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Background and aims Immunoglobulin 4 (IgG4)-related disease (IgG4-RD) is a lymphoproliferative disorder characterized by elevated serum IgG4 levels and tissue infiltration of IgG4-positive plasma cells. We analyzed the serum proteins, whose levels varied based on the disease state and treatment. Materials and methods Serum proteins from patients with IgG4-related disease and healthy subjects were resolved using two-dimensional electrophoresis, silver-stained, and scanned. Alternatively, the proteins were labeled with Cy2, Cy3, and Cy5 before electrophoresis. The proteins, whose expression differed significantly between patients and healthy individuals, and between before and after steroid treatment, were identified and validated using enzyme-linked immunosorbent assays. Results Pre-treatment sera from patients with IgG4-related disease was characterized by increased levels of immunoglobulins such as IgG1, IgG4; inflammatory factors such as α-1 antitrypsin (A1AT); and proteins associated with immune system regulation such as clusterin and leucine-rich α-2-glycoprotein (LRG-1). The serum levels of A1AT, LRG-1 and clusterin, during treatment with prednisolone for up to 12 months revealed that LRG-1 levels were halved after 1 month of treatment, comparable to those in healthy subjects; LRG-1 levels remained normal until the end of treatment. Conclusion LRG-1 could serve as a novel biomarker of IgG4-related diseases.

Nonalcoholic steatohepatitis (NASH), the aggressive form of the most common chronic liver disease nonalcoholic fatty liver disease, is characterized by inflammation and damage in the liver. Although hepatocyte injury and cell death have been identified as cardinal pathological features of NASH, its pathogenesis has not yet been elucidated in detail. Immortalized cell lines and primary cultured cells have been used as in vitro models of NASH. However, these cells have several disadvantages, such as specialized characteristics by immortalization or limited growth potential. To overcome these difficulties and develop a strategy to analyze the pathology of NASH, we employed hepatocyte-like cells differentiated from human induced pluripotent stem cells (hiPSC-HLCs) as an in vitro model of NASH to clarify the intracellular effects of glyceraldehyde-derived advanced glycation end-products (AGEs), also named toxic AGEs (TAGE). The viability of hiPSC-HLCs decreased with the accumulation of TAGE in the cells, which was consistent with previous findings on human hepatocellular carcinoma cells and human primary cultured hepatocytes. In addition, the TAGE accumulation up-regulated the expression of inflammation-related genes (interleukin 6, interleukin 8, and monocyte chemoattractant protein-1) in hiPSC-HLCs. These results indicated that the accumulation of TAGE induced hiPSC-HLC cytotoxicity and inflammation, which are features of the pathology of NASH. Therefore, we suggest the use of hiPSC-HLCs as an important strategy for analyses of the pathology of NASH.

The habitual and excessive consumption of sugar (i.e., sucrose and high-fructose corn syrup, HFCS) is associated with the onset and progression of lifestyle-related diseases (LSRD). Advanced glycation end-products (AGEs) have recently been the focus of research on the factors contributing to LSRD. Approaches that inhibit the effects of AGEs may be used to prevent and/or treat LSRD; however, since the structures of AGEs vary depending on the type of reducing sugars or carbonyl compounds to which they respond, difficulties are associated with verifying that AGEs are an etiological factor. Cytotoxic AGEs derived from glyceraldehyde, a triose intermediate in the metabolism of glucose and fructose, have been implicated in LSRD and are called toxic AGEs (TAGE). A dietary imbalance (the habitual and excessive intake of sucrose, HFCS, or dietary AGEs) promotes the generation/accumulation of TAGE in vivo. Elevated circulating levels of TAGE have been detected in non-diabetics and diabetics, indicating a strong relationship between the generation/accumulation of TAGE in vivo and the onset and progression of LSRD. We herein outline current findings on “TAGE as a new target” for human health.

