Dexamethasone exacerbates cisplatin-induced muscle atrophy.

Dexamethasone for antiemetic therapy is typically administered with anticancer drugs such as cisplatin. We previously reported that cisplatin upregulates the muscle-specific E3 ubiquitin ligase genes, namely muscle ring-finger protein 1 (MuRF1) and atrophy gene-1 (atrogin-1), and promotes muscle atrophy in mice. It is well known that dexamethasone causes upregulation of MuRF1 and Atrogin-1 expression in skeletal muscles. Although it is speculated that a combination of dexamethasone and cisplatin worsens muscle atrophy, there are no reports based on research. We thereby investigated the effects of cisplatin and dexamethasone, alone or in combination, on the expression of MuRF1 and Atrogin-1 in murine skeletal muscles and C2C12 myotubes. Mice were intraperitoneally injected with cisplatin or the vehicle control once daily for 4 days. Dexamethasone or the vehicle control was subcutaneously administered 30 minutes prior to the administration of cisplatin. Dexamethasone enhanced MuRF1 and Atrogin-1 gene expression upregulated by cisplatin in murine quadriceps muscles and C2C12 myotubes. Cisplatin-caused upregulation of myostatin and downregulation of IGF-1 gene expression were also enhanced by co-administration of dexamethasone in murine quadriceps muscles and C2C12 myotubes. This study shows that the combination treatment of cisplatin and dexamethasone exacerbated muscle atrophy in mice. Therefore, this treatment regimen might exacerbate muscle atrophy in cancer patients.

Effect of acute treadmill exercise on cisplatin-induced muscle atrophy in the mouse.

Cisplatin, a platinum-based anti-cancer drug, is one of the most effective broad-spectrum anti-cancer agents used against various cancers. It has been recently suggested that low skeletal muscle mass is predictive of mortality in patients with cancer. Although several molecules produced by the actual tumor itself contribute to skeletal muscle impairment, we recently suggested that the administration of cisplatin could increase levels of muscle RING finger-1 (MuRF1) and atrogin-1, possibly leading to muscle atrophy in the mouse. Exercise is an important factor that induces muscle protein synthesis and muscle hypertrophy by enhancing the positive effects of the Akt/mTOR/p70S6 kinase pathway. In the present study, we therefore investigated the effect of treadmill exercise on cisplatin-induced muscle atrophy. C57BL/6J mice were treated with cisplatin (3 mg/kg, i.p.) or saline for four consecutive days. On day 4, the quadriceps and gastrocnemius muscles were isolated from the mice. The animals in the treadmill exercise groups were forced to run on a motorized treadmill for 20 min once a day for 9 days. In addition to muscle mass, the decrease in myofiber diameter associated with cisplatin administration was significantly restored by treadmill exercise. This exercise also significantly attenuated cisplatin-induced upregulation of MuRF1 and atrogin-1 in quadriceps and gastrocnemius muscle. The decreased Akt, p70S6 kinase, and Foxo3a phosphorylation observed with cisplatin treatment was significantly recovered by treadmill exercise in both the muscles. In the present study, myostatin (Mstn) gene expression, upregulated by cisplatin administration, was also attenuated by treadmill exercise. These findings suggest that treadmill exercise could attenuate cisplatin-induced muscle atrophy, at least partially, and could improve prognosis.

Active Ingredients of Hange-shashin-to, Baicalelin and 6-Gingerol, Inhibit 5-Fluorouracil-Induced Upregulation of CXCL1 in the Colon to Attenuate Diarrhea Development.

