Analyses of the possible anti-tumor effect of yokukansan.

The Kampo medicine yokukansan (YKS) has a wide variety of properties such as anxiolytic, anti-inflammatory and analgesic effects, and is also thought to regulate tumor suppression. In this study, we investigated the anti-tumor effect of YKS. We used Lewis lung carcinoma (LLC)-bearing mice that were fed food pellets containing YKS and then performed a fecal microbiota analysis, a microarray analysis for microRNAs (miRNAs) and an in vitro anti-tumor assay. The fecal microbiota analysis revealed that treatment with YKS partly reversed changes in the microbiota composition due to LLC implantation. Furthermore, a miRNA array analysis using blood serum showed that treatment with YKS restored the levels of miR-133a-3p/133b-3p, miR-1a-3p and miR-342-3p following LLC implantation to normal levels. A TargetScan analysis revealed that the epidermal growth factor receptor 1 signaling pathway is one of the major target pathways for these miRNAs. Furthermore, treatment with YKS restored the levels of miR-200b-3p and miR-200c-3p, a recognized mediator of cancer progression and controller of emotion, in the hypothalamus of mice bearing LLC. An in vitro assay revealed that a mixture of pachymic acid, saikosaponins a and d and isoliquiritigenin, which are all contained in YKS, exerted direct and additive anti-tumor effects. The present findings constitute novel evidence that YKS may exert an anti-tumor effect by reversing changes in the fecal microbiota and miRNAs circulating in the blood serum and hypothalamus, and the compounds found in YKS could have direct and additive anti-tumor effects.

Hypothalamus region-specific global gene expression profiling in early stages of central endocrine disruption in rat neonates injected with estradiol benzoate or flutamide.

To identify genes linked to early stages of disruption of brain sexual differentiation, hypothalamic region-specific microarray analyses were performed using a microdissection technique with neonatal rats exposed to endocrine-acting drugs. To validate the methodology, the expression fidelity of microarrays was first examined with two-round amplified antisense RNAs (aRNAs) from methacarn-fixed paraffin-embedded tissue (PET) in comparison with expression in unfixed frozen tissue (UFT). Decline of expression fidelity when compared with the 1x-amplified aRNAs from UFTs was found as a result of the preferential amplification of the 3' side of mRNAs in the second round in vitro transcription. However, expression patterns for the 2x-amplified aRNAs were mostly identical between methacarn-fixed PET and UFT, suggesting no obvious influence of methacarn fixation and subsequent paraffin embedding on expression levels. Next, in the main experiment, neonatal rats at birth were treated subcutaneously either with estradiol benzoate (EB; 10 microg/pup) or flutamide (FA; 250 microg/pup), and medial preoptic area (MPOA)-specific microarray analysis was performed 24 h later using 2x-amplified aRNAs from methacarn-fixed PET. Numbers of genes showing constitutively high expression in the MPOA predominated in males, implying a link with male-type growth supported by perinatal testosterone. Around 60% of genes showing sex differences in expression demonstrated altered levels after EB treatment in females, suggesting an involvement of genes necessary for brain sexual differentiation. When compared with EB, FA affected a rather small number of genes, but fluctuation was mostly observed in females, as with EB. Moreover, many selected genes common to EB and FA showed down-regulation in females with both drugs, suggesting a common mechanism for endocrine center disruption in females, at least at early stages of post-natal development.

Effects of aromatase inhibitors on human osteoblast and osteoblast-like cells: a possible androgenic bone protective effects induced by exemestane.

Effects of aromatase inhibitors (AIs) on the human skeletal system due to systemic estrogen depletion are becoming clinically important due to their increasing use as an adjuvant therapy in postmenopausal women with breast cancer. However, possible effects of AIs on human bone cells have remained largely unknown. We therefore studied effects of AIs including the steroidal AI, exemestane (EXE), and non-steroidal AIs, Aromatase Inhibitor I (AI-I) and aminoglutethimide (AGM), on a human osteoblast. We employed a human osteoblast cell line, hFOB, which maintains relatively physiological status of estrogen and androgen pathways of human osteoblasts, i.e., expression of aromatase, androgen receptor (AR), and estrogen receptor (ER) beta. We also employed osteoblast-like cell lines, Saos-2 and MG-63 which expressed aromatase, AR, and ERalpha/beta in order to further evaluate the mechanisms of effects of AIs on osteoblasts. There was a significant increment in the number of the cells following 72 h treatment with EXE in hFOB and Saos-2 but not in MG-63, in which the level of AR mRNA was lower than that in hFOB and Saos-2. Alkaline phosphatase activity was also increased by EXE treatment in hFOB and Saos-2. Pretreatment with the AR blocker, flutamide, partially inhibited the effect of EXE. AI-I exerted no effects on osteoblast cell proliferation and AGM diminished the number of the cells. hFOB converted androstenedione into E2 and testosterone (TST). Both EXE and AI-I decreased E2 level and increased TST level. In a microarray analysis, gene profile patterns following treatment with EXE demonstrated similar patterns as with DHT but not with E2 treatment. The genes induced by EXE treatment were related to cell proliferation, differentiation which includes genes encoding cytoskeleton proteins. We also examined the expression levels of these genes using quantitative RT-PCR in hFOB and Saos-2 treated with EXE and DHT and with/without flutamide. HOXD11 gene known as bone morphogenesis factor and osteoblast growth-related genes were induced by EXE treatment as well as DHT treatment in both hFOB and Saos-2. These results indicated that the steroidal aromatase inhibitor, EXE, stimulated hFOB cell proliferation via both AR dependent and independent pathways.