Hideki Arimochi

Betulinic acid augments the inhibitory effects of vincristine on growth and lung metastasis of B16F10 melanoma cells in mice.

We examined the antitumour effect of a combination of betulinic acid (BA) and vincristine (VCR) on murine melanoma B16F10 cells in vitro and in vivo. Betulinic acid, a pentacyclic triterpene, showed a synergistic cytotoxic effect on melanoma cells by combinational use of VCR. Betulinic acid and VCR induced cell cycle arrest at different points (BA at G1 phase and VCR at G2/M phase) and caused apoptosis in B16F10 melanoma cells. In the in vivo study, VCR inhibited metastasis of tumour cells to the lung. The addition of BA to VCR augmented suppression of the experimental lung metastasis of melanoma cells in C57BL/6 mice. The number of lung nodules of more than 1 mm in diameter in mice treated with BA and VCR was less than that in mice treated with VCR alone. These results suggest that BA is an effective supplement for enhancing the chemotherapeutic effect on malignant melanoma.

Reduced Diversity and Imbalance of Fecal Microbiota in Patients with Ulcerative Colitis

BackgroundClinical observations and experimental colitis models have indicated the importance of intestinal bacteria in the etiology of ulcerative colitis (UC), but a causative bacterial agent has not been identified.AimTo determine how intestinal bacteria are associated with UC, fecal microbiota and other components were compared for UC patients and healthy adults.MethodsFresh feces were collected from 48 UC patients. Fecal microbiota were analyzed by use of terminal-restriction fragment length polymorphism (T-RFLP), real-time PCR, and culture. The concentrations of organic acids, indole, and ammonia, and pH and moisture, which are indicators of the intestinal environment, were measured and compared with healthy control data.ResultsT-RFLP data divided the UC patients into four clusters; one cluster was obtained for healthy subjects. The diversity of fecal microbiota was significantly lower in UC patients. There were significantly fewer Bacteroides and Clostridium subcluster XIVab, and the amount of Enterococcus was higher in UC patients than in healthy subjects. The fecal concentration of organic acids was significantly lower in UC patients who were in remission.ConclusionUC patients have imbalances in the intestinal environment—less diversity of fecal microbiota, lower levels of major anaerobic bacteria (Bacteroides and Clostridium subcluster XIVab), and a lower concentration of organic acids.

Desipramine induces apoptotic cell death through nonmitochondrial and mitochondrial pathways in different types of human colon carcinoma cells

Cytotoxic effects of desipramine on human colon carcinoma HT29 and HCT116 cells were examined. Desipramine reduced the viability of HT29 cells in a concentration-dependent manner, but failed to cause any significant change in the viability of HCT116 cells by the concentration up to 50 µmol/l, at which an approximately 60% reduction of the viability of HT29 cells was observed. Despite their different sensitivities, desipramine caused the nonoxidative apoptotic damage to both of them. In contrast to HT29 cells, desipramine might cause the apoptotic death of HCT116 cells through the disturbance of mitochondrial function. These results suggest that desipramine may cause the nonoxidative apoptotic damage to different types of human colon carcinoma cells through either a nonmitochondrial or a mitochondrial pathway, which may confer the different sensitivities to this drug on these tumor cells.

Inhibitory effects of fermented brown rice on induction of acute colitis by dextran sulfate sodium in rats.

Although the pathogenic mechanisms of inflammatory bowel diseases are not fully understood, colonic microbiota may affect the induction of colonic inflammation, and some probiotics and prebiotics have been reported to suppress colitis. The inhibitory effects of brown rice fermented by Aspergillus oryzae (FBRA), a fiber-rich food, on the induction of acute colitis by dextran sulfate sodium (DSS) were examined. Feeding a 5% and 10% FBRA-containing diet significantly decreased the ulcer and erosion area in the rat colon stained with Alcian blue. In another experiment, 10% FBRA feeding decreased the ulcer index (percentage of the total length of ulcers in the full length of the colon) and colitis score, which were determined by macroscopic observation. It also decreased myeloperoxidase activity in the colonic mucosa. Viable cell numbers of Lactobacillus in the feces decreased after DSS administration and was reversely correlated with severity of colitis, while the cell number of Enterobacteriaceae increased after DSS treatment and was positively correlated with colitis severity. These results indicate that FBRA has a suppressive effect on the induction of colitis by DSS and suggest FBRA-mediated modification of colonic microbiota.

