Shinichi Harada

Induction of various blood-brain barrier properties in non-neural endothelial cells by close apposition to co-cultured astrocytes

Vascular endothelial cells (EC) exhibit organ‐to‐organ heterogeneity in their functions and morphologies. In particular, brain capillary EC have unique characteristics exemplified by the blood‐brain barrier (BBB). The formation and the maintenance of BBB have been ascribed to EC responses to inductive signal(s) or factor(s) from astrocytes that encircle microvessels in the central nervous system. These EC responses were demonstrated in numerous in vivo studies, exemplified by those of Janzer and Raff (Nature 325:253, 1987) and Tout et al. (Neuroscience 55:291, 1993) showing that transplanted astrocytes induced BBB properties in non‐neural vascular EC. In this study, we constructed a heterologous co‐culture system, in which rat fetal brain astrocytes were cultivated on one surface of a porous membrane and human umbilical vein EC on the opposite surface. Electron microscopic examination revealed that astrocytes passed their endfeet through the pores, making contact with EC. In this system, γ‐glutamyltranspeptidase (γ‐GTP) activity in EC was found to be significantly increased by contacting astrocytes in a density‐and time‐dependent manner, but not when the astrocyte feeder layer was apart from EC or replaced by COS cells; astrocyte‐derived extracellular matrix partially activated γ‐GTP. mRNAs for some of the representative BBB markers, including transferrin receptor, P‐glycoprotein, brain‐type glucose transporter(GLUT‐1), and γ‐GTP were also demonstrated by reverse transcription‐polymerase chain reaction to be upregulated in EC co‐cultured with astrocytes. Astrocyte inductions of close membrane apposition resembling a zonula occludens and of an increase in the content of mitochondria in EC were also noted in electron micrographs. Furthermore, an increased barrier activity against inulin was conferred on EC when they were lined with astrocytes. The results obtained with this heterologous co‐culture system thus indicate that through contact with their feet, astrocytes are capable of transdifferentiating non‐neural EC into the brain type, endowing them with the BBB properties. Glia 19:13–26, 1997

Extended longevity of Caenorhabditis elegans by knocking in extra copies of hsp70F, a homolog of mot-2 (mortalin)/mthsp70/Grp75

The Caenorhabditis elegans homolog of mortalin/mthsp70/Grp75 (called mot‐2 hereafter) was isolated by screening of a nematode cDNA library with mouse mot‐2 cDNA. The isolated clone matched to hsp70F of C. elegans. Analysis with two of the antibodies raised against hsp70F revealed that unlike mammalian mot‐2, it is heat inducible. Transient induction of hsp70F by heat shock led to a slight (<13%) extension in the C. elegans life span. The transgenic worms that constitutively over‐expressed hsp70F predominantly in muscle showed life span extension (∼43% for mean and ∼45% for maximum life span) as compared to the wild‐type and green fluorescent protein‐transgenic worms. Life span extension of human cells was obtained by over‐expression of mot‐2 [Kaul et al. (2000) FEBS Lett. 474, 159–164]. Our results show, for the first time, that this member of the hsp70 family governs the longevity of worms and thus there are common pathways that determine mammalian and worm longevity.

Placenta growth factor (PlGF) mRNA expression in brain tumors

To investigate the relationship between placenta growth factor (PlGF) and brain tumor angiogenesis, we screened 36 primary and 3 metastatic brain tumors. We examined the expression of PlGF mRNA with respect to vasculature of various tumors which was determined by preoperative angiography. The expression of genes of the other angiogenic factors, vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (bFGF) was also tested, and compared to that of PlGF. The primary tumors consisted of 16 meningiomas, 7 gliomas, 7 schwannomas, 4 pituitary adenomas, 1 germinoma, and 1 choriocarcinoma. Using a quantitative reverse transcription-polymerase chain reaction, the mRNA for PlGF149 and PlGF170 were detected in 25 out of 39 (64.1%) brain tumors. In primary brain tumors, PlGF mRNA was expressed in all the hypervascular tumors, but only in 5 of 16 hypovascular tumors (31.3%). None of the 3 metastatic hypervascular tumors expressed PlGF mRNA. The VEGF and bFGF mRNA expression was both detected in 87.2% of the tumors examined. We conducted hypoxic experiments with cultured U-251MG human glioma cells to determine the mechanism of PlGF gene regulation. As the atmospheric oxygen concentration was decreased, the PlGF mRNA level in the U-251MG cells was markedly increased. These results suggest that PlGF may contribute to the pathogenesis of brain tumor angiogenesis.