5-Fluorouracil (5-FU) is widely used as an anti cancer drug and is known to cause severe diarrhea. Recently we suggested that levels of chemokine (C-X-C motif) ligand 1 (CXCL1) and neutrophil recruitment in the colonic mucosa were drastically increased by the 5-FU administration in mice. Hange-shashin-to (HST) is prescribed in Japan for treat gastritis, stomatitis, and inflammatory diarrhea. We therefore examined the effects of HST and its active ingredients on 5-FU-induced CXCL1 upregulation in cultured colon tissue, and also examined the effects of HST on 5-FU-induced diarrhea development in the mouse. The distal colon isolated from the mouse was incubated with 5-FU and HST. Mice were given 5-FU (50 mg/kg, intraperitoneally (i.p.)) daily for four days. HST (300 mg/kg, per os (p.o.)) was administered 30 min before mice received 5-FU. mRNA levels of CXCL1 in the colon were examined using quantitative RT-PCR. 5-FU enhanced CXCL1 mRNA in the colon but the effect by 5-FU was markedly suppressed by application of HST and its active ingredients, baicalein and 6-gingerol. Nuclear factor kappa B (NF-κB) was activated by 5-FU treatment in cultured colon tissue, which was also suppressed by HST and the combination of baicalein and 6-gingerol. Furthermore, HST reduced 5-FU-induced diarrhea development. Under such experimental condition, CXCL1 gene, protein levels of neutrophil elastase and myeloperoxidase upregulation induced by 5-FU in the colon was attenuated by HST. These findings suggest that HST, especially baicalein and 6-gingerol, prevent the development of neutrophil recruitment and diarrhea by the inhibition of NF-κB activity.

Role of peptide YY in 5-fluorouracil-induced reduction of dietary intake.

5-fluorouracil (5-FU) is part of the standard care for cancer treatment but is associated with high incidences of appetite loss and reduced food intake, which may contribute to chemotherapy-induced cachexia (weakness and wasting of tissue). The role of gastrointestinal satiety hormones in chemotherapy-induced appetite loss has not been intensively investigated. Peptide YY (PYY) and glucagon-like peptide (GLP)-1 are important signals of gastrointestinal satiety, so this study examined the roles of these gut hormones in 5-FU-induced reduction of dietary intake. Mice were given 5-FU (50 mg/kg, intraperitoneal [i.p.]) every day for 4 consecutive days. Gene expression levels of proglucagon (Pro-Gcg), a precursor of GLP-1, and PYY in the colon were examined by real-time RT-PCR. Serum levels of GLP-1 and PYY were measured by enzyme-linked immunosorbent assay. Some mice were pretreated with the GLP-1 receptor antagonist exendin9-39 (1 mg/kg) or the neuropeptide Y type 2 (NPY2) receptor antagonist BIIE0246 (2 mg/kg) via the i.p. route 30 minutes before 5-FU administration. Mice receiving 5-FU exhibited a significant reduction in food intake that was correlated with body weight loss. These mice also showed significantly enhanced expression levels of mRNAs encoding pro-GLP-1 and PYY in the transverse and distal colon as well as elevated serum concentrations of GLP-1 and PYY compared to vehicle-treated controls. The 5-FU-induced reduction in food intake was attenuated by BIIE0246 but not by exendin9-39. These data suggest that administration of a NPY2 receptor antagonist may be effective for attenuating the anorexia caused by 5-FU chemotherapy.

Imprinted DNA methylation of the H19 ICR is established and maintained in vivo in the absence of Kaiso.

BACKGROUND: Paternal allele-specific DNA methylation of the imprinting control region (H19 ICR) controls genomic imprinting at the Igf2/H19 locus. We previously demonstrated that the mouse H19 ICR transgene acquires imprinted DNA methylation in preimplantation mouse embryos. This activity is also present in the endogenous H19 ICR and protects it from genome-wide reprogramming after fertilization. We also identified a 118-bp sequence within the H19 ICR that is responsible for post-fertilization imprinted methylation. Two mutations, one in the five RCTG motifs and the other a 36-bp deletion both in the 118-bp segment, caused complete and partial loss, respectively, of methylation following paternal transmission in each transgenic mouse. Interestingly, these mutations overlap with the binding site for the transcription factor Kaiso, which is reportedly involved in maintaining paternal methylation at the human H19 ICR (IC1) in cultured cells. In this study, we investigated if Kaiso regulates imprinted DNA methylation of the H19 ICR in vivo. RESULTS: Neither Kaiso deletion nor mutation of Kaiso binding sites in the 118-bp region affected DNA methylation of the mouse H19 ICR transgene. The endogenous mouse H19 ICR was methylated in a wild-type manner in Kaiso-null mutant mice. Additionally, the human IC1 transgene acquired imprinted DNA methylation after fertilization in the absence of Kaiso. CONCLUSIONS: Our results indicate that Kaiso is not essential for either post-fertilization imprinted DNA methylation of the transgenic H19 ICR in mouse or for methylation imprinting of the endogenous mouse H19 ICR.