Histone deacetylase inhibitors promote neurosteroid-mediated cell differentiation and enhance serotonin-stimulated brain-derived neurotrophic factor gene expression in rat C6 glioma cells

Progesterone treatment has previously been reported to promote the differentiation of glial cells probably through the production of 5α‐reduced neurosteroids, resulting in the enhancement of serotonin‐stimulated brain‐derived neurotrophic factor (BDNF) gene expression, which is considered to contribute to the survival, regeneration, and plasticity of neuronal cells in the brain and hence has been suggested to improve mood disorders and other symptoms in depressive patients. Based on these previous observations, the effects on glial cells of histone deacetylase (HDAC) inhibitors, which are known as agents promoting cell differentiation, were examined using rat C6 glioma cells as a model for in vitro studies. Consequently, trichostatin A (TSA), sodium butyrate (NaB), and valproic acid (VPA) stimulated glial fibrillary acidic protein (GFAP) gene expression, and their stimulatory effects on GFAP gene expression were inhibited by treatment of these cells with finasteride, an inhibitor of the enzyme producing 5α‐reduced neurosteroids. In addition, HDAC inhibitors enhanced serotonin‐stimulated BDNF gene expression, the enhancement of which could be abolished by the inhibition of 5α‐reduced neurosteroid production in the glioma cells. These results suggest that HDAC inhibitors may be able to promote the differentiation of rat C6 glioma cells through the production of 5α‐reduced neurosteroids, resulting in the enhancement of serotonin‐stimulated BDNF gene expression as a consequence of promoting their differentiation, indicating the possibility that differentiated glial cells may be implicated in preserving the integrity of neural networks as well as improving the function of neuronal cells in the brain.

Possible involvement of 5α-reduced neurosteroids in adrenergic and serotonergic stimulation of GFAP gene expression in rat C6 glioma cells

Influence of adrenergic and serotonergic stimulation on glial fibrillary acidic protein (GFAP) gene expression in rat C6 glioma cells was first examined as an in vitro model experiment for investigating the neuronal regulation of glial cell differentiation. Stimulation of these cells with isoproterenol and serotonin elevated GFAP mRNA levels followed by an increase in its protein contents, thus suggesting that both adrenergic and serotonergic stimulation might induce the differentiation of the glioma cells. In addition, progesterone and its 5alpha-reduced metabolite dihydroprogesterone also elevated GFAP mRNA levels in rat C6 glioma cells, consistent with their stimulatory actions on GFAP gene expression observed in rat astrocytes. Further studies showed that the elevation of GFAP mRNA levels induced by isoproterenol and serotonin as well as progesterone was abolished by pretreatment of the glioma cells with finasteride, an inhibitor of 5alpha-reduced steroid production. Moreover, the stimulatory actions of isoproterenol and serotonin on GFAP gene expression were inhibited by pretreatment with a GABA(A) receptor antagonist bicuculline and a progesterone receptor antagonist RU486. These findings suggest that both adrenergic and serotonergic stimulation may indirectly activate GFAP gene expression probably through the production of 5alpha-reduced steroid metabolites in rat C6 glioma cells, proposing the possibility that 5alpha-reduced neurosteroids may play a potential role in the neuronal regulation of glial cell differentiation.

Genetic Variation in 16S‐23S rDNA Internal Transcribed Spacer Regions and the Possible Use of This Genetic Variation for Molecular Diagnosis of Bacteroides Species

The structural variation in 16S‐23S rDNA internal transcribed spacer regions (ITS) among Bacteroides species was assessed by PCR amplification and sequencing analysis, and its possible use for molecular diagnosis of these species was evaluated. Ninety strains of the genus Bacteroides, including the species B. distasonis, B. eggerthii, B. fragilis, B. ovatus, B. thetaiotaomicron, B. uniformis and B. vulgatus, produced one to three ITS amplification products with sizes ranging from 615 to 810 bp. Some Bacteroides strains could be differentiated at species level on the basis of ITS amplification patterns and restriction fragment length polymorphism (RFLP) analysis using a four‐nucleotide‐recognizing enzyme, Msp I. The results of sequence analysis of ITS amplification products revealed genes for Ile‐tRNA and Ala‐tRNA in all strains tested. The nucleotide sequence, except for that in tRNA‐coding regions, was highly variable and characteristic for each species, but a common sequence among B. fragilis, B. thetaiotaomicron and B. ovatus was observed. A digoxigenin‐labeled oligonucleotide probe (named FOT1), which was designed from this conserved sequence, specifically hybridized to the ITS amplification products from B. fragilis, B. thetaiotaomicron and B. ovatus. These results suggest that the ITS region is a useful target for the development of rapid and accurate techniques for identification of Bacteroides species.