Advanced glycosylation end products stimulate the growth but inhibit the prostacyclin-producing ability of endothelial cells through interactions with their receptors

The influence of advanced glycosylation end products (AGE) on endothelial cells was investigated. When human umbilical endothelial cells were cultured with AGE‐bovine serum albumin, viable cell number as well as DNA synthesis was significantly stimulated, whereas prostacyclin production by the endothelial cells was decreased. Antisense oligodeoxyribonucleotides against mRNA coding for AGE receptor were found to reverse both the AGE‐induced growth stimulation and the inhibition of prostacyclin production in endothelial cells. These results thus suggest that AGE ligand‐receptor interactions in endothelial cells can promote angiogenesis and thrombogenesis, leading to the development of diabetic vascular complications.

Gene amplification of ESR1 in breast cancers-fact or fiction? A fluorescence in situ hybridization and multiplex ligation-dependent probe amplification study

Oestrogen receptor‐alpha (ERα), encoded by the ESR1 gene located on 6q25, is a nuclear transcription factor. Since it was reported in 2007 that more than 20% of breast cancers show ESR1 gene amplification, there has been considerable controversy about its frequency and clinical significance. We set out to assess the frequency and levels of ESR1 amplification in breast cancers. In a total of 106 breast needle biopsy specimens examined by immunohistochemistry, 78 tumours contained more than 10% ERα‐positive cancer cells. In fluorescence in situ hybridization (FISH) analysis with an ESR1‐specific probe, variously extended ESR1 signals were found in ERα‐expressing cells. Some of these were indistinguishable from large clustered signals generally accepted to mean high‐level gene amplification in homogeneously staining regions (HSRs), and could be considered to represent gene amplification. However, with RNase treatment, the ‘HSR‐like’ signals changed to small compact signals, and are thus thought to represent concentrated RNA. FISH using two differently labelled probes corresponding to the non‐overlapping 5′‐ and 3′‐end portions of the ESR1 gene on touch smears showed a preserved spatial relationship of the 3′ to 5′ sequence of ESR1, therefore strongly suggesting that the RNA consisted of primary transcripts. Using touch smears obtained from 51 fresh tumours, precise enumeration of ESR1 signals with a correction by the number of centromere 6 on FISH after RNase A treatment revealed that three tumours (5.9%) had tumour cells with one to three additional copies of ESR1 as predominant subpopulations. This infrequent and low level of gene amplification of ESR1 was also detected as a ‘gain’ of the gene by analysis with multiplex ligation‐dependent probe amplification (MLPA). The consistent results from immunohistochemistry, FISH, and MLPA in the present study settle the long‐standing debate concerning gene amplification of ESR1 in breast carcinoma. Copyright

Expression of Vascular Endothelial Growth Factor D Is Associated with Lymph Node Metastasis in Human Colorectal Carcinoma

Objective: Expression of vascular endothelial growth factor (VEGF)-D by tumors is associated with metastasis to lymph nodes in mice. However, there are few reports concerning the clinical significance of VEGF-D protein in human carcinoma. Methods: After confirming production of VEGF-D by eight colorectal carcinoma cell lines, we investigated relationships between the expression of VEGF-D protein, lymph node metastasis and postoperative survival in 83 colorectal carcinoma patients. mRNA levels in cell lines were evaluated using the real-time reverse transcriptase-polymerase chain reaction, and protein was detected by Western blotting in cell lines and by immunohistochemistry in resected tissues using an antibody recognizing the processed form of the molecule. Results: Immunohistochemistry showed VEGF-D-positive staining in 26 of the 83 carcinomas (31%). There was a significant relationship between the presence of VEGF-D protein and the incidence of lymph node metastasis (p < 0.01). Multivariate logistic regression analysis revealed that VEGF-D protein expression was an independent factor affecting lymph node metastasis (p < 0.01). Nonetheless, the presence or absence of VEGF-D protein had no significant impact on the survival of the patients (p = 0.15). Conclusion: These results suggest that the expression of VEGF-D protein could be useful in predicting the nodal status of colorectal carcinoma patients.

Angiotensin II enhances epithelial-to-mesenchymal transition through the interaction between activated hepatic stellate cells and the stromal cell-derived factor-1/CXCR4 axis in intrahepatic cholangiocarcinoma

