Previous studies have reported that a strong correlation between the estimated cumulative thermal exposure in the crystalline lens and the incidence of nuclear cataracts; however, the precise relationship between temperature and cataracts remains to be fully elucidated. In the present study, the shotgun liquid chromatography/mass spectroscopy‑based global proteomic approach was applied to investigate cataract‑inducing factors in lens cultured at normal (35.0˚C) and slightly warmer (37.5˚C) conditions. In the rat lens, 190 proteins (total) were identified. Of these, 48 proteins (25.3%) were found in lenses cultured at both 35.0˚C and 37.5˚C. Moreover, 85 proteins (44.7%) were unique to lenses cultured at 35.0˚C, while 57 proteins (30.0%) were unique to lenses cultured at 37.5˚C. Protein expression changes in rat lenses cultured at 37.5˚C were examined using a label‑free semiquantitative approach that uses spectral counting and Gene Ontology analysis. Filensin and vimentin protein expression, key factors in maintaining lens structure, were decreased. These findings may serve as a valuable indicator for elucidating the relationship between temperature and the onset of nuclear cataracts.

Neokestose is considered to have a prebiotic function. However, the physiological activity of neokestose remains unknown. Neokestose has a blastose, a sucrose analog, in its structure. We previously demonstrated that oral administration of blastose to diabetic rats suppressed the increase in plasma glucose (PG) concentration after sucrose administration. Therefore, neokestose might have a similar effect. In this study, we investigated the effects of neokestose on PG concentrations and the mechanism of its action. We first administered neokestose orally to streptozotocin-induced diabetic rats and observed that the expected consequent increase in PG concentration was significantly suppressed. Next, we examined the inhibitory effect of neokestose on glycosidase activity, but observed only a slight inhibitory effect. Therefore, we hypothesized that neokestose might be hydrolyzed by gastric acid to produce blastose. We performed an acid hydrolysis of neokestose using artificial gastric juice. After acid hydrolysis, peaks corresponding to neokestose and its decomposition products including blastose were observed. Therefore, we suggest that neokestose and blastose, a decomposition product, synergistically inhibit glycosidase activity. These findings support the potential use of neokestose as a useful functional oligosaccharide that can help manage plasma glucose concentrations in patients with diabetes mellitus.

Maple syrup is a natural sweetener consumed worldwide. Active ingredients of maple syrup possess antitumor effects; however, these ingredients are phenolic compounds. The present study aimed to investigate components other than phenolic compounds that may have antitumor effects against colorectal cancer (CRC). Cell proliferation assays demonstrated that treatment with the more than 10,000 molecular weight fraction significantly inhibited viability in DLD‑1 cells. Therefore, we hypothesized that the protein components of maple syrup may be the active ingredients in maple syrup. We obtained protein components from maple syrup by ammonium sulfate precipitation, and treatment with the protein fraction of maple syrup (MSpf) was found to exhibit a potential antitumor effect. MSpf‑treated DLD‑1 colon adenocarcinoma cells exhibited significantly decreased proliferation, migration and invasion. In addition, upregulation of LC3A and E‑cadherin and downregulation of MMP‑9 expression levels were observed following MSpf treatment. Investigation of the components of MSpf suggested that it was primarily formed of advanced glycation end products (AGEs). Therefore, whether AGEs in MSpf affected the STAT3 pathway through the binding to its receptor, receptor of AGE (RAGE), was assessed. MSpf treatment was associated with decreased RAGE expression and STAT3 phosphorylation. Finally, to determine whether autophagy contributed to the inhibitory effect of cell proliferation following MSpf treatment, the effect of MSpf treatment on autophagy induction following bafilomycin A1 treatment, a specific autophagy inhibitor, was assessed. The inhibitory effect of MSpf treatment on cell proliferation was enhanced through the inhibition of autophagy by bafilomycin A1 treatment. These results suggested that AGEs in MSpf suppressed cell proliferation and epithelial‑mesenchymal transition through inhibition of the STAT3 signaling pathway through decreased RAGE expression. Therefore, AGEs in MSpf may be potential compounds for the development of antitumor drugs for the treatment of CRC with fewer adverse effects compared with existing antitumor drugs.

In the original publication [...].

Edible plant-derived nanovesicles have been explored as effective materials for preventing colorectal cancer (CRC) incidence, dependent on gene status, as a K-Ras-activating mutation via the macropinocytosis pathway. Approximately 70% of CRC harbors the p53 mutation, which is strongly associated with a poor prognosis for CRC. However, it has not been revealed whether p53 inactivation activates the macropinocytosis pathway or not. In this study, we investigated parental cells, wild-type or null for p53 treated with Citrus limon L.-derived nanovesicles, as potential materials for CRC prevention. Using ultracentrifugation, we obtained C. limon L.-derived nanovesicles, the diameters of which were approximately 100 nm, similar to that of the exosomes derived from mammalian cells. C. limon L.-derived nanovesicles showed inhibitory effects on cell growth in not p53-wild, but also in p53-inactivated CRC cells. Furthermore, we revealed that the macropinocytosis pathway is activated by p53 inactivation and C. limon L.-derived nanovesicles were up taken via the macropinocytosis pathway. Notably, although C. limon L.-derived nanovesicles contained citrate, the inhibitory effects of citrate were not dependent on the p53 status. We thus provide a novel mechanism for the growth inhibition of C. limon L.-derived nanovesicles via macropinocytosis and expect to develop a functional food product containing them for preventing p53-inactivation CRC incidence.

Fructosyl oligosaccharides, including fructo-oligosaccharide (FOS), are gaining popularity as functional oligosaccharides and have been found in various natural products. Our previous study suggested that maple syrup contains an unidentified fructosyl oligosaccharide. Because these saccharides cannot be detected with high sensitivity using derivatization methods, they must be detected directly. As a result, an analytical method based on charged aerosol detection (CAD) that can detect saccharides directly was optimized in order to avoid relying on these structures and physical properties to clarify the profile of fructosyl oligosaccharides in maple syrup. This analytical method is simple and can analyze up to hepta-saccharides in 30 min. This analytical method was also reliable and reproducible with high validation values. It was used to determine the content of saccharides in maple syrup, which revealed that it contained not only fructose, glucose, and sucrose but also FOS such as 1-kestose and nystose. Furthermore, we discovered a fructosyl oligosaccharide called neokestose in maple syrup, which has only been found in a few natural foods. These findings help to shed light on the saccharides profile of maple syrup.

