Yoko Kawamoto

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.

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.

Insulin-like growth factor 2 mRNA-binding protein-3 as a marker for distinguishing between cutaneous squamous cell carcinoma and keratoacanthoma

In the histopathological diagnosis of cutaneous tumors, the differential diagnosis of squamous cell carcinoma (SCC) with crateriform architecture and keratoacanthoma (KA) is often difficult so an accurate understanding of the biological features and the identification of reliable markers of SCC and KA are crucial issues. Insulin-like growth factor 2 mRNA-binding protein-3 (IGF2BP3, also known as IMP3) is thought of as a bona fide oncofetal protein, which is overexpressed and is involved in cell proliferation, migration, and invasion in several kinds of tumors. However, the role of IMP3 in cutaneous SCC and KA has not been well studied. Therefore, we focused on studying the biological functions of IMP3 in SCC and KA. In human skin SCC cell lines, HSC-1 and HSC-5, and the human keratinocyte cell line, HaCaT, IMP3 mRNA levels were significantly higher than that of normal human skin. The knockdown of IMP3 expression reduced the proliferation of HSC-1, and significantly reduced invasion by HSC-1 and HSC-5. In contrast, the knockdown of IMP3 did not significantly affect invasion by HaCaT cells. In immunohistochemical studies of SCC and KA tissues, the Ki-67 labeling index (LI) of the suprabasal cell layer was significantly higher in SCC, compared with KA tissues and the tumor-free margin (TFM) adjacent to SCC and KA. Most SCC tissues stained strongly positive for IMP3, but KA tissues and TFM were mostly negative for IMP3. The Ki-67 LI of the IMP3-positive group was significantly higher than that of the IMP3-negative group in the suprabasal cell layer of SCC. These results suggest that IMP3 plays an important role in proliferation and, more significantly, in the invasion of SCC, and may be a suitable marker for the histopathological diagnosis of SCC with a crateriform architecture and KA. Furthermore, IMP3 may potentially be a new therapeutic target for SCC.

Acinar Cell Cystadenoma of the Pancreas in a Cat

Cystic tumours of the pancreas are heterogeneous lesions with a spectrum of morphology and biological behaviour in people. These are poorly characterized in animals. A multicystic tumour of the pancreas was identified in an 11-year-old, female, mixed breed cat. The tumour was 5.5 cm in diameter and the largest cysts were 1.5 cm in diameter. Microscopically, the cysts were lined by single layered or pseudostratified, flat, cuboidal or columnar epithelial cells that occasionally formed papillary structures with a thin fibrous core. The tumour cells had eosinophilic granules in the apical cytoplasm, similar to zymogen granules, and the nuclei were uniform in size and shape. Mitotic figures were not observed. Immunohistochemically, the tumour cells expressed trypsin, but not cytokeratin 7. A diagnosis of acinar cell cystadenoma of the pancreas was made and this is the first report of this tumour in a cat.

Morphological and cytoskeletal alterations of nervous system tumor cells with different culturing methods.

Cell culture is one of the most important methods of research in molecular and cellular biology, and various culture systems have been developed, including two-dimensional (2D), three-dimensional (3D) and floating culture systems. In the present study, we examined morphological changes and different expression patterns of cytoskeletal proteins in three different types of nervous system tumor cells grown in 2D, 3D and floating cell cultures. A172, KG-1-C and IMR-32 cells showed marked morphological changes, depending on the cell culture methods. F-actin expression was clearly observed at the level of the cells nearest the plate surface in 2D and 3D cultures. On the other hand, expression of F-actin was weak in the floating culture system. α-tubulin was detected in the cytoplasm of cells in 2D culture, but in floating and 3D cultures, α-tubulin was expressed in the peripheral regions of spheres and spheroids. In conclusion, this study demonstrated that nervous system tumor cells showed different alterations in morphology, and different cytoskeletal protein expression patterns, depending on the culture methods.