In Vitro Cytotoxic Effect of Ethanol Extract Prepared From Sporophyll of Undaria pinnatifida on Human Colorectal Cancer Cells

Brown seaweed Undaria pinnatifida (Harvey) Suringar is popular as a foodstuff, and used for medical care in East Asian countries. The major components of this seaweed are shown to benefit hypertension and hyperlipidemia, and considered to reduce the risks of infarction and ischemic diseases. Furthermore, the intake of dietary fiber of seaweeds is considered to prevent the production and proliferation of cancer in the gastrointestinal tract. The direct effect of an ethanol extract prepared from Undaria pinnatifida sporophyll (mekabu) on HCT116 human colorectal cancer cells was examined, and the mekabu extract was shown to induce the non‐oxidative apoptotic damage to the cells, thus resulting in the reduction of their viabilities in a concentration‐dependent manner. Moreover, the cytotoxic effects of carcinostatic drugs, such as 5‐fluorouracil (5‐FU) and irinotecan (CPT‐11), were observed only in the medium containing sera, while the mekabu extract could effectively reduce the cell viabilities even in the serum‐free medium. These findings suggest that the mekabu extract may contain a potential active substance inducing the non‐oxidative apoptotic cell death probably through a mechanism different from those of 5‐FU and CPT‐11, and hence mekabu is possibly useful as an auxiliary drug to the chemotherapy of colorectal cancer. Copyright

Adrenergic activation of steroid 5α-reductase gene expression in rat C6 glioma cells

Steroid 5α-reductase (5α-R) is well known as the enzyme converting progesterone and other steroid hormones to their 5α-reduced metabolites and has been reported to be localized in both neuronal and glial cells in the brain. Previously, the enzyme activity in glial cells has been shown to be enhanced either by coculturing with neuronal cells or by adding the conditioned medium of neuronal cells, suggesting a possible implication of neuro-glial interactions in the regulation of neurosteroid metabolism in the brain. In the present studies, the effects of adrenergic agonists on 5α-R mRNA and protein levels in rat C6 glioma cells were examined as one of the model experiments for investigating the influence of neuronal activity on the expression of 5α-R gene in the glial cell. The direct challenge of β-adrenergic agonists to glioma cells resulted in the rapid and transient elevation of 5α-R mRNA levels through the activation of the cyclic AMP (cAMP)/protein kinase A-mediated signaling pathway. Further studies showed that cAMP-induced 5α-R mRNA expression was completely abolished by pretreatment of cells with actinomycin D and also indicated that the elevation of 5α-R mRNA levels was accompanied by an increase in enzyme protein in the cells. These findings provide strong evidence that the stimulation of β-adrenergic receptors might induce the transcriptional activation of 5α-R gene expression in glial cells, proposing the possibility that neuronal activity might be involved in the production of neuroactive 5α-reduced steroids in the brain.

The ARNT–STAT3 axis regulates the differentiation of intestinal intraepithelial TCRαβ + CD8αα + cells

Intestinal intraepithelial T cells contribute to the regulation of inflammatory responses in the intestine; however, the molecular basis for their development and maintenance is unknown. The aryl hydrocarbon receptor complexes with the aryl hydrocarbon receptor nuclear translocator (ARNT) and senses environmental factors, including gut microbiota. Here, we identify ARNT as a critical regulator of the differentiation of TCRαβ(+)CD8αα(+) intestinal intraepithelial T cells. Mice deficient in either ARNT or aryl hydrocarbon receptor show a greater than- eight-fold reduction in the number of TCRαβ(+)CD8αα(+) intestinal intraepithelial T cells. The number of TCRαβ(+)CD8αα(+) intestinal intraepithelial T cells is increased by treatment with an aryl hydrocarbon receptor agonist in germ-free mice and is decreased by antibiotic treatment. The Arnt-deficient precursors of TCRαβ(+)CD8αα(+) intestinal intraepithelial T cells express low amounts of STAT3 and fail to differentiate towards the TCRαβ(+)CD8αα(+) cell fate after IL-15 stimulation, a deficiency that is overcome by overexpression of Stat3. These data demonstrate that the ARNT-STAT3 axis is a critical regulator of TCRαβ(+)CD8αα(+) intestinal intraepithelial T-cell development and differentiation.