The tricarboxylic acid (TCA) cycle is one of the most important pathways of energy metabolism, and the profiles of its components are influenced by factors such as diseases and diets. Therefore, the differences in metabolic profile of TCA cycle between healthy and cancer cells have been the focus of studies to understand pathological conditions. In this study, we developed a quantitative method to measure TCA cycle metabolites using LC-MS/MS to obtain useful metabolic profiles for development of diagnostic and therapeutic methods for cancer. We successfully analyzed 11 TCA cycle metabolites by LC MS/MS with high reproducibility by using a PFP column with 0.5% formic acid as a mobile phase. Next, we analyzed the concentration of TCA cycle metabolites in human cell lines (HaCaT: normal skin keratinocytes; A431: skin squamous carcinoma cells; SW480: colorectal cancer cells). We observed reduced concentration of succinate and increased concentration of citrate, 2-hydroxyglutarate, and glutamine in A431 cells as compared with HaCaT cells. On the other hand, decreased concentration of isocitrate, fumarate, and α-ketoglutarate and increased concentration of malate, glutamine, and glutamate in A431 cells were observed in comparison with SW480 cells. These findings suggested the possibility of identifying disease-specific metabolites and/or organ-specific metabolites by using this targeted metabolomic analysis.

The authors wish to make the following corrections to this paper [...].

Circulating tumor cells (CTCs) are involved in metastasis; thus, one of the most important approaches for identifying metastatic cancer is to detect CTCs in blood. In the present study, we examined whether directly analyzing cells with capillary electrophoresis (CE) could distinguish cancer cells from normal cells, based on differences in cell surface glycosylation. We compared human colorectal cancer (CRC) cell lines to a normal colon epithelium cell line. Our results demonstrated that direct CE analysis could successfully distinguish between CRC and normal cells with high reproducibility, based on migration times. We found that the weighted-average migration time was significantly shorter for CRC cells than for normal cells. Next, we observed changes in the electrophoretic behaviors of CRC cells by adding five different types of lectins. When Aleuria aurantia lectin was added, migration delays were observed in CRC cells, but not in normal colon cells. Therefore, by focusing on shifts in migration time after adding specific lectins, we could distinguish cancer cells from normal cells. These findings suggested that this diagnostic method of directly analyzing cells with CE after adding specific lectin(s) could be useful for detecting the difference in the sugar moieties on a surface of normal and cancer cells.

Enhanced expression of cyclophilin A (CypA) in colorectal cancer (CRC) was reported; however, how CypA influences CRC progression is not clear. Therefore, we examine the effects of CypA on CRC cell progression. Knockdown of CypA in SW480 cells significantly inhibited cell migration and invasion but had no effect on cell proliferation. In addition, upregulation of E-cadherin and downregulation of N-cadherin and Snail expression were observed by CypA knockdown. These results suggested that CypA knockdown inhibited cell migration and invasion by suppressing epithelial-mesenchymal transition. CypA knockdown was also associated with increased p38 phosphorylation, and the p38 inhibitor treatment led to increase in the number of invasive CypA-knockdown SW480 cells. Therefore, CypA may be a potential therapeutic target in preventing CRC metastasis.

It is important to elucidate how retinal stimulation leads to retinal protection and dysfunction. The current study aimed to identify factors that are up- and downregulated in the retinas of streptozotocin (STZ)-induced diabetic rats with acute retinal dysfunction. Retinal function was measured and changes in protein expressions were determined using electroretinograms (ERGs) and liquid chromatography/mass spectroscopy-based shotgun proteomics, respectively. The results revealed that the plasma glucose levels of STZ rats were markedly higher when compared with normal rats. Furthermore, levels of a-waves, b-waves and oscillatory potential amplitudes on ERGs in STZ rats were decreased compared with healthy animals. With use of shotgun proteomics, 391 proteins were identified in the retinas of normal rats and 541 proteins were found in the retinas of STZ rats. Of the 560 proteins identified in rat retinas, 372 (66.4%) were present in both normal and STZ rats. Of these, 19 (3.39%) were unique to normal rats and 169 (30.1%) were unique to STZ rats. Gene Ontology analysis was performed on the candidate proteins that were differentially regulated in the retinas of STZ rats and focused on those classified as 'protein binding', which serve important roles in retinal neurodegeneration. The results revealed an excessive expression of retinol-binding protein 1 (RBP1) and a negative expression of rod outer segment membrane protein 1 (Rom-1) in the retinas of STZ rats. Therefore, retinal function may be decreased with STZ-induced injury, and expressions of Rom-1 and RBP1 may be altered in the retinas of STZ rats.

The incidence of diabetes mellitus (DM) is increasing rapidly and is associated with changes in dietary habits. Although restrictions in the use of sweeteners may prevent the development of DM, this might reduce the quality of life of patients with DM. Therefore, there has been a great deal of research into alternative sweeteners. In the search for such sweeteners, we analyzed the carbohydrate content of maple syrup and identified a novel oligosaccharide composed of fructose and glucose, linked at the C-4 of glucose and the C-6 of fructose. This oligosaccharide inhibited the release of fructose from sucrose by invertase (IC50: 1.17 mmol/L) and the decomposition of maltose by α-(1-4) glucosidase (IC50: 1.72 mmol/L). In addition, when orally administered together with sucrose to rats with DM, the subsequent plasma glucose concentrations were significantly lower than if the rats had been administered sucrose alone, without having any effect on the insulin concentration. These findings suggest that this novel oligosaccharide might represent a useful alternative sweetener for inclusion in the diet of patients with DM and may also have therapeutic benefits.

Maple syrup is a natural sweetener that is consumed worldwide. It has been previously reported that dark-colored maple syrup exerts an inhibitory effect on colorectal cancer (CRC) proliferation and invasion. In the present study, the underlying mechanism of CRC cell growth inhibition was examined with dark-colored maple syrup treatment using a shotgun liquid chromatography-tandem mass spectrometry-based global proteomic approach. Applying a semi-quantitative method based on spectral counting, 388 proteins were identified with expression changes of >1.5-fold following dark-colored maple syrup treatment. Gene Ontology analysis revealed that these proteins possessed cell cycle-associated functions. It was also indicated that CRC cells treated with dark-colored maple syrup exhibited decreased proliferating cell nuclear antigen (PCNA) expression and S-phase cell cycle arrest. Dark-colored maple syrup treatment also resulted in altered expression of cell cycle-associated genes, including cyclin-dependent kinase (CDK)4 and CDK6. In conclusion, these data suggested that dark-colored maple syrup induced S-phase cell cycle arrest in CRC cells by reducing the expression of PCNA and regulating cell cycle-associated genes. These findings suggest that dark-colored maple syrup may be a source of compounds for the development of novel drugs for colorectal cancer treatment.