In diabetic patients, the metabolism of excess glucose increases the toxicity of the aldehyde group of sugar. Aldehydes, including glyceraldehyde (GA), react with intracellular proteins to form advanced glycation end-products (AGEs), which deteriorate bone quality and cause osteoporosis. One of the causes of osteoporotic fractures is impaired osteoblast osteogenesis; however, the cytotoxic effects of aldehydes and the subsequent formation of AGEs in osteoblasts have not yet been examined in detail. Therefore, the present study investigated the cytotoxicity of intracellular GA and GA-derived AGEs, named toxic AGEs (TAGE), in the mouse osteoblastic cell line MC3T3-E1. Treatment with GA induced MC3T3-E1 cell death, which was accompanied by TAGE modifications in several intracellular proteins. Furthermore, the downregulated expression of Runx2, a transcription factor essential for osteoblast differentiation, and collagen correlated with the accumulation of TAGE. The GA treatment also reduced the normal protein levels of collagen in cells, suggesting that collagen may be modified by TAGE and form an abnormal structure. Collectively, the present results show for the first time that GA and TAGE exert cytotoxic effects in osteoblasts, inhibit osteoblastic differentiation, and decrease the amount of normal collagen. The suppression of GA production and associated accumulation of TAGE has potential as a novel therapeutic target for osteoporosis under hyperglycemic conditions.

The habitual intake of large amounts of sugar, which has been implicated in the onset/progression of lifestyle-related diseases (LSRD), induces the excessive production of glyceraldehyde (GA), an intermediate of sugar metabolism, in neuronal cells, hepatocytes, and cardiomyocytes. Reactions between GA and intracellular proteins produce toxic advanced glycation end-products (toxic AGEs, TAGE), the accumulation of which contributes to various diseases, such as Alzheimer’s disease, non-alcoholic steatohepatitis, and cardiovascular disease. The cellular leakage of TAGE affects the surrounding cells via the receptor for AGEs (RAGE), thereby promoting the onset/progression of LSRD. We demonstrated that the intracellular accumulation of TAGE triggered numerous cellular disorders, and also that TAGE leaked into the extracellular space, thereby increasing extracellular TAGE levels in circulating fluids. Intracellular signaling and the production of reactive oxygen species are affected by extracellular TAGE and RAGE interactions, which, in turn, facilitate the intracellular generation of TAGE, all of which may contribute to the pathological changes observed in LSRD. In this review, we discuss the relationships between intracellular TAGE levels and numerous types of cell damage. The novel concept of the “TAGE theory” is expected to open new perspectives for research into LSRD.

Background: The death of pancreatic islet β-cells (β-cells), which are the insulin-producing cells, promote the pathology in both Type 1 and Type 2 diabetes mellitus (DM) (T1DM and T2DM), and they are protected by autophagy which is one of the mechanisms of cell survival. Recently, that some advanced glycation end-products (AGEs), such as methylglyoxial-derived AGEs and Nε-carboxymethyllysine, induced the death of β-cells were revealed. In contrast, we had reported AGEs derived from glyceraldehyde (GA, the metabolism intermediate of glucose and fructose) are considered to be toxic AGEs (TAGE) due to their cytotoxicity and role in the pathogenesis of T2DM. More, serum levels of TAGE are elevated in patients with T1 and T2DM, where they exert cytotoxicity. Aim: We researched the cytotoxicity of intracellular and extracellular TAGE in β-cells and the possibility that intracellular TAGE were associated with autophagy. Methods: 1.4E7 cells (a human β-cell line) were treated with GA, and analyzed viability, quantity of TAGE, microtubule-associated protein 1 light chain 3 (LC3)-I, LC3-II, and p62. We also examined the viability of 1.4E7 cells treated with TAGE-modified bovine serum albumin, a model of TAGE in the blood. Results: Intracellular TAGE induced death of 1.4E7 cells, decrease of LC3-I, LC3-II, and p62. Extracellular TAGE didn’t show cytotoxicity in the physiological concentration. Conclusion: Intracellular TAGE induced death of β-cells more strongly than extracellular TAGE, and may suppress autophagy via reduction of LC3-I, LC3-II, and p62 to inhibit the degradation of them.

