Mitsuru Chiba

Exosomes secreted from human colorectal cancer cell lines contain mRNAs, microRNAs and natural antisense RNAs, that can transfer into the human hepatoma HepG2 and lung cancer A549 cell lines.

Exosomes are microvesicles that are released from various cells into the extracellular space. It has been reported that the components within exosomes vary according to the type of secreted cell. In the present study, we investigated the tetraspanin family proteins CD63, CD9 and CD81 as useful collection markers of exosomes derived from the three colorectal cancer (CRC) cell lines HCT-15, SW480 and WiDr. In addition, we aimed to detect the mRNAs, microRNAs and natural antisense RNAs within the exosomes secreted from the three CRC cell lines. Furthermore, we examined whether exosomes containing their RNAs were transferred into the hepatoma cell line HepG2 and lung cancer cell line A549. CD81 was detected in exosomes secreted from the three CRC cell lines. This result indicates that CD81 can be a collection marker of exosomes derived from the three CRC cell lines. When the RNA species within exosomes derived from the three CRC cell lines were examined, the mRNAs of housekeeping genes such as ACTB and GAPDH, the microRNAs such as miR-21, miR-192 and miR-221, and the natural antisense RNAs of LRRC24, MDM2 and CDKN1A genes, were detected. We discovered their natural antisense RNAs within exosomes for the first time in the present study. Furthermore, PKH67-labeled exosomes derived from the CRC cell lines were taken up into HepG2 and A549 cells. These findings indicate that the intracellular RNAs enclosed within exosomes are secreted to the outside, and exosomes derived from the CRC cell lines are transferred into HepG2 and A549 cells. In conclusion, we reveal that exosomes derived from the CRC cell lines contain mRNAs, microRNAs and natural antisense RNAs, and can be delivered into HepG2 and A549 cells. These findings indicate that exosomal RNAs can shuttle between cells, and may be involved in the regulation of gene expression in recipient cells.

Role of platelets on liver regeneration after 90% hepatectomy in mice

BACKGROUND/AIMS Mortality after 90% partial hepatectomy in mice was associated with severe acute liver failure. Recently, we revealed that platelets have a strong promotional effect on hepatic regeneration. In the present study, we investigated the effect of thrombocytosis on liver regeneration after 90% hepatectomy in mice. METHODS For thrombocytosis induction PEG-rHuMGDF was injected 5 days before operation. Hepatectomy, sparing only the caudate lobe, was performed in normal and thrombocytotic BALB/c mice. Survival rate, platelet number, liver weight/body weight ratio, proliferating cell nuclear antigen, serum parameters, signal transduction and overexpressed genes were examined. RESULTS Platelet number was significantly higher in thrombocytotic group. All mice in normal group died within 30 h after hepatectomy. Survival rate in thrombocytotic group was 6/11 at 30 h and 3/11 one week after hepatectomy. Activation of Akt and STAT3 signaling pathways in thrombocytotic group was observed earlier and recognized to be stronger compared to normal group. Cell cycle, signaling pathways, metabolism and transport genes were significantly overexpressed in thrombocytotic group up to 24h after hepatectomy. CONCLUSIONS Under the thrombocytotic condition, liver regeneration occurred even in 90% hepatectomized mice. Platelets contribute to cell cycle progression and metabolic pathways in addition to preventing acute liver failure.

Novel L-amino acid oxidase with antibacterial activity against methicillin-resistant Staphylococcus aureus isolated from epidermal mucus of the flounder Platichthys stellatus

Fish produce mucus substances as a defensive outer barrier against environmental xenobiotics and predators. Recently, we found a bioactive protein in the mucus layer of the flounder Platichthys stellatus, which showed antibacterial activity against Staphylococcus epidermidis, Staphylococcus aureus and methicillin‐resistant S. aureus. In this study, we isolated and identified the antibacterial protein from the mucus components of P. stellatus using a series of column chromatography steps. We then performed gel electrophoresis and cDNA cloning to characterize the protein. The antibacterial protein in the mucus had a molecular mass of approximately 52 kDa with an isoelectric point of 5.3, and cDNA sequencing showed that it corresponded completely with the peptide sequence of antibacterial protein from the gill. A BLAST search suggested that the cDNA encoded an antibacterial protein sharing identity with a number of l‐amino acid oxidases (LAAOs) and possessing several conserved motifs found in flavoproteins. RT‐PCR using a specific primer, and immunohistochemical analysis with anti‐LAAO IgG, demonstrated tissue‐specific expression and localization in the gill. Moreover, the anti‐LAAO IgG was able to neutralize the antibacterial activity of the protein against methicillin‐resistant S. aureus. Thus, we demonstrated that this antibacterial protein, identified from P. stellatus‐derived epidermal mucus, is a novel LAAO‐like protein with antibacterial activity, similar to snake LAAOs.

Anti-inflammatory effect of proteoglycan and progesterone on human uterine cervical fibroblasts.