Diabetes mellitus is a widespread metabolic disorder, and long-term hyperglycemia in diabetics leads to diabetic keratopathy. In the present study, we used a shotgun liquid chromatography/mass spectrometry-based global proteomic approach using the cornea of streptozotocin-induced diabetic (STZ) rats to examine the mechanisms of delayed corneal wound healing in diabetic keratopathy. Applying a label-free quantitation method based on spectral counting, we identified 188 proteins that showed expression changes of >2.0-fold in the cornea of STZ rats. In particular, the level of lumican expression in the cornea of STZ rats was higher than that of the normal rats. In the cornea of the normal rat, the expression level of lumican was elevated during the wound healing process, and it returned to the same expression level as before cornea injury after the wound was healed completely. On the other hand, a high expression level of lumican in the cornea of STZ rats was still maintained even after the wound was healed completely. In addition, adhesion deficiency in corneal basal cells and Bowman's membrane was observed in the STZ rat. Thus, abnormally overexpressed lumican may lead to adhesion deficiency in the cornea of STZ rats.

Collagen peptides (CPs), derived by hydrolyzing collagen with chemicals or enzymes, are often used as functional materials, due to their various bioactivities and high bioavailability. A previous study by our group reported that collagen from soft‑shelled turtle, Pelodiscus sinensis, induces keratinocytes to undergo epithelial‑mesenchymal transition and facilitates wound healing. Therefore, CPs derived from soft‑shelled turtle collagen may have useful effects on the skin. In the present study, the functional effects of CPs on human skin were examined by analyzing CP‑treated human keratinocytes with a shotgun liquid chromatography/mass spectrometry‑based global proteomic approach. A semi‑quantitative method based on spectral counting was applied and 211 proteins that exhibited >2‑fold changes in expression after CP treatment were successfully identified. Based on a Gene Ontology analysis, the functions of these proteins were indicated to be closely linked with protein processing. In addition, CP treatment significantly increased the expression of calpain‑1, a calcium‑dependent intracellular cysteine protease. Furthermore, CP‑treated keratinocytes exhibited elevated interleukin (IL)‑1α and IL‑8 expression and reduced IL‑6 expression. CPs also induced the expression of proteins implicated in cell‑cell adhesion and the skin barrier. Therefore, CPs from soft‑shelled turtle may provide significant benefits for maintaining the biological environment of the skin, and may be useful as components of pharmaceuticals and medical products.

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.

Sericin is a major constituent of silk produced by silkworms. We previously found that the instillation of sericin enhanced the proliferation of corneal epithelial cells, and acted to promote corneal wound healing in both normal and diabetic model rats. However, the mechanisms by which sericin promotes the proliferation of corneal cells have not been established. In this study, we investigated the effects of sericin on Akt and ERK activation in a human corneal epithelial cell line (HCE-T cells) and rat debrided corneal epithelium. Although Akt phosphorylation was not detected following the treatment of HCE-T cells with sericin, ERK1/2 phosphorylation was enhanced. The growth of HCE-T cells treated with sericin was significantly increased, with the cell growth of sericin-treated HCE-T cells being 1.7-fold higher in comparison with vehicle-treated HCE-T cells. On the other hand, both of an ERK inhibitor U0126 (non-specific specific inhibitor) and SCH772984 (specific inhibitor) attenuated the enhanced cell growth by sericin, and the growth level in the case of co-treatment with sericin and ERK1/2 inhibitor was similar to that of cells treated with ERK1/2 inhibitor alone. In an in vivo study using rat debrided corneal epithelium, the corneal wound healing rate was enhanced by the instillation of sericin, and this enhancement was also attenuated by the instillation of U0126. In addition, the corneal wound healing rate in rats co-instilled with sericin and U0126 was similar to that following the instillation of U0126 alone. In conclusion, we found that the instillation of sericin enhanced cell proliferation via the activation of the MAPK/ERK pathway, resulting in the promotion of corneal wound healing in rat eyes. These findings provide significant information for designing further studies to develop potent corneal wound-healing drugs.

Streptozotocin (STZ)-induced diabetic rats (STZ rats) were used to investigate diabetic cataracts. In the current study, a shotgun liquid chromatography (LC)/mass spectrom-etry (MS)-based global proteomic analysis method was used to examine the mechanism of lens opacification as a result of hyperglycemia in STZ rats. The 6-week old Wistar rats were injected with STZ for 2 days (100 mg/kg/day, i.p.) and housed for 3 weeks. The plasma glucose levels were identified to be significantly higher when compared with the normal rats and insulin was not detected in the STZ rats. Furthermore, opacification of the cortical epithelium was observed in the lenses of STZ rats. A total of 235 proteins were identified in the lenses of the STZ rats and 229 in the lenses of the normal rats. A label-free semi-quantitative method, based on spectral counting, identified 52 proteins that were differentially expressed in the lenses of STZ rats compared with normal rats. In particular, superoxide dismutase, which is a critical antioxidant enzyme that detoxifies superoxide through redox cycling, was downregulated when analyzed by the semi-quantitative method. In addition, phosphorylated-p38, which is important in the signaling pathway involved in the oxidative stress response, was significantly increased in the lenses of STZ rats when compared with normal rats (P< 0.05). Thus, the changes in protein expression were evaluated in the lenses of STZ rats using a shotgun LC/MS-based global proteomic analysis approach, and a decrease in antioxidant enzymes and an increase in oxidative stress were identified in the lenses of STZ rats. Further studies are required to examine the role of these proteins in the onset or progression of diabetic cataracts.

Soft-shelled turtles (Pelodiscus sinensis) are widely distributed in some Asian countries, and we previously reported that soft-shelled turtle tissue could be a useful material for collagen. In the present study, we performed shotgun liquid chromatography (LC)/mass spectrometry (MS)-based global proteomic analysis of collagen-administered human keratinocytes to examine the functional effects of collagen from soft-shelled turtle on human skin. Using a semiquantitative method based on spectral counting, we were able to successfully identify 187 proteins with expression levels that were changed more than twofold by the administration of collagen from soft-shelled turtle. Based on Gene Ontology analysis, the functions of these proteins closely correlated with cell-cell adhesion. In addition, epithelial-mesenchymal transition was induced by the administration of collagen from soft-shelled turtle through the down-regulation of E-cadherin expression. Moreover, collagen-administered keratinocytes significantly facilitated wound healing compared with nontreated cells in an in vitro scratch wound healing assay. These findings suggest that collagen from soft-shelled turtle provides significant benefits for skin wound healing and may be a useful material for pharmaceuticals and medical care products. © 2017 The Authors Journal of Biomedical Materials Research Part B: Applied Biomaterials Published by Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 2403-2413, 2018.

