Kiyoko Kawahara

Nestin is a novel target for suppressing pancreatic cancer cell migration, invasion and metastasis.

Nestin, is a class VI intermediate filament (IF) that is expressed in 30% of pancreatic ductal adenocarcinoma (PDAC) cases, and its expression in PDAC positively correlates with peripancreatic invasion. An expression vector carrying a short hairpin RNA (shRNA) targeting nestin was stably transfected into PANC-1 and PK-45H human pancreatic cancer cells, which express high nestin levels. Alterations in morphology and alignment of actin filaments and α-tubulin were examined by phase-contrast and immunocytochemistry. Effects on cell growth, migration in scratch and Boyden chamber assays, invasion, cell adhesion, and in vivo growth were determined. Differences in mRNA levels were examined by arrays. Nestin shRNA-transfected cells exhibited decreased nestin expression, a sheet-like appearance with tight cell-cell adhesion, increased expression of filamentous F-actin and E-cadherin, and attenuated migration and invasion, both of which were enhanced following nestin re-expression. Expression of α-tubulin, and in vitro cell growth and adhesion were not altered by nestin down-regulation, whereas hepatic metastases were decreased. Thus, nestin plays important roles in pancreatic cancer cell migration, invasion and metastasis by selectively modulating the expression of actin and cell adhesion molecules, and may therefore be a novel therapeutic target in PDAC. See commentary: Pancreatic cancer with Nest-in tendencies

Expression of lumican in human colorectal cancer cells

Lumican is a member of a small leucine‐rich proteoglycan family and its overexpression in human breast cancer tissues is reported to influence the growth of cancer cells. In the present study, we aimed to clarify the expression of lumican mRNA and its protein in human colorectal cancer cell lines and their localization in normal and cancerous colorectal tissues. Reverse transcription–polymerase chain reaction and western blot analysis revealed lumican mRNA and its protein expression in COLO 205, DLD‐1, HCT‐15, SW 480 and WiDr colorectal cancer cell lines. The lumican in colorectal cancer cells had non‐sulfated or poorly sulfated polylactosamine side chains. Based on its immunoreactivity, the lumican protein was found to be localized in fibroblasts and stromal tissues of normal colorectal tissues, but not in colorectal epithelial cells. In colorectal cancer tissues, the lumican was strongly localized in cancer cells in eight of 12 cancer cases. The lumican protein was also localized in epithelial cells with mild reactive dysplasia and fibroblasts adjacent to cancer cells. Lumican mRNA was expressed in cancer cells and adjacent fibroblasts, and epithelial cells. These findings may indicate that the lumican protein synthesized by cancer cells, fibroblasts and epithelial cells with mild reactive dysplasia found adjacent to cancer cells may affect the growth of human colorectal cancer cells.

Neuroepithelial stem cell marker nestin regulates the migration, invasion and growth of human gliomas

Nestin, a class VI intermediate filament protein, was originally described as a neuronal stem cell marker during central nervous system development. Nestin is expressed in gliomas, and its expression levels are higher in gliomas with high WHO histopathological classification grades than in those with low grades. In the present study, we examined whether nestin regulates the biological activities of human glioma cells. Immunohistochemically, the nestin expression patterns in 10 human glioblastoma patients were examined. The expression levels of nestin in A172, a human high-grade glioma cell line, and KG-1-C, a human low-grade glioma cell line, were examined using real-time PCR, Western blot and immunofluorescence analyses. An expression vector carrying a short hairpin RNA targeting nestin was stably transfected into A172 (Sh) cells. The effects of decreased expression levels of nestin in Sh cells on cell growth, migration, invasion, adhesion to extracellular matrices and fibrillar actin expression on three-dimensional culture plates were examined. The nestin expression vector was transiently transfected into KG-1-C (Nes) cells, and the effects of the nestin overexpression on cell growth and migration were examined. Nestin was expressed in the cytoplasm of the glioblastoma cells in all cases examined. Sh cells showed marked decreases in the expression levels of nestin mRNA and protein, and the growth rate of Sh cells was lower than that of sham (Sc) cells. In contrast, the adhesion activity of Sh cells to types I and IV collagens, fibronectin and laminin was higher than that of Sc cells. Fibrillar actin was clearly detected at the periphery of colonies of Sh cells at the attachment sites on three-dimensional culture plates. The migration and invasion of Sh cells were markedly inhibited compared with those of Sc cells. In contrast, the levels of nestin expression markedly increased in the Nes cells, which were transiently transfected with the nestin expression vector. The growth rate and motility of Nes cells were higher than those of the mock cells. In conclusion, nestin plays important roles in cell growth, migration, invasion and adhesion to extra-cellular matrices in glioma cells. Nestin may serve as a novel candidate for molecular-targeted therapy for gliomas, including glioblastomas.