Mosaic Analysis with a Repressible Cell Marker (MARCM) Clone Generation in Drosophila Dendritic Arborization Neurons.

Mosaic analysis with a repressible cell marker (MARCM) is used in Drosophila research to create labeled homozygous mutant clones of cells in an otherwise heterozygous fly. It allows the study of the effect of embryonically lethal genes and the determination of cell autonomy for a mutant phenotype. When used in dendritic arborization (da) neurons with a fluorescent protein targeted to the plasma membrane, MARCM allows the identification of homozygous mutant neurons and clear imaging of the dendrite arbor in both live and fixed preparations. Previous protocols that outlined experimental procedures to create MARCM clones in da neurons used a heat shock promoter to drive Flippase (FLP) expression; such an approach requires laborious embryo collection and heat shock steps, and it creates clones in other tissues besides the da neurons. The updated protocol described here outlines the use of FLP expression driven by a sensory organ precursor promoter (SOP-FLP); it requires no embryo collection or manipulation steps and creates clones exclusively in the peripheral sensory neuron lineage.

Use of DeTerm for Automated Drosophila Dendrite Arbor Terminal Counts.

Neurons have a complex dendritic architecture that governs information flow through a circuit. Manual quantification of dendritic arbor morphometrics is time-consuming and can be inaccurate. Automated quantification systems such as DeTerm help to overcome these limitations. DeTerm is a software tool that automatically recognizes dendrite branch terminals with high precision. It uses an artificial neural network to label the terminals, count them, and provide each terminal's positional data. DeTerm can recognize the dendritic terminals of Drosophila dendritic arborization (da) neurons, and it can also examine other types of neurons, including mouse Purkinje cells. It is freely available and works on Mac, Windows, and Linux. Here, we describe the use of DeTerm.

Mounting of Embryos, Larvae, and Pupae for Live Drosophila Dendritic Arborization Neuron Imaging.

Live imaging approaches are essential for monitoring how neurons go through a coordinated series of differentiation steps in their native mechanical and chemical environment. These imaging approaches also allow the study of dynamic subcellular processes such as cytoskeleton remodeling and the movement of organelles. Drosophila dendritic arborization (da) neurons are a powerful experimental system for studying the dendrite arbor in live animals. da neurons are located on the internal surface of the body wall and, therefore, are easily accessible for imaging. Moreover, many genetic tools target da neurons to disrupt genes or proteins of interest and allow the investigator to visualize fluorescent markers and endogenously tagged proteins in the neurons. This protocol introduces methods for preparing and mounting intact Drosophila embryos, larvae, and pupae, allowing live imaging of dynamic cellular processes in da neurons.

Study of Dendrite Differentiation Using Drosophila Dendritic Arborization Neurons.

Neurons receive, process, and integrate inputs. These operations are organized by dendrite arbor morphology, and the dendritic arborization (da) neurons of the Drosophila peripheral sensory nervous system are an excellent experimental model for examining the differentiation processes that build and shape the dendrite arbor. Studies in da neurons are enabled by a wealth of fly genetic tools that allow targeted neuron manipulation and labeling of the neuron's cytoskeletal or organellar components. Moreover, as da neuron dendrite arbors cover the body wall, they are highly accessible for live imaging analysis of arbor patterning. Here, we outline the structure and function of different da neuron types and give examples of how they are used to elucidate central mechanisms of dendritic arbor formation.

A novel function of the human chaperonin CCT epsilon subunit in yeast.

A malfunction in the yeast HAC1 causes the unfolding-protein response in the endoplasmic reticulum, resulting in stress-sensitive and inositol auxotrophic phenotypes. Chaperonin-containing TCP1 (CCT) is necessary for the folding of actin and tubulin in the cytosol. The introduction of the truncated human CCT epsilon subunit into yeast cells of which hac1 was disrupted clearly suppressed not only its inositol auxotrophic phenotype but also its stress-sensitive phenotype.

Theoretical study on isomerization of α-acids: A DFT calculation.