Phosphorylation of Thr1495 of nestin in a mouse model of cerebral ischemia and reperfusion damage

Nestin, a class VI intermediate filament protein, is expressed by neuronal progenitor cells in the subventricular zone (SVZ). In the present study, we analyzed the nestin expression and phosphorylation levels in nerve cells in a mouse model of cerebral ischemia and reperfusion. C57BL/6 mice were subjected to three‐vessel occlusion for 14 min, and were killed either 1 or 4 days after the procedure. The percentages of cells in the SVZ that were positive for nestin, Thr1495‐phosphorylated nestin or Ki67 did not significantly differ between the ischemic reperfusion and sham groups. Conversely, in the striatum and cornu ammonis 2 (CA2) regions, the mice at 4 days after ischemic reperfusion showed significantly higher numbers and percentages of nerve cells that were positive for nestin, Thr1495‐phosphorylated nestin and Ki67 compared to results from the other groups. To our knowledge, this is the first description of phosphorylated nestin expression in neural progenitor cells in the SVZ of adult mice. In this cerebral ischemia and reperfusion mouse model, cells positive for Thr1495‐phosphorylated nestin were increased in the striatum and CA2 field of the hippocampus; suggesting that nestin phosphorylation may play an important role in mitotically active neuronal progenitor cells.

2-Deoxy-d-glucose increases GFAT1 phosphorylation resulting in endoplasmic reticulum-related apoptosis via disruption of protein N-glycosylation in pancreatic cancer cells

The glycolytic inhibitor 2-deoxy-d-glucose (2DG) causes energy starvation, affecting cell viability in a wide range of cancer cell lines. To determine the action of 2DG in pancreatic cancer, we performed proteomic analysis of pancreatic cancer cell line after 2DG treatment. Eighty proteins showed differential expression and among these, proteins involved in phosphohexose metabolism were upregulated. Up-regulation of glutamine: fructose 6-phosphate aminotransferase 1 (GFAT1), which belongs to the hexosamine biosynthesis pathway (HBP) that produces uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) to maintain glycoprotein, was validated by evaluation of mRNA and protein levels. Therefore, we assessed the amounts of total N-glycoproteins. Unexpectedly, we found a reduction of total N-glycoproteins and phosphorylation of GFAT1 by AMP-activated protein kinase (AMPK). These data may shed light on HBP dysfunction. Furthermore, we found endoplasmic reticulum (ER) stress accompanied by increased expression of ER stress markers, such as glucose response protein 78 (GRP78) and C/EBP-homologous protein (CHOP), in 2DG-treated cells. Moreover, the additive activation of AMPK by metformin (Met) synergistically enhanced the reduction of protein N-glycosylation and cell growth inhibition in the presence of 2DG. These results suggest that 2DG reduces N-glycosylation of proteins following the increase of phosphorylation of GFAT1 and results in the inhibition of cell growth mediated by ER stress in pancreatic cancer cells.

Abstract 3750: Inhibition of phosphorylation of nestin decrease pancreatic cancer cell growth.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background: Nestin is a class VI intermediate filament, first described as a central nervous system (CNS) progenitor/stem cell marker and recently reported as a tumor stem cell marker in several malignant tumors, including glioblastoma and melanoma. In the normal pancreas, nestin is expressed in exocrine progenitor cells. Previously, we have reported that 30% of pancreatic cancer cases expressed nestin in cancer cells, and its expression correlated with nerve invasion and the presence of cancer cells in the tumor resection margins (Kawamoto, et al. Hum Pathol 2009). Knockdown of nestin exhibited a sheet-like appearance with tight cell-cell adhesion, increased expression of filamentous-actin and E-cadherin, and attenuated migration and invasion, both of which recovered following nestin re-expression (Matsuda, et al. Cancer Biol Ther 2011). Furthermore, knockdown of nestin suppressed sphere-forming ability, and altered the expression levels of stemness-related genes such as NANOG and transcription factor 4 (Matsuda, et al. AACR 2012). Therefore, nestin is considered to be a novel therapeutic target for pancreatic cancer via suppression of cell migration, invasion, metastasis and stemness. To develop nestin-targeting therapy, we focused on the phosphorylation of nestin protein. Phosphorylation of nestin at two different sites of progenitor cells of the CNS and myocytes has been reported to regulate the cell cycle via activation of cyclin-dependent kinase 5 and cdc2 kinase (Sahlgren, et al. Mol Cell Biol 2003). In this study, we analyzed the expression and roles of nestin phosphorylation in pancreatic cancer cells. Methods: Expression levels of phosphorylated nestin in pancreatic cancer cells were determined by flow cytometer, fluorescent staining and Western blot analyses. To assess the functions of phosphorylated nestin, we prepared three mutated nestin expression vectors which possess mutations at two different phosphorylated sites, both in nestin. A human pancreatic cancer cell line, MIA PaCa-2, which express very low levels of nestin was transfected with the mutated or wild-type nestin-expression vector. Results: Expression level of phosphorylated nestin was higher in M-phase cells than S- or G0/G1-phase cells. Phosphorylated nestin was strongly and diffusely localized in M-phase cells, while it was weakly detected in the nuclei of cells in other phases. Mutated nestin in phosphorylated site-transfected cells showed lower levels of cell growth, migration and invasion than wild-type nestin-transfected cells. Furthermore, mutated nestin-transfected cells had a tendency towards increasesed multinuclear cells. Conclusion: Phosphorylation of nestin affects the cell growth, migration and invasion of pancreatic cancer cells, and phosphorylated nestin may play important roles in the regulation of the cell cycle. Inhibition of phosphorylated nestin may be a novel target for pancreatic cancer treatment. Citation Format: Yoko Matsuda, Hisashi Yoshimura, Taeko Suzuki, Zenya Naito, Kiyoko Kawahara, Yuji Yanagisawa, Yoko Kawamoto, Toshiyuki Ishiwata. Inhibition of phosphorylation of nestin decrease pancreatic cancer cell growth. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3750. doi:10.1158/1538-7445.AM2013-3750