Overexpressed fibroblast growth factor receptor 2 in the invasive front of colorectal cancer: A potential therapeutic target in colorectal cancer

Fibroblast growth factor receptor 2 (FGFR2) is considered a novel therapeutic target for various cancer. We used a silencing strategy to clarify the effect of reduced FGFR2 expression in human colorectal cancer (CRC) cells. The invasive front of cancer cells exhibited stronger FGFR2 expression than the surface area of the cancers. FGFR2 shRNA-transfected LoVo cells inhibited cell migration, invasion and tumor growth in vitro and in vivo. Thus, FGFR2 plays important roles in CRC progression in association with tumor cell migration, invasion and growth, and FGFR2 might be a novel therapeutic target for CRC.

Comparison of Fixation Methods for Preservation of Morphology, RNAs, and Proteins From Paraffin-Embedded Human Cancer Cell-Implanted Mouse Models

Xenograft transplantation of human tumor cells into immunodeficient mice is an important method to clarify the roles of specific molecules or chemicals in vivo. Recently, this method has been reported as a definitive examination to identify tumor stem cells. In this study, the authors compared the morphology and the quality and quantity of ribonucleic acid (RNA) and protein in paraffin-embedded tissues of nude mice implanted with human uterine cervical cancer cells, followed by fixation with commonly used fixatives, including 4% paraformaldehyde (PFA), 10% neutral buffered formalin (NBF), 20% NBF, and 99% ethanol (EtOH). The quality of the isolated RNA from PFA- and NBF-fixed paraffin-embedded tissues was high, while EtOH-fixed tissues showed degradation of RNA. NBF-fixed tissues showed excellent quality of morphology, but EtOH-fixed tissues showed contraction of cells. Immunohistochemical results showed differences depending on fixations. The 99% EtOH-fixed samples showed decreases of Ki-67 and VEGF-A immunoreactivities, but improved cytokeratin immunoreactivity. This study indicated that formalin fixation is better than alcohol fixation for RNA preservation in paraffin-embedded cancer cell implantation models. Immunohistochemical results differed markedly depending on fixation materials and antibodies; therefore, suitable fixations are needed to quantify and compare the results of immunohistochemical staining on cancer cell implanted nude mice tissues.

Secreted 70 kDa lumican stimulates growth and inhibits invasion of human pancreatic cancer

Lumican expression in the stromal tissues of pancreatic ductal adenocarcinoma (PDAC) correlates with tumor invasion, and tends to correlate with poor prognosis. We used gene transfection techniques to examine the biological roles of lumican secreted from PDAC cells. Lumican-transfected PANC-1 cells secreted a 70-kDa lumican protein and had an active ERK pathway. Transfection stimulated PANC-1 cell growth, increased cell adhesion to laminin, inhibited cell invasion, and decreased active matrix metalloproteinase-9. Down-regulation of lumican using siRNA resulted in opposite cell behavior. Thus, the 70-kDa lumican secreted by PDAC cells plays important roles in cell growth and invasion.

Morphological and cytoskeletal changes of pancreatic cancer cells in three-dimensional spheroidal culture

Three-dimensional (3D) cell cultures are expected to mimic in vivo environments. We used a NanoCulture plate to determine the spheroid-forming ability of pancreatic ductal adenocarcinoma (PDAC) cell lines and compared the morphology and expression of cytoskeletal proteins of PDAC cells to those in two-dimensional (2D) cultures. All examined PDAC cells grew as monolayers in 2D culture. PANC-1 and KLM-1 formed spheroids in 3D culture, but PK-45H and MIAPaCa-2 did not. Strong expression of F-actin was observed in the cells attached to the surface of the plate, which formed cell projections in 3D culture. F-actin was detected on the grids of the NanoCulture plate in PANC-1 cells but not in PK-45H. The levels of tubulin expression in cells were higher in 3D culture than in 2D culture. The expression level of E-cadherin mRNA in PANC-1 and KLM-1 was higher than that in PK-45H and MIAPaCa-2. In conclusion, PDAC cells showed morphological changes, spheroid formation, and alterations of cytoskeletal proteins in 3D culture. E-cadherin might be one of the key molecules involved in spheroid formation of PDAC cells. The 3D spheroidal culture system was a useful method for cell imaging with contrast-phase microscopy and confocal microscopy.

Enhanced expression of lumican inhibited the attachment and growth of human embryonic kidney 293 cells.