Maple syrup is a natural sweetener that is commonly consumed worldwide. While maple syrup mainly comprises sucrose, it also contains phytochemicals that present various biological effects. Maple syrup is made by boiling down sap, and its color and composition vary in accordance with the sap collection season. Typically, seasonal progression is associated with darker syrup color, and antioxidant activity is proportional to the increasingly dark color. The authors previously reported that maple syrup demonstrated inhibitory effects on colorectal cancer cell growth and invasion, which correlated with darker maple syrup color. In the present study, they examined the effects of two different grades of maple syrup on gastrointestinal cancer cell proliferation, to investigate whether the dark-color maple syrup was suitable as a phytomedicine for gastrointestinal cancer treatment. Administration of dark-color maple syrup significantly inhibited gastrointestinal cancer cell growth as compared to non-treated cancer cells. Moreover, administration of dark-color maple syrup clearly inhibited protein kinase B (AKT) phosphorylation and did not impact mitogen-associated protein kinase phosphorylation. These data suggested that dark-color maple syrup may inhibit cell proliferation through suppression of AKT activation and, thus, may be suitable as a phytomedicine for gastrointestinal cancer treatment.

The prognosis of hepatocellular carcinoma (HCC) is unfavorable following complete tumor resection. The aim of the present study was to identify a molecule able to predict HCC prognosis through comprehensive protein profiling and to elucidate its clinicopathological significance. Comprehensive protein profiling of HCC was performed by liquid chromatography-tandem mass spectrometry. Through the bioinformatic analysis of proteins expressed differentially in HCC and non-HCC tissues, protein disulfide-isomerase A3 (PDIA3) was identified as a candidate for the prediction of prognosis. PDIA3 expression was subsequently examined in 86 cases of HCC by immunostaining and associations between PDIA3 expression levels and clinicopathological characteristics were evaluated. The Ki-67 index and apoptotic cell death of carcinoma cells were examined by immunostaining and terminal deoxynucleotidyl transferase dUTP nick-end labeling assay in 24 cases. The results demonstrated that PDIA3 was expressed in all 86 HCC cases; 56 HCC cases (65%) exhibited high expression of PDIA3 and 30 (35%) exhibited low expression. The disease-free and overall survival times of HCC patients with high PDIA3 expression were significantly shorter than in HCC patients with low expression. Furthermore, increased expression of PDIA3 was associated with an elevated Ki-67 index, indicating increased cancer cell proliferation and a reduction in apoptotic cell death. Taken together, these results suggest that PDIA3 expression is associated with tumor proliferation and decreased apoptosis in HCC, and that increased expression of PDIA3 predicts poor prognosis. PDIA3 may therefore be a key molecule in the development of novel targeting therapies for patients with HCC.

Colorectal cancer (CRC) is one of the most common cancers worldwide, and many patients are already at an advanced stage when they are diagnosed. Therefore, novel biomarkers for early detection of colorectal cancer are required. In this study, we performed a global shotgun proteomic analysis using formalin-fixed and paraffin-embedded (FFPE) CRC tissue. We identified 84 candidate proteins whose expression levels were differentially expressed in cancer and non-cancer regions. A label-free semiquantitative method based on spectral counting and gene ontology (GO) analysis led to a total of 21 candidate proteins that could potentially be detected in blood. Validation studies revealed cyclophilin A, annexin A2, and aldolase A mRNA and protein expression levels were significantly higher in cancer regions than in non-cancer regions. Moreover, an in vitro study showed that secretion of aldolase A into the culture medium was clearly suppressed in CRC cells compared to normal colon epithelium. These findings suggest that decreased aldolase A in blood may be a novel biomarker for the early detection of CRC.

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.

Soft-shelled turtles (Pelodiscus sinensis) are widely distributed in some Asian countries, and parts of this turtle contain abundant collagen. In this study, we optimized a method for extracting collagen from the soft-shelled turtle. We used three types of solvent and four extraction conditions to determine an effective collagen extraction method, which was extraction at 37 degrees C with acetic acid after hydrochloric acid pretreatment. Next, we extracted collagen from three regions in the soft-shelled turtle: muscle, skin, and an area of soft tissue in the periphery of the turtle shell known in Japan and China as the "emperor." We determined that emperor tissue yielded the highest concentration and purity of collagen. We then optimized the pretreatment method for extraction from emperor tissue by using formic acid instead of hydrochloric acid, and the amount of extracted collagen increased by approximately 1.3-fold. Finally, we identified the optimal solvent out of four types of organic acid for collagen extraction from emperor tissue; the amount of extracted collagen from emperor tissue increased approximately 3-fold when citric acid was used as the extraction solvent instead of acetic acid. Emperor tissue can regenerate; thus, it is possible to obtain collagen from the emperor repeatedly without killing the turtle. Our findings suggest that the emperor tissue of soft-shelled turtles may be a good source of collagen for pharmaceutical and cosmetic applications.

Nuclear HMGB1 that contains 3 cysteine residues is acetylated and secreted to the extracellular space, promoting inflammation via multiple molecules such as RAGE and TLR4. We thus evaluated and characterized the redox state-dependent effects of peripheral HMGB1 on nociception. Intraplantar (i.pl.) administration of bovine thymus-derived HMGB1 (bt-HMGB1), all-thiol HMGB1 (at-HMGB1) or disulfide HMGB1 (ds-HMGB1) caused long-lasting mechanical hyperalgesia in mice. The hyperalgesia following i.pl. bt-HMGB1 or at-HMGB1 was attenuated by RAGE inhibitors, while the ds-HMGB1-induced hyperalgesia was abolished by a TLR4 antagonist. Thus, nociceptive processing by peripheral HMGB1 is considered dependent on its redox states. (C) 2016 Japanese Pharmacological Society. Production and hosting by Elsevier B.V.