Abstract 249: Inhibition of nestin using siRNA as a novel therapeutic option for pancreatic cancer.

Introduction: Nestin is a member of the class VI intermediate filaments and it was first described as a central nervous system (CNS) progenitor/stem cell marker. Nestin is highly expressed in various cancers and was recently reported as a tumor stem cell marker for glioblastoma and melanoma. Previously,we have reported that 30% of pancreatic cancer cases expressed nestin in cancer cells, and its expression correlated with nerve invasion and the presence of cancer cells in the tumor resection margins (Kawamoto, et al. Hum Pathol 2009). Knockdown of nestin exhibited a sheet-like appearance with tight cell-cell adhesion, increased expression of filamentous actin and E-cadherin, and attenuated migration and invasion (Matsuda, et al. Cancer Biol Ther 2011). These findings suggest that nestin is a novel therapeutic target for pancreatic cancer. In this study, we examined the effects of siRNA targeting nestin on pancreatic cancer cell growth migration, and invasion in vitro and in vivo. Methods: Two types of siRNA targeting nestin, which binds to different sites of nestin mRNA, were prepared and transfected to PANC-1 and PK-45H cells. Cell growth, migration, invasion and sphere formation abilities of these cells were analyzed in vitro. We also determined the synergistic effects of gemcitabine in pancreatic cancer cells under treatment with siRNA targeting nestin. For an in vivo study, we constructed luciferase-gene-transfected pancreatic cancer cells and orthotopically implanted the cells into the pancreas of NOD/Shi-scid/IL-2Rγ null (NOG) mice. We injected siRNA into the tail vein of NOG mice once per week with Invivofectamine. Every week, luciferin was injected into the peritoneal cavity of NOG mice and images of primary and metastases were taken using IVIS. After 6 weeks, animals were sacrificed, and the primary and metastatic tumors were macro and microscopically examined. Results: siRNA targeting nestin inhibited the growth, migration, invasion and sphere-forming ability of both PANC-1 and PK-45H cells. Treatment with gemcitabine, in addition to the treatment with siRNA targeting nestin in pancreatic cancer cells, decreased cell viability as compared with only gemcitabine, siRNA targeting nestin or control siRNA- treated pancreatic cancer cells. In the orthotopic implantation model, siRNA targeting nestin significantly decreased primary tumor formation in the pancreas, as compared with negative control siRNA groups. Furthermore, nestin siRNA markedly suppressed the formation of metastatic nodules in the liver and lungs. Conclusion: In pancreatic cancer cells, siRNA targeting nestin inhibited cell growth, migration, invasion and sphere formation in vitro, and primary tumor growth and metastatic tumor formation in vivo. Furthermore, siRNA targeting nestin had synergistic effects with gemcitabine. Inhibition of nestin using siRNA may be a novel therapeutic optiont for pancreatic cancer treatment. Citation Format: Toshiyuki Ishiwata, Hisashi Yoshimura, Taeko Suzuki, Yuji Yanagisawa, Yoko Kawamoto, Kiyoko Kawahara, Zenya Naito, Murray Korc, Yoko Matsuda. Inhibition of nestin using siRNA as a novel therapeutic option for pancreatic cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 249. doi:10.1158/1538-7445.AM2013-249