The anhydrofructose pathway is an alternate pathway for glycogen degradation by α-1,4-glucan lyase. The sugar 1,5-anhydro-D-fructose (1,5-AF) acts as the central intermediate of this pathway, but its physiological role of in mammals is unclear. Glycation reactions forming advanced glycation end-products (AGEs) are important in the development of complications of diabetes mellitus. We hypothesized that 1,5-AF may contribute to cellular damage by forming 1,5-AF-derived AGEs (AF-AGEs) with intracellular proteins. To clarify the role of 1,5-AF in protein modification, we created a novel antibody targeting AF-AGEs. Serum albumin modified by AF-AGEs was prepared by incubating rabbit serum albumin (RSA) or bovine serum albumin (BSA) with 1,5-AF. After immunizing rabbits with AF-AGEs-RSA, affinity chromatography of anti-AF-AGE antiserum was performed on a Sepharose 4B column coupled with AF-AGEs-BSA or N-(carboxymethyl)/N-(carboxyethyl)lysine-BSA. A novel immunopurified anti-AF-AGE antibody was obtained and was characterized using a competitive enzyme-linked immunosorbent assay. Then an AF-AGEs assay was established using this immunopurified antibody. This assay was able to detect AF-AGEs in human and animal serum samples. Finally, intracellular accumulation of AF-AGEs was shown to be associated with damage to cultured hepatocytes (HepG2 cells). This is the first report about in vivo detection of AF-AGEs with a novel structural epitope.

Glucocorticoids (GCs) potently induce T-cell apoptosis in a GC receptor (GR)-dependent manner and are used to control lymphocyte function in clinical practice. However, its downstream pathways remain controversial. Here, we showed that GC-induced transcript 1 (GLCCI1) is a novel downstream molecule of the GC-GR cascade that acts as an antiapoptotic mediator in thymic T cells. GLCCI1 was highly phosphorylated and colocalized with microtubules in GLCCI1-transfected human embryonic kidney QBI293A cells. GR-dependent up-regulation of GLCCI1 was associated with GC-induced proapoptotic events in a cultured thymocyte cell line. However, GLCCI1 knockdown in a thymocyte cell line led to apoptosis. Consistently, transgenic mice overexpressing human GLCCI1 displayed enlarged thymi that consisted of larger numbers of thymocytes. Further molecular characterization showed that GLCCI1 bound to both dynein light chain LC8-type 1 (LC8) and its functional kinase, p21-protein activated kinase 1 (PAK1), thereby inhibiting the kinase activity of PAK1 toward LC8 phosphorylation, a crucial event in apoptotic signaling. GLCCI1 induction facilitated LC8 dimer formation and reduced Bim expression. Thus, GLCCI1 is a candidate factor involved in apoptosis regulation of thymic T cells.-Kiuchi, Z., Nishibori, Y., Kutsuna, S., Kotani, M., Hada, I., Kimura, T., Fukutomi, T., Fukuhara, D., Ito-Nitta, N., Kudo, A., Takata, T., Ishigaki, Y., Tomosugi, N., Tanaka, H., Matsushima, S., Ogasawara, S., Hirayama, Y., Takematsu, H., Yan, K. GLCCI1 is a novel protector against glucocorticoid-induced apoptosis in T cells.

Excessive intake of glucose and fructose in beverages and foods containing high-fructose corn syrup (HFCS) plays a significant role in the progression of lifestyle-related diseases (LSRD). Glyceraldehyde-derived advanced glycation end-products (AGEs), which have been designated as toxic AGEs (TAGE), are involved in LSRD progression. Understanding of the mechanisms underlying the effects of TAGE on gene expression in the kidneys remains limited. In this study, DNA microarray analysis and quantitative real-time polymerase chain reaction (PCR) were used to investigate whether HFCS-consuming Wister rats generated increased intracellular serum TAGE levels, as well as the potential role of TAGE in liver and kidney dysfunction. HFCS consumption resulted in significant accumulation of TAGE in the serum and liver of rats, and induced changes in gene expression in the kidneys without TAGE accumulation or upregulation of receptor for AGEs (RAGE) upregulation. Changes in specific gene expression profiles in the kidney were more correlated with TAGE levels in the liver tissue than in the serum. These findings suggest a direct or indirect interaction may be present between the liver and kidneys that does not involve serum TAGE or RAGE. The involvement of internal signal transduction factors such as exosomes or cytokines without IL-1&beta, and TNF-&alpha, is suggested to contribute to the observed changes in kidney gene expression.

