimately induced malignant mesothelioma in ～50% of both wild-type and DMT1Tg mice (23/47 and 14/28, respectively); this effect was marginally (p?=?0.069) delayed in DMT1Tg mice, promoting survival. The promotional effect of nitrilotriacetic acid was limited, and the liver showed significantly higher iron content both in DMT1Tg mice and after crocidolite exposure. The results indicate that global DMT1 overexpression causes decreased superoxide generation upon stimulation in inflammatory cells, which presumably delayed the promotional stage of crocidolite-induced mesothelial carcinogenesis. DMT1Tg mice with low-stamina inflammatory cells may be helpful to evaluate the involvement of inflammation in various pathologies.     

Mass Isotopomer Analysis of Metabolically Labeled Nucleotide Sugars and N- and O-Glycans for Tracing Nucleotide Sugar Metabolisms

Nucleotide sugars are the donor substrates of various glycosyltransferases, and an important building block in N- and O-glycan biosynthesis. Their intercellular concentrations are regulated by cellular metabolic states including diseases such as cancer and diabetes. To investigate the fate of UDP-GlcNAc, we developed a tracing method for UDP-GlcNAc synthesis and use, and GlcNAc utilization using ¹³C₆-glucose and ¹³C₂-glucosamine, respectively, followed by the analysis of mass isotopomers using LC-MS.Metabolic labeling of cultured cells with ¹³C₆-glucose and the analysis of isotopomers of UDP-HexNAc (UDP-GlcNAc plus UDP-GalNAc) and CMP-NeuAc revealed the relative contributions of metabolic pathways leading to UDP-GlcNAc synthesis and use. In pancreatic insulinoma cells, the labeling efficiency of a ¹³C₆-glucose motif in CMP-NeuAc was lower compared with that in hepatoma cells.Using ¹³C₂-glucosamine, the diversity of the labeling efficiency was observed in each sugar residue of N- and O-glycans on the basis of isotopomer analysis. In the insulinoma cells, the low labeling efficiencies were found for sialic acids as well as tri- and tetra-sialo N-glycans, whereas asialo N-glycans were found to be abundant. Essentially no significant difference in secreted hyaluronic acids was found among hepatoma and insulinoma cell lines. This indicates that metabolic flows are responsible for the low sialylation in the insulinoma cells. Our strategy should be useful for systematically tracing each stage of cellular GlcNAc metabolism.Protein glycosylation, which is the most abundant post-translational modification, has important roles in many biological processes by modulating conformation and stability, whereas its dysregulation is associated with various diseases such as diabetes and cancer (1, 2). Glycosylation is regulated by various factors including glucose metabolism, the availability and localization of nucleotide sugars, and the expression and localization of glycosyltransferases (3, 4). Thus, ideally all of these components should be considered when detecting changes in a dynamic fashion; namely, it is necessary not only to take a snapshot but also to make movies of the dynamic changes in glycan metabolism.Glucose is used by living cells as an energy source via the glycolytic pathway as well as a carbon source for various metabolites including nucleotide sugars (e.g. UDP-GlcNAc and CMP-NeuAc). These nucleotide sugars are transported into the Golgi apparatus, and added to various glycans on proteins. UDP-GlcNAc is the donor substrate for N-acetylglucosaminyl (GlcNAc)¹ transferases; alternatively, it is used in the cytosol for O-GlcNAc modification (i.e. O-GlcNAcylation) of intracellular proteins (5). The UDP-GlcNAc synthetic pathway is complex as it is a converging point of glucose, nucleotide, fatty acid and amino acid metabolic pathways. Thus, the metabolic flow of glucose modulates the branching patterns of N-glycans via UDP-GlcNAc concentrations because many of the key GlcNAc transferases that determine the branching patterns have widely different K_m values for UDP-GlcNAc ranging from 0.04 mm to 11 mm (6, 7). Indeed, it was demonstrated that the branching formation of N-glycans in T cells is stimulated by the supply from the hexosamine pathway, whereby it regulates autoimmune reactions promoted by T cells (8).UDP-GlcNAc is also used for the synthesis of CMP-NeuAc, the donor substrate for sialyltransferases (9). The CMP-NeuAc concentration is controlled by the feedback inhibition of UDP-GlcNAc epimerase/ManNAc kinase by the final product CMP-NeuAc, and hence a high CMP-NeuAc level reduces metabolic flow in CMP-NeuAc de novo synthesis (10). However, there is still only limited information about how the levels of nucleotide sugars dynamically change in response to the environmental cues, and how such changes are reflected in the glycosylation of proteins.Stable isotope labeling is a promising approach to quantify metabolic changes in response to external cues (11, 12). For example, the use of nuclear magnetic resonance to obtain isotopomer signals of metabolically labeled molecules has been applied to trace the flux in glycolysis and fatty acid metabolism (13). An approach based on the mass isotopomers of labeled metabolites with ¹³C₆-glucose has been developed to monitor the UDP-GlcNAc synthetic pathway (13–15). The method based on the labeling ratio of each metabolite related to UDP-GlcNAc synthesis has clarified the contribution of each metabolic pathway (14). Moseley reported a novel deconvolution method for modeling UDP-GlcNAc mass isotopomers (15).Previous studies into the use of nucleotide sugars in glycosylation have relied on the specific detection of metabolically radiolabeled glycans (16). It is possible not only to deduce the glycan structures but also to trace their relative contributions to glycan synthesis without MS. On the other hand, mass isotopomer analysis of glycans labeled with stable isotope provides the ratios of labeled versus unlabeled molecules from MS spectra and structural details of the glycans. However, there are only a limited number of publications reporting the application of stable isotope labeling of glycans for monitoring the dynamics of glycans (17). To date, there have been no reports describing a systematic method for tracing cellular GlcNAc biosynthesis and use based on mass isotopomer analysis.The aim of this study was to extend our knowledge of the synthesis and metabolism of UDP-GlcNAc as well as its use in the synthesis of CMP-NeuAc, N- and O-glycans. We recently developed a conventional HPLC method for simultaneous determination of nucleotide sugars including unstable CMP-NeuAc (18). We first established an LC-MS method for isotopomer analysis of ¹³C₆-glucose labeled nucleotide sugars for tracing UDP-GlcNAc metabolism from synthesis to use, because previous methods were not suitable for estimating UDP-GlcNAc use in CMP-NeuAc de novo synthesis (15). We also established a method for isotopomer analysis of labeled N- and O-glycan to monitor the metabolic flow of hexosamine into glycans. Using these two methods, we demonstrated the differences in the use of hexosamines between hepatoma and pancreatic insulinoma cell lines. Our approach may be useful for identifying a metabolic “bottleneck” that governs the turnover speed and patterns of cellular glycosylation, which may be relevant for various applications including glycoprotein engineering and discovery of disease biomarkers.     