Epithelial ovarian cancer (EOC) consists of four major subtypes: clear cell carcinoma (CCC), endometrioid adenocarcinoma (EA), mucinous adenocarcinoma (MA) and serous adenocarcinoma (SA). Relative to the other subtypes, the prognosis of CCC is poor due to a high recurrence rate and chemotherapy resistance, but CCC-specific biomarkers have yet to be identified. With the aim of identifying diagnostic and treatment biomarkers for CCC, we analyzed 96 cases of EOC (32 CCC, 13 EA, 19 MA, 32 SA) using liquid chromatography/mass spectrometry (LC/MS) followed by immunohistochemistry (IHC) and quantitative reverse transcription PCR (RT-qPCR). Semi-quantification of protein differences between subtypes showed upregulation of 150 proteins and downregulation of 30 proteins in CCC relative to the other subtypes. Based on hierarchical clustering that revealed a marked distinction in the expression levels of cystatin B (CYTB) and Annexin A4 (ANXA4) in CCC relative to the other subtypes, we focused the study on CYTB and ANXA4 expression in E0Cs by IHC, RT-qPCR and western blot analyses using tissue specimens and cultured cells. As a result, compared to the other subtypes, CCC showed significantly high expression levels of CYTB and ANXA4 in the analyses. To examine the possibility of CYTB and ANXA4 as serum diagnostic biomarkers of CCC, we checked the protein levels in conditioned media and cell lysates using culture cells. Compared with the other subtypes, CCC cell lines showed a significantly higher level of expression of CYTB in both conditioned media and cell lysates, while ANXA4 showed a higher level of expression in cell lysates only. Our results demonstrate that CYTB and ANXA4 overexpression may be related to carcinogenesis and histopathological differentiation of CCC. CYTB may be a secreted protein, and may serve as a potential serum diagnostic biomarker of CCC, while ANXA4 may be useful as an intracellular marker.

Maple syrup is a natural sweetener consumed by individuals of all ages throughout the world. Maple syrup contains not only carbohydrates such as sucrose but also various components such as organic acids, amino acids, vitamins and phenolic compounds. Recent studies have shown that these phenolic compounds in maple syrup may possess various activities such as decreasing the blood glucose level and an anticancer effect. In this study, we examined the effect of three types of maple syrup, classified by color, on the cell proliferation, migration and invasion of colorectal cancer (CRC) cells in order to investigate whether the maple syrup is suitable as a phytomedicine for cancer treatment. CRC cells that were administered maple syrup showed significantly lower growth rates than cells that were administered sucrose. In addition, administration of maple syrup to CRC cells caused inhibition of cell invasion, while there was no effect on cell migration. Administration of maple syrup clearly inhibited AKT phosphorylation, while there was no effect on ERK phosphorylation. These data suggest that maple syrup might inhibit cell proliferation and invasion through suppression of AKT activation and be suitable as a phytomedicine for CRC treatment, with fewer adverse effects than traditional chemotherapy.

We investigate whether maple syrup is a suitable sweetener in the management of type 2 diabetes using the Otsuka Long-Evans Tokushima Fatty (OLETF) rat. The enhancement in plasma glucose (PG) and glucose absorption in the small intestine were lower after the oral administration of maple syrup than after sucrose administration in OLETF rats, and no significant differences were observed in insulin levels. These data suggested that maple syrup might inhibit the absorption of glucose from the small intestine and preventing the enhancement of PG in OLETF rats. Therefore, maple syrup might help in the prevention of type 2 diabetes.

Lumican, a member of the small leucine-rich proteoglycan family, regulates the assembly and diameter of collagen fibers in the extracellular matrix of various tissues. The lumican expression correlates with pathological conditions and the growth and metastasis of various malignancies. In cutaneous neoplasms, the lumican expression is lower in advanced-stage malignant melanomas that invade the dermis than in early-stage melanomas. Furthermore, we have recently reported that the expression pattern of lumican is different from that of actinic keratosis and the Bowen disease. Lumican is positive in the poroid cells of intraepidermal sweat ducts therefore, we examined the expression patterns of lumican in acanthotic-type seborrheic keratosis and Pinkus-type poroma followed by clonal-type seborrheic keratosis and hidroacanthoma simplex. The neoplastic cells of acanthotic-type seborrheic keratosis exhibited positive immunostaining in only 1 of 31 cases (3.23%), whereas the poroid cells of Pinkus-type poroma exhibited positive immunoreactivity in 26 of 28 patients (92.8%). In the hidroacanthoma simplex cases, lumican was expressed in poroid cells forming intraepidermal nests in 22 of 28 patients (78.6%), whereas the neoplastic cells in most cases of clonal-type seborrheic keratosis were negative for lumican. In some seborrheic keratosis cases that were positive for lumican in neoplastic cells, lumican was observed in squamoid cells but not in basaloid cells. Therefore, it is necessary to evaluate the immunoreactivity of lumican in seborrheic keratosis and in basaloid cells. These findings suggest that lumican is a potent differential diagnostic marker that distinguishes hidroacanthoma simplex from clonal-type seborrheic keratosis. © 2014 Lippincott Williams & Wilkins.

Lumican, a member of the small leucine-rich proteoglycan family, regulates the assembly and diameter of collagen fibers in the extracellular matrix of various tissues. Lumican expression correlates with pathological conditions, including skin fragility, corneal opacification, and corneal and cardiac wound healing. Lumican is overexpressed in tumor cells, including in the breast, colorectal, neuroendocrine cell, uterine cervical, and pancreatic cancers. Lumican expression also correlates with the growth and metastasis of various malignancies. For example, lumican expression is lower in the dermis of malignant melanoma cases than in early-stage melanomas. However, the expression patterns and roles of lumican in nonmelanoma skin cancer have not been elucidated. In this study, we used immunohistochemistry and in situ hybridization to examine the expression patterns of lumican in normal skin, Bowen disease, and actinic keratosis. In normal skin, lumican was expressed in the collagen fibers in the dermis, acrosyringium, follicular epithelium, and sebocytes but not in epidermal keratinocytes. In Bowen disease, lumican was expressed in 34 (91.8%) of 37 patients. Notably, all cases of actinic keratosis were negative for lumican. These findings suggest that lumican plays an important role in the pathogenesis of Bowen disease and actinic keratosis and might be useful as an adjunct to the diagnosis for subtypes of 2 diseases: bowenoid actinic keratosis and Bowen disease in sun-exposed areas.

Lumican, a member of the class II small leucine-rich proteoglycan family, regulates the assembly and diameter of collagen fibers in the extracellular matrix of various tissues. We previously reported that lumican expression in the stromal tissues of pancreatic ductal adenocarcinoma (PDAC) correlates with tumor invasion, and tends to correlate with poor prognosis. Lumican stimulates growth and inhibits the invasion of a PDAC cell line. In the present study, we performed a global shotgun proteomic analysis using lumican-overexpressing PANC-1 cells and lumican downregulated PANC-1 cells to identify candidate proteins that are regulated by lumican and related to cell growth and invasion in PDAC cells. A total of 448 proteins were identified from lumican-overexpressing PANC-1 and control cells. Additionally, 451 proteins were identified from lumican-downregulated PANC-1 cells and control cells. As a result of semi-quantification based on spectral counting, 174 differentially expressed proteins were identified by lumican upregulation, and 143 differentially expressed proteins were identified by lumican downregulation. The expression levels of 24 proteins, including apoptosis-and invasion-related proteins correlated with lumican expression levels. It is likely that the expression of these proteins is regulated by lumican, and that they are involved in apoptosis and invasion in PDAC. These findings suggest that lumican may be involved in cell growth and invasion through the regulation of these 24 proteins expressed in PDAC.