Hepatocyte cell death is a key process in the pathogenesis of nonalcoholic steatohepatitis (NASH). However, the factors responsible for and mechanisms underlying NASH-related cell death have not yet been elucidated in detail. We herein investigated the effects of intracellular glyceraldehyde (GA)-derived advanced glycation end-products (AGEs), named toxic AGEs (TAGE), on the production of reactive oxygen species (ROS), which have been implicated in the pathogenesis of NASH. Cell death related to intracellular TAGE accumulation was eliminated in the hepatocyte carcinoma cell line HepG2 by the antioxidant effects of N-acetyl-L-cysteine. The intracellular accumulation of TAGE increased ROS production and the expression of Nrf2, including its downstream gene. These results suggest that ROS are produced in association with the accumulation of TAGE and are a direct trigger for cell death. We also investigated the factors responsible for these increases in ROS. Catalase activity did not decrease with the accumulation of TAGE, while mitochondrial membrane depolarization was enhanced in cells treated with GA. These results indicate that TAGE play an important role in mitochondrial abnormalities and increases in ROS production, both of which are characteristic features of NASH. The suppression of TAGE accumulation has potential as a new therapeutic target in the progression of NASH.

Nonalcoholic fatty liver disease (NAFLD) ranges from simple steatosis to nonalcoholic steatohepatitis (NASH), leads to fibrosis and potentially cirrhosis, liver failure, and hepatocellular carcinoma, and is one of the most common causes of liver disease worldwide. NAFLD has also been implicated in other medical conditions such as insulin resistance, obesity, metabolic syndrome, hyperlipemia, hypertension, cardiovascular disease, and diabetes. Continuous hyperglycemia has been implicated in the pathogenesis of diabetic micro- and macro-vascular complications via various metabolic pathways, and numerous hyperglycemia-induced metabolic and hemodynamic conditions exist, including the increased generation of various types of advanced glycation end-products (AGEs). We recently demonstrated that glyceraldehyde-derived AGEs (Glycer-AGEs), the predominant components of toxic AGEs (TAGE), played an important role in the pathogenesis of angiopathy in diabetic patients. Moreover, a growing body of evidence suggests that the interaction between TAGE and the receptor for AGEs may alter intracellular signaling, gene expression, and the release of pro-inflammatory molecules and also elicits the generation of oxidative stress in numerous types of cells including hepatocytes and hepatic stellate cells. Serum levels of TAGE were significantly higher in NASH patients than in those with simple steatosis and healthy controls. Moreover, serum levels of TAGE inversely correlated with adiponectin (adiponectin is produced by adipose tissue and is an anti-inflammatory adipokine that can increase insulin sensitivity). Furthermore, immunohistochemical staining of TAGE showed intense staining in the livers of patients with NASH. Serum levels of TAGE may be a useful biomarker for discriminating NASH from simple steatosis. The administration of atorvastatin (10 mg daily) for 12 months significantly improved NASH-related metabolic parameters and significantly decreased serum levels of TAGE. The steatosis grade and NAFLD activity score were also significantly improved. These results demonstrated that atorvastatin decreased the serum levels of TAGE in NASH patients with dyslipidemia and suggest the usefulness of TAGE as a biomarker for the attenuation of NASH. Serum levels of TAGE were significantly higher in non-B or non-C hepatocellular carcinoma (NBNC-HCC) patients than in NASH subjects without HCC or control subjects. TAGE may be involved in the pathogenesis of NBNC-HCC, and could, therefore, be a biomarker that could discriminate NBNC-HCC from NASH. We propose that serum levels of TAGE are promising novel targets for the diagnosis of and therapeutic interventions against NASH.