AIMS The aim of this study was to compare the anti-inflammatory effect of proteoglycan (PG) with that of progesterone (P) in the cultured fibroblasts from human uterine cervix. MAIN METHODS After obtaining informed consent, the cervix was collected from normal women undergoing total hysterectomy. The cervix was cultured until fibroblasts proliferated and had grown to confluence, then, the fibroblasts were stimulated by lipopolysaccharide (LPS) with or without PG, P and a combination of both; they were cultured for 24-48 h. The anti-inflammatory effects of PG and P were evaluated by the suppression of IL-6 or IL-8 secretion. The expression of the IL-6 or IL-8 gene and the expression of their protein were determined by real-time PCR, and ELISA, respectively. Activation of Toll-like receptor (TLR) 4 was evaluated by Western blotting. KEY FINDINGS LPS markedly enhanced gene and protein expression of IL-6 and IL-8 in human uterine cervical fibroblasts. The up-regulation of the IL-6 or IL-8 gene and protein expression by LPS was significantly suppressed with PG, P and a combination of both. Western blotting revealed that combination of PG and P showed more potent inhibition on LPS-stimulated TLR4 induction than that seen by each. SIGNIFICANCE This study showed that both PG and P have an inhibitory effect on LPS-induced inflammation. This anti-inflammatory effect of PG and P was augmented by co-administration of both, suggesting for the first time that PG has an anti-inflammatory effect on human uterine cervical fibroblasts.

Existence of Pink1 antisense RNAs in mouse and their localization.

PTEN-induced kinase 1 (PINK1), which is identified as the gene transactivated by the tumor suppressor PTEN, has been found to be one of the causative genes in Parkinson’s disease (PD). In order to understand PD, rodent models containing affected Pink1 such as loss-of-function mutations have been exploited. Recently, natural antisense RNA of PINK1 has been demonstrated to be involved in the regulation of the PINK1 locus. However, no antisense RNAs of Pink1 except for human have been reported so far. Therefore, in the present study, while searching for the Pink1 antisense RNAs in mouse, we found that the antisense RNAs are transcribed from a mouse genomic region corresponding to the human region from which the antisense RNAs are produced. Further, we investigated the localization of the antisense RNAs in mouse brain using in situ hybridization; this demonstrated that the antisense RNAs were localized in the regions of brain where the Pink1 mRNA was found. In addition, the mRNA and antisense RNAs were found more densely in the hippocampus than in the other brain regions in newborn and 1-week-old mice, while those RNAs were found uniformly in the mouse brain regions of embryo day (E) 14, E17, and 8-weeks-old.

αCGRP and βCGRP transcript amount in mouse tissues of various developmental stages and their tissue expression sites

The calcitonin gene-related peptides (CGRP), alphaCGRP and betaCGRP, have been implicated to play various roles in primates and rodent. However, since the expression information has been limited, in the present study, we measured the amount of gene expression in mouse brain, liver, kidney, heart, and testis at embryonic day (E) 14, E17, postnatal day (P) 1, P7, and adult using real-time PCR, and determined the precise localization of alphaCGRP and betaCGRP sense/antisense transcripts in tissues using in situ hybridization. The sense transcripts of alphaCGRP and betaCGRP were found mainly in brain, and their amount profiles were similar in the course of development: one expression peak was observed at E17 and the other at P7. The amounts of alphaCGRP transcripts were greater than those of betaCGRP transcripts in the range between 3.6 and 31 times. In the E17 and P7 brains, the localization pattern of alphaCGRP sense transcripts was similar with that of alphaCGRP antisense transcripts. Fewer transcripts were found in neuroblasts of E17 corpus callosum, and neuroblasts of P7 corpus callosum, olfactory bulb, plexus chorioideus, and ventriculus lateralis than in other brain areas. The localization pattern of betaCGRP sense and antisense transcripts was similar to that for alphaCGRP except that the betaCGRP antisense transcripts showed spot-like localizations. Additionally, the alphaCGRP sense transcript, and betaCGRP sense and antisense transcripts were found in parafollicular cells (C cells) of E17 thyroid lobe. These findings together indicate that alphaCGRP and betaCGRP have their own roles in the ontogenic process.

Exosomes derived from SW480 colorectal cancer cells promote cell migration in HepG2 hepatocellular cancer cells via the mitogen-activated protein kinase pathway.

Exosomes are membrane-derived extracellular vesicles that have recently been recognized as important mediators of intercellular communication. In the present study, we investigated the effects of exosomes derived from SW480 colorectal cancer cells in recipient HepG2 hepatocellular cancer cells. We demonstrated that SW480-derived exosomes were taken up by the recipient HepG2 cells via dynamin-dependent endocytosis and were localized to the HepG2 lysosomes. In addition, SW480-derived exosomes induced the phosphorylation of extracellular signal-regulated kinase (ERK)1/2 following their uptake into HepG2 cells. Of note, these changes occurred during the early phase after exosome treatment. Furthermore, SW480-derived exosomes promoted the migration of recipient HepG2 cells in a wound-healing assay, which was suppressed by pretreatment with U0126, an upstream inhibitor of ERK1/2. These results indicated that SW480-derived exosomes activated a classical mitogen-activated protein kinase pathway in recipient HepG2 cells via dynamin-dependent endocytosis and subsequently enhanced cell migration by ERK1/2 activation. Our results provide new insights into the regulation of cellular functions by exosomes.