Nodular fasciitis (NF) is a benign, reactive lesion with a self-limiting process. Because NF is rare in the parotid gland and has many cytological similarities to other benign or malignant tumors, cytological misinterpretation is common. The patient, a 30-year-old woman, had a painless mass in her right parotid gland. Fine needle aspiration cytology (FNAC) was performed. Spindle cells with basophilic and well-demarcated cytoplasm were observed in a mucoid-like background. The mucoid-like substance was metachromatic and appeared to be the matrix of PA. Histopathologically, spindle-shaped cells with intervening birefringent mature collagen were arranged in short irregular bundles. Prominent mucoid-like matrixes as well as few infiltrating neutrophils and lymphocytes were found in the background. Lesional cells were positive for CD10 and -catenin in the cytoplasm, but negative for cytokeratin, the S-100 protein, CD34, and neurofilament. Ultimately, this patient was diagnosed with NF. In FNAC of the parotid gland region, distinguishing NF from other real tumors is important for deciding treatment strategies. Diagn. Cytopathol. 2013;41:829-833. (c) 2013 Wiley Periodicals, Inc.

Nestin expression reportedly correlates with aggressive growth, metastasis, poor prognosis and presence of cancer stem cells (CSCs) in various tumors. In this study, we determined the expression and role of nestin in cervical intraepithelial neoplasia (CIN) and cervical cancer. We performed immunohistochemical and in situ hybridization analyses of nestin in 26 cases for each stage of CIN and 55 cervical cancer tissue samples. To examine the role of nestin in cervical cancer cells, we stably transfected expression vectors containing nestin cDNA into ME-180 cells. We studied the effects of increased nestin expression on cell proliferation, cell motility, invasion as well as sphere and soft agar formation. Nestin was not localized in the squamous epithelium in normal cervical tissues, but it was weakly expressed in the basal squamous epithelium of CIN 1. In CIN 2, nestin was localized to the basal to lower 2/3 of the squamous epithelium, whereas in CIN 3, it was localized to the majority of the squamous epithelium. Nestin was detected in all cases of invasive cervical cancer. Nestin mRNA was expressed in both ME-180 and CaSki cells. Growth rate, cell motility and invasion ability of stably nestin-transfected ME-180 cells were not different from empty vector-transfected ME-180 (mock cells). However, the nestin-transfected ME-180 cells formed more colonies and spheres compared to the mock cells. These findings suggest that nestin plays important roles in carcinogenesis and tumor formation of cervical cancer cells. Nestin may closely correlate with regulation of CSCs.

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. (C) 2012 Elsevier Ireland Ltd. All rights reserved.

In pancreatic ductal adenocarcinoma (PDAC), the fibroblast growth factor receptor 1 (FGFR-1) IIIb isoform correlates with the inhibition of cancer cell proliferation, migration, and invasion, whereas FGFR-1 IIIc enhances cancer cell proliferation. The FGFR-2 IIIb isoform is expressed in PDAC, and its expression correlates with increased venous invasion. We examined the role of FGFR-2 IIIc in PDAC. FGFR-2 IIIc was expressed in all six pancreatic cancer cell lines examined and was highest in PANC-1 cells. FGFR-2 IIIc was abundant in the cancer cells from 83 of 117 PDAC cases, which correlated with decreased duration to development of liver metastasis after surgery. FGFR-2 IIIc-transfected cells exhibited increased proliferation in vitro and formed larger subcutaneous and orthotopic tumors, the latter producing more liver metastases. Moreover, FGF-2 exerted a more rapid stimulatory effect on the levels of phosphorylated extracellular signal-regulated kinase (p-ERK) in FGFR-2 IIIc stably transfected PANC-1 cells, compared with control cells. FGFR-2 IIIc-transfected cells also formed more spheres and contained more side population cells. Suppression of FGFR-2 IIIc expression inhibited the proliferation of PANC-1 cells, whereas an anti-FGFR-2 IIIc antibody inhibited the proliferation and migration of PANC-1 cells. Thus, high FGFR-2 IIIc levels in PDAC contribute to disease aggressiveness and confer to pancreatic cancer cells features suggestive of cancer stem cells, indicating that FGFR-2 IIIc may be a novel and important therapeutic target in PDAC. (Am J Pathol 2012,180:1928-1941; DOI: 10.1016/j.ajpath.2012.01.020)

Keratinocyte growth factor (KGF), also known as fibroblast growth factor-7, is mainly synthesized by mesenchymal cells. KGF modulates proliferation, differentiation, migration and adhesion to extracellular matrices of epithelial cells that specifically express the KGF receptor (KGFR). We previously reported that KGF is expressed in cancer cells and adjacent stromal fibroblasts in human pancreatic cancer tissues. Furthermore, KGF is thought to stimulate the growth of certain pancreatic cancer cell lines. The aim of the present study was to examine whether the mitogen-activated protein kinase (MAPK) pathway contributes to exogenous KGF-induced pancreatic cancer cell growth. Recombinant human KGF (rhKGF) was administered to MIA PaCa-2 cells, which expressed KGFR and negligible levels of KGF. Cell growth rates in MIA PaCa-2 cells were significantly increased in a dose-dependent manner following the addition of rhKGF. In the MAPK pathway, phosphorylation of extracellular signal-regulated kinase (ERK) in MIA PaCa-2 cells was increased in a dose-dependent manner, and phosphorylation of p38 was slightly increased following the administration of 100 ng/ml rhKGF. In contrast, JNK was not phosphorylated following the addition of rhKGF in MIA PaCa-2 cells. U0126, a specific inhibitor of ERK activation, decreased the rhKGF-induced phosphorylation of ERK and the growth rates of MIA PaCa-2 cells. These findings indicated that phosphorylation of the ERK signaling pathway plays a significant role in exogenous KGF-induced pancreatic cancer cell growth.

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. (C) 2011 Elsevier Ireland Ltd. All rights reserved.

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-I-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.

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 313 cultures. On the other hand, expression of F-actin was weak in the floating culture system. a-tubulin was detected in the cytoplasm of cells in 2D culture, but in floating and 3D cultures, a-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.

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 alpha 5 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 50 kDa lumican protein plays important roles in the inhibition of HEK cell attachment and growth, and it might inhibit the activation of integrin pathways. (C) 2010 Elsevier Inc. All rights reserved.