Hepatocyte cell death is a key feature of nonalcoholic steatohepatitis (NASH); however, the pathogenesis of NASH currently remains unclear. We aimed to investigate the effects of intracellular glyceraldehyde (GA)-derived advanced glycation end-products (GA-AGEs) on human hepatocyte cell death. The accumulation of intracellular GA-AGEs has been associated with the induction of DNA damage and hepatocyte necrotic cell death. Among intracellular GA-AGEs, caspase-3 has been identified as a GA-AGE-modified protein with abrogated protein function. Furthermore, the activation of caspase-3 and induction of hepatocyte apoptosis by camptothecin, a DNA-damaging agent, was suppressed by a treatment with GA. These results suggest the inhibitory effects of GA-AGE-modified caspase-3 on the induction of DNA-damage-induced apoptosis, which is associated with hepatocyte necrosis. Therefore, the suppression of necrosis, the inflammatory form of cell death, by the accumulation of GA-AGEs and GA-AGE-modified caspase-3 may represent a novel therapeutic target for the pathogenesis of NASH.

Non-alcoholic fatty liver disease (NAFLD) and alcoholic liver disease (ALD) are among the most common causes of chronic liver diseases in the westernized world. NAFLD and ALD are frequently accompanied by extrahepatic complications, including hepatocellular carcinoma and cardiovascular diseases, which have a negative impact on patient survival. The chronic ingestion of an excessive daily diet containing sugar/high-fructose corn syrup increases the level of the fructose/glucose metabolite, glyceraldehyde (GA), while the chronic consumption of an excessive number of alcoholic beverages increases the level of the alcohol metabolite, acetaldehyde (AA) in the liver. GA and AA are known to react non-enzymatically with the ε- or α-amino groups of proteins, thereby generating advanced glycation end-products (AGEs, GA-AGEs, and AA-AGEs, respectively) in vivo. The interaction between GA-AGEs and the receptor for AGEs (RAGE) alters intracellular signaling, gene expression, and the release of pro-inflammatory molecules and also elicits the production of reactive oxygen species by human hepatocytes and hepatic stellate cells, all of which may contribute to the pathological changes associated with chronic liver diseases. We herein discuss the pathophysiological roles of GA-AGEs and AA-AGEs (toxic AGEs, TAGE) and a related novel theory for preventing the onset/progression of NAFLD and ALD.

Objective Saikokeishito (TJ-10) is a Kampo (traditional Japanese herbal) medicine, clinically used for hundreds of years in East Asia. Among its various mechanisms elucidated so far, TJ-10 inhibits the production of transforming growth factor-β1 (TGF-β1) and development of pancreatic fibrosis in vivo. Oxidative damage of normal human dermal fibroblasts (NHDFs) in the corium is a cause of human dermal senescence. Our aim was to determine whether TJ-10 protects NHDFs from premature senescence by hydrogen peroxide (H 2 O 2 ). Methods Premature senescence was induced in NHDFs by 200 μmol/L H 2 O 2 for 4 h. Cell viability and the expressions of p53, AMP-activated protein kinase α1 (AMPKα1), AMPKα2, and 14-3-3 protein sigma (14-3-3 σ) were measured in NHDFs treated with TJ-10 for 48 h before exposure to H 2 O 2 for 4 h. Results Cell viability after treatment with 200 μmol/L H 2 O 2 for 4 h was similar (about 80%) to after pre-treatment with TJ-10. Ascorbic acid as a control did not protect NHDFs from damage by 200 μmol/L H 2 O 2 . Treatment with 200 μmol/L H 2 O 2 tended to up-regulate p53 and to down-regulate SIRT1 and AMPKα1, but had no effect on AMPKα2 and 14-3-3 σ expression. Pretreatment with TJ-10 inhibited H 2 O 2 -induced up-regulation of p53 and enhanced AMPKα1 expression. Conclusion It is suggested that Saikokeishito has a protective effect on oxidative stress-induced senescence of NHDFs.