Secretion of small/microRNAs including miR-638 into extracellular spaces by sphingomyelin phosphodiesterase 3

A recent study demonstrated that intracellular small/microRNAs are released from cells, and some of these extracellular RNAs are embedded in vesicles, such as ceramide-rich exosomes, on lipid-bilayer membranes. In the present study, we examined the effects of sphingomyelin phosphodiesterase 3 (SMPD3), which generates ceramide from sphingomyelin, on the release of small/microRNAs from intracellular to extracellular spaces. In these experiments, SW480 human colorectal and HuH-7 human hepatocellular cancer cells were cultured for 48 h in serum-free media. Culture supernatants were then collected, and floating cells and debris were removed by centrifugation and filtration through a 0.22-μm filter. Extracellular small RNAs in purified culture supernatants were stable for 4 weeks at room temperature, after 20 freeze-thaw cycles and exposure to pH 2.0, and were resistant to ribonuclease A degradation. Amino acid sequence analyses of SMPD3 showed high homology between mammals, indicating evolutionary conservation. Therefore, to investigate the mechanisms of cellular small/microRNA export, SW480 and HuH-7 cells were treated with the SMPD3 inhibitor GW4869 in serum-free media. Culture supernatants were collected for microarray and/or reverse transcription quantitative polymerase chain reaction (RT-qPCR) experiments. The number of microRNAs in culture supernatants was decreased following treatment with GW4869. Among these, extracellular and intracellular miR-638 were dose-dependently decreased and increased, respectively. These data suggest that SMPD3 plays an important role in the release of microRNAs into extracellular spaces.

Phytoestrogenic activity of blackcurrant (Ribes nigrum) anthocyanins is mediated through estrogen receptor alpha

SCOPE Blackcurrants (Ribes nigrum L., Grossulariaceae) contain high amounts of anthocyanin polyphenols, which have antioxidant and anti-carcinogenic health benefits. This study analyzed the potential phytoestrogenic effects of blackcurrant extract (BCE) in breast cancer (MCF-7) and human endometrial cancer (Ishikawa) cell lines that over-express estrogen receptor alpha (ERα), as well as in immature female rats. METHODS AND RESULTS Microarray analysis and Ingenuity® Pathway Analysis showed that BCE activated the ERα pathway, whereas quantitative-PCR confirmed that BCE and four types of anthocyanins up-regulated genes downstream of ERα. BCE (0.1-1.0 μg/mL) and anthocyanins (0.1-10 μM) induced MCF-7 cell proliferation; however, this effect was blocked by ER antagonist fulvestrant. Flow cytometry showed that anthocyanins reduced and increased the number of MCF-7 cells in the G0/G1 and G2/M phases, respectively. Anthocyanins stimulated ERα transcriptional activity in human ERα reporter assays and induced alkaline phosphatase activity in Ishikawa cells. Competition assays and in silico analysis indicated that anthocyanins bind to ERα. Finally, BCE focally induced stratification of columnar epithelial cells in the rat uterus and increased cytoplasmic mucin levels in these cells. CONCLUSION These results suggest that blackcurrant anthocyanins act as phytoestrogens in vitro and in vivo.

Identification of up-regulated and down-regulated cis-natural antisense transcripts in the human B lymphoblastic cell line IM-9 after X-ray irradiation

Ionizing radiation (IR) causes DNA injury and induces multiple signal mechanisms, including the regulation of DNA repair, the cell cycle and gene expression through the activation of p53-related pathways. Cis-natural antisense transcripts (cis-NATs), which are transcribed from the DNA strand opposite to that for mRNA of the gene, are recognized as important regulators of gene expression in eukaryotic cells, but the effects on cis-NAT expression by IR are unknown to date. Therefore, we investigated the effects of X-ray irradiation on cis-NAT expression together with mRNA expression using a human B lymphoblast cell line (IM-9), a custom-microarray and strand-specific RT-qPCR. Eighteen, 33 and 106 mRNAs were demonstrated to be differentially expressed in IM-9 cells after 1, 2 and 4 Gy irradiation, respectively, as compared to 0 Gy by microarray analysis (fold change, FC >2.0). On the other hand, 10, 22 and 43 NATs were demonstrated to be differentially expressed in IM-9 cells after 1, 2 and 4 Gy irradiation, respectively, as compared to 0 Gy by microarray analysis (FC >2.0). Among these mRNAs/NATs, the IR dose-dependent up-regulation of mRNAs and cis-NATs of MDM2 and CDKN1A were confirmed by strand-specific RT-qPCR. Additionally, the cis-NATs of MDM2 were indicated to be localized in the cytoplasm, while cis-NATs of CDKN1A were located in the nucleus and cytoplasm. In conclusion, the radiation-responsive cis-NATs in conjunction with mRNAs were identified for the first time in the present study. It is possible that these cis-NATs regulate the gene expression in a post-transcriptional fashion. The IR dose-dependent up- and down-regulation of these mRNAs/cis-NATs may be a marker for ionizing radiation.