We previously reported that hepatic stellate cells (HSCs) activated by angiotensin II (AngII) facilitate stromal fibrosis and tumor progression in intrahepatic cholangiocarcinoma (ICC). AngII has been known as a growth factor which can promote epithelial-to-mesenchymal transition (EMT) in renal epithelial cells, alveolar epithelial cells and peritoneal mesothelial cells. However, in the past, the relationship between AngII and stromal cell-derived factor-1 (SDF-1) in the microenvironment around cancer and the role of AngII on EMT of cancer cells has not been reported in detail. SDF-1 and its specific receptor, CXCR4, are now receiving attention as a mechanism of cell progression and metastasis. In this study, we examined whether activated HSCs promote tumor fibrogenesis, tumor progression and distant metastasis by mediating EMT via the AngII/AngII type 1 receptor (AT-1) and the SDF-1/CXCR4 axis. Two human ICC cell lines and a human HSC line, LI-90, express CXCR4. Significantly higher concentration of SDF-1α was released into the supernatant of LI-90 cells to which AngII had been added. SDF-1α increased the proliferative activity of HSCs and enhanced the activation of HSCs as a growth factor. Furthermore, addition of SDF-1α and AngII enhanced the increase of the migratory capability and vimentin expression, reduced E-cadherin expression, and translocated the expression of β-catenin into the nucleus and cytoplasm in ICC cells. Co-culture with HSCs also enhanced the migratory capability of ICC cells. These findings suggest that SDF-1α, released from activated HSCs and AngII, play important roles in cancer progression, tumor fibrogenesis, and migration in autocrine and paracrine fashion by mediating EMT. Our mechanistic findings may provide pivotal insights into the molecular mechanism of the AngII and SDF-1α-initiated signaling pathway that regulates fibrogenesis in cancerous stroma, tumor progression and meta-stasis of tumor cells expressing AT-1 and CXCR4.

The role of human peritoneal mesothelial cells in the fibrosis and progression of gastric cancer

Peritoneal dissemination is the most frequent metastatic pattern of scirrhous gastric cancer. However, despite extensive research effort, disease outcomes have not improved sufficiently. Tumor progression and metastasis result from interactions between cancer and various cells in the stroma, including endothelial cells, immune cells and fibroblasts. Fibroblasts have been particularly well studied; they are known to change into carcinoma-associated fibroblasts (CAFs) and produce transforming growth factor β (TGF-β), which mediates cancer-stroma interactions. Here, we investigated whether TGF-β derived from cancer cells in the peritoneal microenvironment activates human peritoneal mesothelial cells (HPMCs), leading to the progression and fibrosis of gastric cancer. We found that activated HPMCs (a-HPMCs) took on a spindle shape formation, decreased the expression of E-cadherin and increased that of α-SMA. Furthermore, a-HPMCs became more invasive and upregulated proliferation of human gastric cancer-derived MKN45 cells following direct cell-cell contact. Notably, MKN45 cells co-cultured with a-HPMCs also acquired anchorage-independent cell growth and decreased expression of E-cadherin in vitro. To measure the effects of the co-culture in vivo, we developed a mouse xenograft model into which different culture products were subcutaneously injected. The largest tumors were observed in mice that had been given MKN45 cells co-cultured with a-HPMCs. Furthermore, these tumors contained HPMC-derived fibrous tissue. Thus, the epithelial-mesenchymal transition (EMT) of HPMCs appears to drive peritoneal dissemination and tumor fibrosis.

The Angiotensin II type 1 receptor blocker candesartan suppresses proliferation and fibrosis in gastric cancer

Gastric cancer with peritoneal dissemination has poor clinical prognosis because of the presence of rich stromal fibrosis and acquired drug resistance. Recently, Angiotensin II type I receptor blockers such as candesartan have attracted attention for their potential anti-fibrotic activity. We examined whether candesartan could attenuate tumor proliferation and fibrosis through the interaction between gastric cancer cell line (MKN45) cells and human peritoneal mesothelial cells. Candesartan significantly reduced TGF-β1 expression and epithelial-to-mesenchymal transition-like change, while tumor proliferation and stromal fibrosis were impaired. Targeting the Angiotensin II signaling pathway may therefore be an efficient strategy for treatment of tumor proliferation and fibrosis.

Valproic acid, a histone deacetylase inhibitor, enhances radiosensitivity in esophageal squamous cell carcinoma

Histone deacetylase (HDAC) inhibitors have been shown to enhance radiation response in various cancer cell lines. Valproic acid (VPA) has been used in clinical practice for the treatment of epilepsy and other seizure disorders and is also one of the most represented HDAC inhibitors. The aim of this study was to evaluate the radiosensitizing ability of VPA and its mechanisms in four esophageal squamous cell carcinoma (ESCC) cell lines (TE9, TE10, TE11 and TE14). VPA inhibited the viability of all ESCC cells in a dose-dependent manner. The 50% inhibitory concentration (IC50) value of VPA in each cell line was between 1.02-2.15 mM, which is higher than clinically used safe concentrations. VPA induced the hyperacetylation of histones H3 and H4, as well as apoptosis and had a radiosensitizing effect on all four ESCC cell lines at a concentration of 0.5 mM which is equivalent to the therapeutic plasma concentration of anti-epilepsy therapy in humans. The radiosensitization was accompanied by an increase in γH2AX levels, indicating the presence of double-strand breaks (DSBs), and decrease in Rad51 expression, a DSB repair protein. These results suggest that a clinically safe dose of VPA can enhance radiation-induced cytotoxicity in human ESCC cells by chromatin decondensation with histone hyperacetylation and downregulation of Rad51. In conclusion, VPA appears to be a safe and promising radiosensitizer for esophageal cancer radiotherapy.