Non-alcoholic fatty liver disease (NAFLD) is currently the most common feature of chronic liver disease. Non-alcoholic steatohepatitis (NASH) is a severe form of NAFLD, and one of its risk factors is hyperglycemia. The chronic ingestion of excessive amounts of high-fructose corn syrup is associated with an increased prevalence of fatty liver. Under hyperglycemic conditions, advanced glycation end-products (AGEs) are generated through a non-enzymatic glycation reaction between the ketone or aldehyde groups of sugars and amino groups of proteins. Glyceraldehyde (GA) is a metabolic intermediate of sugars, and GA-derived AGEs (known as toxic AGEs (TAGE)) have been implicated in the development of NASH. TAGE accumulates more in serum or liver tissue in NASH patients than in healthy controls or patients with simple steatosis. Furthermore, the TAGE precursor, GA, causes cell damage through protein dysfunctions by TAGE modifications and induces necrotic-type hepatocyte death. Intracellular TAGE may leak outside of necrotic-type cells. Extracellular TAGE then induce inflammatory or fibrotic responses related to the pathology of NASH in surrounding cells, including hepatocytes and hepatic stellate cells. This review focuses on the contribution of TAGE to the pathology of NASH, particularly hepatic cell death related to NASH.

Molecular targeted drugs, such as mTORC1 inhibitors, have been clinically popularized for advanced renal cell carcinoma treatment but metastasis is still a serious concern. mTORC2 has several important functions, including HIF-2α activation in malignant cells. HIF-2α suppresses E-cadherin expression, which is associated with tumor invasion and metastasis. We investigated whether mTORC2 regulates E-cadherin expression and controls cell motility during HIF-2α down-regulation in renal cell carcinoma cells.We used PP242, a dual inhibitor of mTORC1/mTORC2 and the mTORC1 specific inhibitor rapamycin. E-cadherin expression in 786-O cells was examined using real-time polymerase chain reaction, Western blot and immunocytochemical staining. Cell motility was analyzed by time-lapse microscopy and wound healing assay.High E-cadherin expression was found in RCC4/VHL cells but low levels were found in VHL defective RCC4 and 786-O cells. HIF-2α expression was suppressed only in RCC4/VHL cells. In 786-O cells HIF-2α inhibition induced by the dual mTORC1/C2 inhibitor PP242 (0.05 to 0.5 μmol/L) resulted in a dose dependent increase in E-cadherin expression and the restored E-cadherin was localized at cell-to-cell junctions. Treatment with the mTORC1 inhibitor rapamycin resulted in no significant change. The migration of PP242 treated cells was significantly suppressed compared with those treated with rapamycin.Results show that mTORC2 might regulate E-cadherin expression and suppress cell motility by controlling the mTORC2-HIF-2α signaling pathway. The dual inhibitor of mTORC1/C2 as a cadherin regulatory agent may be a novel therapeutic strategy with tumoricidal agents for advanced renal cell carcinoma.