The α-acids contained in hops are one of the ingredients of beer. The isomerization of α-acids produces iso-α-acids, the main source of bitterness in beer. In this study, the isomerization mechanism of the α-acid, cohumulone, was elucidated by using density functional theory in conjunction with the polarizable continuum model or 3D-RISM integral equation theory of liquids. The calculated reaction diagram is consistent with experimental results; the activation free energy difference between the cis and trans isomers is in good agreement with the experimental estimate. The activation energy difference results from solvation energy. Additionally, a calculation of NMR chemical shifts showed that the proton position of isocohumulone is different from that proposed previously. The effect of Mg2+ cation on the isomerization was also investigated. Both the activation and reaction free energy are stabilized by the presence of Mg2+, which is consistent with experimental results. Water solvation reduces the activation free energy.

Curcumin Inhibits 5-Fluorouracil-induced Up-regulation of CXCL1 and CXCL2 of the Colon Associated with Attenuation of Diarrhoea Development.

The compound 5-fluorouracil (5-FU) is used in cancer chemotherapy and is known to cause diarrhoea. We recently reported that chemokine (C-X-C motif) ligand 1 (CXCL1) and neutrophils in the colonic mucosa were markedly increased by the administration of 5-FU in mice. Curcumin has anti-inflammatory, antitumour and antioxidant properties. Therefore, we examined the effect of curcumin on 5-FU-induced diarrhoea development and CXCL1 and CXCL2 up-regulation in the colon. Mice were given 5-FU (50 mg/kg, i.p.) daily for 4 days. Curcumin (100 or 300 mg/kg, p.o.) was administered on the day before the first administration of 5-FU and administered 30 min. before the administration of 5-FU. Gene expression levels of CXCL1 and CXCL2 in the colon were examined by real-time RT-PCR. Curcumin reduced the 5-FU-induced diarrhoea development. Under this condition, the CXCL1 and CXCL2 gene up-regulated by 5-FU administration was inhibited by curcumin. The gene expression of CXCL1 and CXCL2 was also enhanced by 5-FU application in vitro. The 5-FU-induced up-regulated CXCL1 and CXCL2 gene expressions were inhibited by curcumin, Bay-117082 and bortezomib, nuclear factor kappa B (NF-κB) inhibitors, C646, a p300/cyclic adenosine monophosphate response element-binding protein-histone acetyltransferase (HAT) inhibitor. In conclusion, these findings suggested that curcumin prevented the development of diarrhoea by inhibiting NF-κB and HAT activation.

Preparation of O/I1-type Emulsions and S/I1-type Dispersions Encapsulating UV-Absorbing Agents.

Oil-in-cubic phase (O/I1) emulsions encapsulating the cosmetic UV absorbing agents 2-ethylhexyl 4-methoxycinnamate (EHMC), 2-ethylhexyl 2-cyano-3,3-diphenylacrylate (octocrylene, OCR) and 1-(4-tertbutylphenyl)-3-(4-methoxyphenyl)-1,3-propanedione (Avobenzone, TBMP) were prepared by vortex mixing accompanied by a heating-cooling process. A ternary phase diagram in a water/C12EO25/EHMC system at 25°C was constructed and the two-phase equilibrium of an oil phase and an I1 phase, which is necessary to prepare the O/I1-type emulsions, was confirmed. Also, the melting of the I1 phase into a fluid micellar solution phase was confirmed, allowing emulsification by a heating-cooling process. The O/I1-type emulsions were formulated in the ternary system as well as a quaternary system. The four-component system contained an additional cosolvent, isopropyl myristate (IPM). The use of the cosolvent allows the use of reduced amounts of EHMC, which is desirable because EHMC can cause temporary skin irritation. Formulation of the O/I1-type emulsions with other UV absorbing agents (OCR and TBMP) was also possible using the same emulsification method. When IPM was changed to tripalmitin, which has a melting point greater than room temperature, a solid-oil dispersion in I1 phase was formed. We have termed this a "solidin-cubic phase (S/I1) type dispersion". These novel emulsions have not been reported previously. The UV absorbability of the O/I1-type emulsions and S/I1-type dispersions that encapsulate the UV absorbing agents was confirmed by measurement of UV absorption spectra.