Keratinocyte growth factor (KGF), which is also called fibroblast growth factor (FGF)-7, belongs to the FGF family. KGF is not commonly produced by human cancer cells, but the KGF receptor (KGFR) is expressed in most cancer cells and particularly highly expressed in well-differentiated types of cancer. Recently, it has been reported that vascular endothelial growth factor (VEGF)-A expression is induced by KGF in pancreatic cancer cells. VEGF-A is produced by some cancer cells and plays important roles in the angiogenesis and metastasis of cancer cells including those in the colorectum. In this study, we examined whether recombinant human KGF (rhKGF) induces major angiogenic growth factors including VEGF-A, FGF-2 and hepatocyte growth factor (HGF) in human colorectal cancer cells (HCT-15), which express a high level of KGFR, but a low or negligible level of KGF. rhKGF significantly increased the VEGF-A expression level in a serum-free medium of HCT-15 cells, but FGF-2 and HGF expression levels were too low to detect. Furthermore, the expression levels of the angiogenic growth factors were evaluated in KGF-transfected HCT-15 cells, which were induced to stably overexpress KGF by KGF gene transfection and mock-transfected cells (Mock). KGF and VEGF-A expression levels in the cells and the protein concentrations in serum-free medium were significantly higher in KGF-transfected HCT-15 cells than in Mock cells. In contrast, the FGF-2 and HGF mRNA expression levels were not significantly different between KGF-transfected HCT-15 cells and Mock cells and the protein concentrations in scrum-free medium of the cells were below the detection level. These findings suggest that administration of rhKGF and over-expression of endogenous KGF genes in colorectal cancer cells increase VEGF-A production and may relate to angiogenesis in cancer.

Lumican is a member of a small leucine-rich proteoglycan family and is highly expressed in several types of cancer cells and/or stromal tissue. Lumican expression in the cytoplasm in advanced colorectal cancer correlates with poor patient prognosis. The expression of lumican in stromal tissues is associated with a high tumor grade, a low estrogen receptor expression level, and Young age in breast cancer and is associated with tumor invasion and advanced stage in pancreatic cancer. In this Study, we examined the expression and role of lumican in lung cancer including adenocarcinoma (ADC) and squamous cell carcinoma (SqCC). Immunohistochemically, lumican was weakly expressed in vascular smooth muscle cells, perivascular and peribronchial connective tissues and bronchial epithelium of normal lung tissues. In lung cancer tissues, lumican was localized in the cytoplasm of cancer cells and/or stromal tissues adjacent to cancer cells. In ADC. the expression level of lumican in cancer cells correlated with pleural invasion and larger tumor size, but that of lumican in stromal tissues did not correlate with clinicopathological factors. In SqCC, the expression level of lumican in cancer cells con-elated with formation of a keratinized pattern, and stromal lumican expression correlated with vascular invasion. In SqCC and ADC, the expression level of lumican in cancer cells did not correlate with patient prognosis. In lung cancer cell lines, lumican mRNA and protein were expressed in LC-1/Sq and EBC-1 cells established from SqCC, and A549, RERF-LC-KJ and PC-3 cells from ADC. The molecular weight of lumican extracted from the cytoplasm of lung cancer cells differed from that in the culture medium owing to glycosylation of the protein. These findings suggest that the

Formation of covalently bound protein adducts with lithocholic acid (LCA) might explain LCA's known carcinogenic properties and hepatotoxicity. We performed studies aimed at isolating and identifying hepatic proteins tagged with LCA, presumably via the epsilon-amino group of lysine residues. Antibodies recognizing the 3 alpha-hydroxy-5 beta-steroid moiety of LCA were generated by immunizing rabbits with immunogens in which the carboxyl group of LCA was coupled to BSA via a 6-aminohexanoic acid and/or succinic acid spacer. The resulting antibodies reacted with N-alpha (t-butoxycarbonyl)-L-lysine-epsilon-LCA, the amidated and nonamidated forms of LCA, as well as synthetically prepared LCA adducts with ovalbumin and lysozyme. Proteins tagged with LCA in the liver of bile duct-ligated rats were isolated by immunoprecipitation using these antibodies. Proteins were isolated by two-dimensional electrophoresis, and their structure was identified using matrix-assisted laser desorption ionization time-of-flight mass spectrometry and computer-assisted programs. Proteins labeled with LCA were Rab-3, Rab-12, Rab-16, and M-Ras. Rab proteins are Ras-like small GTP binding proteins that regulate vesicle trafficking pathways. The covalent binding of the Rab proteins with LCA may influence vesicular transport or binding of vesicles to their cognate membrane and may contribute to LCA-induced liver toxicity.

Reactive metabolic-modified proteins have been proposed to play an important role in the mechanism(s) of the hepatotoxicity and colon cancer of lithocholic acid (LCA). To identify cellular proteins chemically modified with LCA, we have generated a monoclonal antibody that recognizes the 3 alpha-hydroxy-5 beta-steroid moiety of LCA. The spleen cells from a BALB/c mouse, which was immunized with an immunogen in which the side chain of LCA was coupled to bovine serum albumin (BSA) via a succinic acid spacer, was fused with SP2/0 myeloma cells to generate antibody-secreting hybridoma clones. The resulting monoclonal antibody (gamma 2b, kappa) was specific to LCA-N-alpha-BOC-lysine as well as the amidated and nonamidated forms of LCA. The immunoblot enabled the detection of LCA residues anchored on BSA and lysozyme. The antibody will be useful for monitoring the generation, localization, and capture of proteins tagged with LCA, which may be the cause of LCA-induced toxicity.

The keratinocyte growth factor receptor (KGFR), also known as FGFR2 IIIb, is mainly localized in epithelial cells and is activated by the keratinocyte growth factor (KGF) that is predominantly synthesized by mesenchymal cells. In this study, we examined the roles of KGFR and KGF in human esophageal cancer (EC). In noncancerous esophageal tissues, KGFR was localized in epithelial cells from the basal region of the epithelium to the lower one-third of the epithelium, and KGF was weakly localized in the basal to parabasal epithelial cells. On the other hand, Ki-67 was localized in the parabasal cells. In EC tissues, KGFR and KGF were expressed in cancer cells in 22 and 37 of 54 patients, respectively. The coexpression of KGFR and KGF in cancer cells was detected in 14 of 54 (26%) patients. Clinicopathologically, KGFR expression correlated with the well-differentiated cell type of EC (p<0.001), and KGF expression correlated with lymphatic invasion and lymph node metastasis (p=0.004 and 0.021, respectively). The coexpression of KGFR and KGF in cancer cells correlated with the well-differentiated cell type of EC (p=0.001). KGFR-positive, KGF-positive and KGFR/KGF coexpression patients tended to have shorter survival rates, but the survival rates were not statistically significantly different (p=0.44, 0.059 and 0.112, respectively). In human EC cell lines (TE-1, TE-8 and TE-11), KGFR mRNA was expressed but no KGF mRNA was detected. The KGFR mRNA level was highest in TE-1 cells, derived from well-differentiated SCC and lowest in TE-8 cells. KGFR was detected in the cancer cell lines by Western blot analysis. Recombinant human KGF significantly stimulated the growth of TE-8 and -11 cells, derived from moderately differentiated SCC, but had no effect on TE-1 cell growth. These results suggest that KGFR expression correlates with the differentiation of a normal esophageal epithelium and the well-differentiated cell type of EC. On the other hand, KGF may induce the growth of some EC cells in a paracrine manner and closely correlates with lymphatic invasion and lymph node metastasis.