Pancreatic cancer is one of the most lethal cancers, with an incidence equaling mortality. It is a heterogeneous group of neoplasms in which pancreatic ductal adenocarcinoma is most common. Pancreatic cancer cannot be cured even if detected early. When treatment is initiated, a suitable method of administration of anticancer drugs must be chosen. Anticancer drugs kill tumor cells. However, side effects including initiation are problematic in anticancer drug therapy. Improved methods for the diagnosis of side effects of pancreatic cancer by using sensitive and specific tumor markers are highly desirable. Therefore, efficient strategies for biomarker discovery are urgently needed. Here, we present an approach based on direct experimental access to proteins released by PANC-1 human pancreatic cancer cells in vitro. A two-dimensional (2-D) map and catalog of this subproteome, herein termed the secretome, were established comprising more than 1,000 proteins observed by '2-D difference in-gel electrophoresis analysis using cyanine dye'. We investigated 22 spots that were 1.20-fold upregulated and 31 spots that were 0.66-fold downregulated by gemcitabine chloride treatment. Proteins in these spots were identified by nano-high-performance liquid chromatography electrospray ionization time of flight mass spectrometry/mass spectrometry. Most secretome constituents were nominally cellular proteins. By mass spectrometry screening, 14-3-3 protein sigma (14-3-3 σ), protein S100-A8, protein S100-A9, galectin-7, lactotransferrin (lactoferrin, LF) precursor, serotransferrin (transferrin) precursor, and vitamin D binding protein precursor were identified. Western blotting confirmed the presence of 14-3-3 σ and LF. We found that upregulation of 14-3-3 σ was associated with apoptosis, and downregulation of LF was found to suppress tumorigenesis.

AIM To determine the possibility that diabetes mellitus promotes pancreatic ductal adenocarcinoma via glyceraldehyde (GA)-derived advanced glycation-end products (GA-AGEs). METHODS PANC-1, a human pancreatic cancer cell line, was treated with 1-4 mmol/L GA for 24 h. The cell viability and intracellular GA-AGEs were measured by WST-8 assay and slot blotting. Moreover, immunostaining of PANC-1 cells with an anti-GA-AGE antibody was performed. Western blotting (WB) was used to analyze the molecular weight of GA-AGEs. Heat shock proteins 90α, 90β, 70, 27 and cleaved caspase-3 were analyzed by WB. In addition, PANC-1 cells were treated with GA-AGEs-bovine serum albumin (GA-AGEs-BSA), as a model of extracellular GA-AGEs, and proliferation of PANC-1 cells was measured. RESULTS In PANC-1 cells, GA induced the production of GA-AGEs and cell death in a dose-dependent manner. PANC-1 cell viability was approximately 40% with a 2 mmol/L GA treatment and decreased to almost 0% with a 4 mmol/L GA treatment (each significant difference was P < 0.01). Cells treated with 2 and 4 mmol/L GA produced 6.4 and 21.2 μg/mg protein of GA-AGEs, respectively (P < 0.05 and P < 0.01). The dose-dependent production of some high-molecular-weight (HMW) complexes of HSP90β, HSP70, and HSP27 was observed following administration of GA. We considered HMW complexes to be dimers and trimers with GA-AGEs-mediated aggregation. Cleaved caspase-3 could not be detected with WB. Furthermore, 10 and 20 μg/mL GA-AGEs-BSA was 27% and 34% greater than that of control cells, respectively (P < 0.05 and P < 0.01). CONCLUSION Although intracellular GA-AGEs induce pancreatic cancer cell death, their secretion and release may promote the proliferation of other pancreatic cancer cells.

Receptor-associated late transducer (RALT) acts as a negative feedback inhibitor of ErbB receptor signaling via physical interaction with ErbB. Although RALT contains a 14-3-3 binding motif (247-RSHSGP-252), little is known about the molecular basis and significance of binding to 14-3-3. Here, we report that 14-3-3 interacts with RALT in H9c2 and COS-7 cells in a Ser-250 phosphorylation-dependent manner. An in vitro kinase assay showed that RALT is a substrate for checkpoint kinase 1 (Chk1). Interaction between ectopically expressed RALT and endogenous 14-3-3 was partially suppressed by pretreatment with the Chk1 inhibitor, UCN-01. In addition, expression of constitutively active Chk1 (Chk11-365 ) resulted in increased phosphorylation of the RALT 14-3-3 binding motif and enhanced the interaction between RALT and 14-3-3θ. Furthermore, fluorescence microscopy revealed that rapid trafficking of RALT to endosome-like vesicle structures was decelerated by coexpression of Chk11-365 , whereas this coexpression had no significant impact on trafficking of the RALT S250A mutant. Finally, a cycloheximide chase assay indicated that coexpression of Chk11-365 decelerated the degradation of ectopically expressed RALT, but not that of the S250A mutant. Collectively, these results suggest that Chk1 plays a role in regulating RALT protein stability by facilitating the interaction between 14-3-3 and RALT.