Pressure-Induced Endocytic Degradation of the Saccharomyces cerevisiae Low-Affinity Tryptophan Permease Tat1 Is Mediated by Rsp5 Ubiquitin Ligase and Functionally Redundant PPxY Motif Proteins

Cells of Saccharomyces cerevisiae express two tryptophan permeases, Tat1 and Tat2, which have different characteristics in terms of their affinity for tryptophan and intracellular localization. Although the high-affinity permease Tat2 has been well documented in terms of its ubiquitin-dependent degradation, the low-affinity permease Tat1 has not yet been characterized fully. Here we show that a high hydrostatic pressure of 25 MPa triggers a degradation of Tat1 which depends on Rsp5 ubiquitin ligase and the EH domain-containing protein End3. Tat1 was resistant to a 3-h cycloheximide treatment, suggesting that it is highly stable under normal growth conditions. The ubiquitination of Tat1 most likely occurs at N-terminal lysines 29 and 31. Simultaneous substitution of arginine for the two lysines prevented Tat1 degradation, but substitution of either of them alone did not, indicating that the roles of lysines 29 and 31 are redundant. When cells were exposed to high pressure, Tat1-GFP was completely lost from the plasma membrane, while substantial amounts of Tat1^K29R-K31R-GFP remained. The HPG1-1 (Rsp5^P514T) and rsp5-ww3 mutations stabilized Tat1 under high pressure, but any one of the rsp5-ww1, rsp5-ww2, and bul1Δ bul2Δ mutations or single deletions of genes encoding arrestin-related trafficking adaptors did not. However, simultaneous loss of 9-arrestins and Bul1/Bul2 prevented Tat1 degradation at 25 MPa. The results suggest that multiple PPxY motif proteins share some essential roles in regulating Tat1 ubiquitination in response to high hydrostatic pressure.     

Noisy Interlimb Coordination Can Be a Main Cause of Freezing of Gait in Patients with Little to No Parkinsonism

Freezing of gait in patients with Parkinson’s disease is associated with several factors, including interlimb incoordination and impaired gait cycle regulation. Gait analysis in patients with Parkinson’s disease is confounded by parkinsonian symptoms such as rigidity. To understand the mechanisms underlying freezing of gait, we compared gait patterns during straight walking between 9 patients with freezing of gait but little to no parkinsonism (freezing patients) and 11 patients with Parkinson’s disease (non-freezing patients). Wireless sensors were used to detect foot contact and toe-off events, and the step phase of each foot contact was calculated by defining one stride cycle of the other leg as 360°. Phase-resetting analysis was performed, whereby the relation between the step phase of one leg and the subsequent phase change in the following step of the other leg was quantified using regression analysis. A small slope of the regression line indicates a forceful correction (phase reset) at every step of the deviation of step phase from the equilibrium phase, usually at around 180°. The slope of this relation was smaller in freezing patients than in non-freezing patients, but the slope exhibited larger step-to-step variability. This indicates that freezing patients executed a forceful but noisy correction of the deviation of step phase, whereas non-freezing patients made a gradual correction of the deviation. Moreover, freezing patients tended to show more variable step phase and stride time than non-freezing patients. Dynamics of a model of two coupled oscillators interacting through a phase resetting mechanism were examined, and indicated that the deterioration of phase reset by noise provoked variability in step phase and stride time. That is, interlimb coordination can affect regulation of the gait cycle. These results suggest that noisy interlimb coordination, which probably caused forceful corrections of step phase deviation, can be a cause of freezing of gait.