Lumican is a member of a small leucine-rich proteoglycan (SLRP) family and it regulates the assembly and diameter of collagen fibers in the extracellular matrix of various tissues. Lumican expression was reported in various kinds of tumor cells. Lumican inhibits the growth of melanoma cells, but the lumican in pancreatic cancer correlated with an advanced stage and retroperitoneal and duodenal invasion. In this study, we clarified whether the enhanced expression of lumican contributes to cellular attachment, growth, colony formation, migration and invasion. HEK 293 cell, stably transfected with lumican cDNA synthesized and secreted a 50 kDa lumican protein at high levels in culture medium. The cells showed a polygonal appearance with long projections and the degree of adhesion of the cells to fibronectin was lower than that of empty vector transfected control cells (mock cells). In contrast, the degree of adhesion of the cells to type I collagen was not different from that of mock cells. The expression levels of alpha5 integrin, the major integrin subunit for fibronectin, were lower in lumican-transfected HEK cells than in mock cells. Furthermore, lumican-transfected HEK cells showed reduced growth rates in vitro and did not form colonies in soft agar. Phosphorylation of AKT, extracellular signal-regulated kinase (ERK) 1/2 and mammalian target of rapamycin (mTOR) decreased in the lumican-transfected HEK cells. Cell migration and invasion were not altered in lumican-transfected HEK cells and mock cells. These findings indicate that the 50kDa lumican protein plays important roles in the inhibition of HEK cell attachment and growth, and it might inhibit the activation of integrin pathways.

Ultrastructural changes and immunohistochemical localization of advanced glycation end products in the heart of streptozotocin-treated Mongolian gerbils.

This study was designed to clarify the developing mechanism of cardiomyopathy and vasculopathy in streptozotocin-treated Mongolian gerbils. Twenty male Mongolian gerbils (MG; 10–12 weeks old) were used, and 150 mg/kg of streptozotocin (STZ) was injected into the left femoral vein. Six control male MG were injected intravenously with normal saline. The animals showed severe hyperglycemia (up to 330 ± 96.4 mg/dl) by 1 week after streptozotocin administration. At 1 week after STZ treatment, cardiomyocytes revealed no significant change, but unclear striated structures were demonstrated in cardiomyocytes at 4 weeks. After 1 year, anisocytosis was observed, and in the perinuclear region granular components were stained positively with periodic acid-Schiff reagent. Ultrastructurally, at 4 weeks and 1 year after STZ treatment, cardiomyocytes were irregular in size, and oval amorphous and lysosomal electron-dense bodies were observed in perinuclear and cytoplasmic regions. In coronary arteries, endothelial and medial cells revealed increased vesicles and intercellular collagen fibrils. Capillaries showed slight swelling of endothelial cells associated with the lamellar thickening of basement membrane and collagen fibrils in the perivascular regions. Immunohistochemically, advanced glycation end products (AGE) were observed in the cytoplasm of vascular and heart cells, and ultrastructurally the reaction products were demonstrated in the endoplasmic reticulum and lysosomes of cardiomyocytes and vascular cells in the STZ-treated Mongolian gerbils. AGE may play an important role not only in angiopathy but also in cardiomyopathy of STZ-treated Mongolian gerbils after STZ treatment.

Expression of keratinocyte growth factor receptor (KGFR/FGFR2 IIIb) in vascular smooth muscle cells.

Keratinocyte growth factor receptor (KGFR), also known as fibroblast growth factor receptor (FGFR)2 IIIb, is located in many types of epithelial cells and is activated by four known ligands (FGF‐1, FGF‐3, FGF‐7 (also known as KGF) and FGF‐10) that are predominantly synthesized by mesenchymal cells. In the early stage of atherosclerosis, vascular smooth muscle cells (VSMC) transform from a contractile to a synthetic phenotype, proliferate and migrate into the intima. Previously, FGF‐7 mRNA expression was reported in VSMC, but KGFR mRNA was not detected. In the present study, we attempted to determine whether KGFR is localized in VSMC cultured from rat aorta and VSMC in human normal and atherosclerotic coronary arteries. Expression of KGFR mRNA and its protein was detected in cultured rat VSMC by reverse transcription–polymerase chain reaction and western blot analysis, respectively. Immunohistochemically, KGFR was localized in the VSMC of the outer layer of the media in normal human coronary arteries. Furthermore, it was localized in the VSMC of the media and thickened intima of atherosclerotic arteries. Recombinant FGF‐7 and/or FGF‐10 proteins stimulated the growth of cultured rat VSMC. These findings indicate that KGFR localized in VSMC may contribute to the proliferation of VSMC in normal and atherosclerotic arteries.