Pancreatic cancer is obstinate and resistant to gemcitabine, a standard chemotherapeutic agent for the disease. We previously showed a therapeutic effect of glycogen synthase kinase-3β (GSK3β) inhibition against gastrointestinal cancer and glioblastoma. Here, we investigated the effect of GSK3β inhibition on pancreatic cancer cell sensitivity to gemcitabine and the underlying molecular mechanism.Expression, phosphorylation, and activity of GSK3β in pancreatic cancer cells (PANC-1) were examined by Western immunoblotting and in vitro kinase assay. The combined effect of gemcitabine and a GSK3β inhibitor (AR-A014418) against PANC-1 cells was examined by isobologram and PANC-1 xenografts in mice. Changes in gene expression in PANC-1 cells following GSK3β inhibition were studied by cDNA microarray and reverse transcription (RT)-PCR.PANC-1 cells showed increased GSK3β expression, phosphorylation at tyrosine 216 (active form), and activity compared with non-neoplastic HEK293 cells. Administration of AR-A014418 at pharmacological doses attenuated proliferation of PANC-1 cells and xenografts, and significantly sensitized them to gemcitabine. Isobologram analysis determined that the combined effect was synergistic. DNA microarray analysis detected GSK3β inhibition-associated changes in gene expression in gemcitabine-treated PANC-1 cells. Among these changes, RT-PCR and Western blotting showed that expression of tumor protein 53-induced nuclear protein 1, a gene regulating cell death and DNA repair, was increased by gemcitabine treatment and substantially decreased by GSK3β inhibition.The results indicate that GSK3β inhibition sensitizes pancreatic cancer cells to gemcitabine with altered expression of genes involved in DNA repair. This study provides insight into the molecular mechanism of gemcitabine resistance and thus a new strategy for pancreatic cancer chemotherapy.

Two monoterpene glycosides, conjugated with gallic acid [globulusin A (1) and B (2)], together with four known compounds, cypellocarpin A (3), eucaglobulin (4), cuniloside (5) and (1S, 2S, 4R)-trans-2-hydroxy-1,8-cineole beta-d-glucopyranoside (6), were isolated from hot-water extracts of the leaves of Eucalyptus globulus. The structures of compounds 1 and 2 were determined by 1D, 2D NMR and MS spectroscopic analyses. The absolute stereochemistry of 1 was determined by correlating the spectroscopic data with those of synthetic compound 6 with a known configuration. Globulusin A (1) and B (2), cypellocarpin A (3) and eucaglobulin (4), scavenged DPPH free radicals and globulusin A (1) showed a higher antioxidant activity than the other tested compounds, with an IC50 of 3.8microM. Globulusin A (1) and eucaglobulin (4) concentration-dependently suppressed inflammatory cytokine production, tumor-necrosis factor-alpha and interleukin-1beta in cultured human myeloma THP-1 cells co-stimulated with phorbol myristate acetate. These compounds also inhibited melanogenesis in cultured murine melanoma B16F1 cells, without any significant cytotoxicity. These results suggested that globulusin A (1) and eucaglobulin (4), which were isolated as antioxidants from E. globulus, also had anti-inflammatory as well as anti-melanogenesis activity.

The new conjugated ketonic fatty acid, porrigenic acid (1), was isolated as a cytotoxic constituent of Pleurocybella porrigens. The structure of 1 was elucidated using spectroscopic methods including 1D and 2D NMR and MS. The absolute stereochemistry of 1 was determined by application of the exciton chirality method. Compound 1 exhibited cytotoxic activity against myeloma THP-1 cells, but did not show any significant toxicity against B16F1 melanoma. This is the first report of the isolation and structural elucidation of the new cytotoxic constituent porrigenic acid (1) from the edible mushroom P. porrigens.