Lumican is a member of a small leucine-rich proteoglycan family and its overexpression has been reported in carcinoid tumor, breast, colorectal, neuroendocrine cell, uterine cervical and pancreatic cancers. The expression of lumican in stromal tissues in breast cancer is associated with a high tumor grade, a low estrogen receptor expression level and young age. Lumican expression in the cytoplasm in advanced colorectal cancer is correlated with a poor prognosis. Lumican expression was previously reported in pancreatic cancer, but the role of lumican in pancreatic cancer is still not well understood. In this study, we aimed to clarify the role of lumican in pancreatic cancer. Reverse-tran scription polymerase chain reaction and Western blot analyses revealed lumican mRNA and protein expression in six pancreatic ductal adenocarcinoma cell lines (i.e. PANC-1, MIA PaCa-2, KLM-1, Capan-1, PK-1 and PK-8). On the basis of its immunoreactivity, lumican was found to be localized in islet cells of normal pancreatic tissues, but not in exocrine cells. In pancreatic cancer tissues, lumican was predominantly localized in the cytoplasm of cancer cells in 30 out of 53 (56.6%) cancer patients, whereas lumican was detected in stromal tissues in 36 out of 53 (67.9%) cancer patients. Lumican expression in pancreatic cancer cells did not correlate with clinicopathological factors, whereas lumican expression in stromal tissues correlated with the female gender, advanced stage, retroperitoneal and duodenal invasion and residual tumor (p=0.030, 0.038, 0.049, 0.049 and 0.048, respectively). Patients with lumican-positive cancer cells tended to survive longer than those with lumican-negative cancer cells (p=0.286), but patients with lumican-positive stromal tissues had shorter survival than those with lumican-negative stromal tissues (p=0.062). These results suggest that lumican in stromal tissues plays an important role in the growth and invasion of pancreatic cancer.

Lumicanは糖鎖修飾されることが知られているが、産生される臓器や細胞により異なる糖鎖修飾を受けることが示唆されている。今回、膵臓癌細胞でのみ産生される特異糖鎖修飾を受けたlumicanの存在について検討するため、消化器癌細胞より産生されるlumicanの精製を試みた。その結果、食道癌と膵臓癌細胞から産生・分泌されるlumicanを精製することができた。また、そのlumicanの発現パターンの違いを検討した所、膵臓癌細胞特異的と思われるlumicanが発現していた。このことから、この膵臓特異的に発現するlumicanを標的とした新たな診断法の開発につながる可能性が考えられた。

ルミカンの発現量と細胞増殖能には正の相関性があり、細胞浸潤能とは負の相関性があることが報告されている。今回、ルミカンが膵癌細胞の増殖や浸潤に関わる機構について検討するため、ルミカン発現調製細胞を用い、それらの細胞の発現タンパク質の変動を網羅的に解析した。その結果、24種類のタンパク質がルミカンの発現と相関性があることが明らかとなった。この中にはアポトーシスのマーカーとして知られているタンパク質やMMP-9の活性化と関連があるタンパク質が含まれていたことから、ルミカンはアポトーシスやこれらのタンパク質の発現に関わることで細胞増殖や浸潤を制御していると考えられた。

線維芽細胞増殖因子受容体2IIIc(FGFR-2IIIc)は、ヒト膵臓癌症例の約70%に発現しており、FGFR-2IIIc陽性症例は術後の肝転移再発までの期間の短縮を認めた。FGFR-2IIIc遺伝子発現ベクターを遺伝子導入した膵臓癌培養細胞は、細胞増殖能の亢進と、皮下移植および同所移植腫瘍の体積の増大が見られた。FGFR-2IIIcに対するポリクローナル抗体を作成し、膵臓癌培養細胞に投与したところ、細胞増殖能と遊走能が抑制された(Ishiwata,Am J Pathol,2012)。FGFR-2の別のアイソフォームであるFGFR-2IIIbへのスプライシングを促進させるepithelial splicing regulatory protein(ESRP)1を遺伝子導入すると、膵臓癌の転移が抑制された。以上より、膵臓癌のFGFR-2IIIcの発現制御は、膵臓癌の治療標的となると考えられた。

浸潤性膵管癌の間質におけるルミカンの発現は、癌の浸潤や予後の悪化と関連があることが報告されている。今回、遺伝子導入法を用いて、培養膵臓癌細胞によって分泌されるルミカンの機能について検討した。ルミカン過剰発現膵癌細胞は、分子量70kDaのルミカンを過剰に分泌し、それに伴いERKが活性化した。また、遺伝子導入したことにより細胞増殖能が亢進するとともにラミニンに対する接着能が増加した。さらに細胞浸潤能が抑制され、それに関連してMMP-9の活性が抑制された。一方、siRNAを用いてルミカンの発現を抑制すると逆の結果が得られた。このことから、膵臓癌細胞によって分泌される70kDaのルミカンは、膵臓癌細胞の増殖や浸潤に重要な役割を果たしていると考えられた。

ケラチノサイト増殖因子(KGF)とその受容体(KGFR)は膵臓癌の増殖・浸潤に重要な役割を果たしている。培養膵臓癌細胞にリコンビナントKGFを投与すると、濃度依存的に膵臓癌細胞の増殖が進行し、それに関連してERKやp38といったMAPKが活性化された。一方、プロテオグリカンの一つであるルミカンの膵臓癌における発現量を調節したところ、ルミカンの発現量と細胞増殖及びERKの活性化に正の相関関係が認められた。KGFとKGFRの結合の安定化にはプロテオグリカンが必要なことから、ルミカンはKGF/KGFR系の活性化を制御することで膵臓癌の増殖を制御している可能性が考えられた。