A Case of Dedifferentiated Melanoma With Lymph Node Metastasis Where Molecular Biological Tests Were Useful for Diagnosis.

Malignant melanoma is known to have an altered phenotype and loss of differentiation markers for melanoma due to metastasis. Here, we report a case in which the expression of the immunohistochemical markers for melanoma was changed due to lymph node metastasis of primary cutaneous malignant melanoma. The patient, a male in his 60s, was diagnosed with malignant melanoma after undergoing excision of a skin mass. The additional excision specimen showed a small number of tumor cell clusters infiltrating the dermis. The biopsied lymph node showed completely different histological findings from those of the skin lesion and consisted of spindle-shaped tumor cells. An immunohistochemical study revealed no significant positive reactions in the lymph node tissue indicative of melanoma. The additional genetic study revealed BRAF V600e mutations in both the primary tumor and a lymph node. Together with the histological findings, the diagnosis was of metastasis of dedifferentiated melanoma to a lymph node. In summary, there is a risk of underestimation or misdiagnosis of melanoma as undifferentiated sarcoma or other tumors when melanoma metastasizes to lymph nodes and findings show a dedifferentiated or undifferentiated tumor. Therefore, as in this case, it is necessary to add a genetic study in order to make a comprehensive judgment.

Interleukin-32 regulates downstream molecules and promotes the invasion of pancreatic cancer cells.

Pancreatic cancer is a malignant neoplasm with high invasiveness and poor prognosis. In a previous study, a highly invasive pancreatic cancer cell line was established and found to feature enhanced interleukin-32 (IL-32) expression. However, whether IL-32 promotes the invasiveness by enhancing or suppressing the expression of IL-32 through regulating downstream molecules was unclear. To investigate the effect of IL-32, cells were established with high levels of expression or downregulated IL-32; their invasive ability was measured using a real-time measurement system and the expression of some candidate downstream molecules involved in invasion was evaluated in the two cell types. The morphological changes in both cell types and the localization of IL-32 expression in pancreatic cancer tissues were studied using immunohistochemistry. Among the several splice variants of IL-32, cells transfected with the ε isoform had increased invasiveness, whereas the IL-32-suppressed cells had reduced invasiveness. Several downstream molecules, whose expression was changed in the two cell types, were monitored. Notably, changes of E-cadherin, CLDN1, CD44, CTGF and Wnt were documented. The morphologies of the two cell types differed from the original cell line. Immunohistochemically, the expression of IL-32 was observed only in tumor cells and not in normal pancreatic cells. In conclusion, IL-32 was found to promote the invasiveness of pancreatic cancer cells by regulating downstream molecules.

Establishment of highly invasive pancreatic cancer cell lines and the expression of IL-32.

Compared to tumors of other organs, pancreatic cancer is highly aggressive; with one of its biological features being that, despite a prominent fibrotic stroma, there is remarkable infiltration of tumor cells. This characteristic is considered to be the main reason for the poor prognosis of patients with pancreatic cancer. Therefore, in order to elucidate the factors that contribute to this high invasiveness, a selective invasion method was used to establish four highly invasive subclones from six human pancreatic cancer cell lines. The results demonstrated that two cell lines did not exhibit enhanced invasiveness. Microarray analysis revealed that, in the highly invasive cell lines, several genes were expressed at high levels, compared with the original cell lines. These highly expressed genes were recognized only in highly invasive cells. Among them, IL-32 was most strongly upregulated in the highly invasive cells, compared with cells with a low invasive potential, as well as the original cells. RT-qPCR and western blot analysis confirmed the high levels of expression of IL-32 in highly invasive cells at the RNA and protein levels. In addition, immunohistochemical analysis of resected surgical materials revealed that the tumor cells expressed IL-32 and, in particular, many IL-32 positive cells were seen at the invasive front of the tumor tissue. IL-32 is a cytokine that is widely involved in the development of cancer and has recently received considerable attention. This cytokine has multiple splice variants and shows a wide variety of behaviors, depending on the tumor type and primary organ. Although some hypotheses have been proposed to explain the activity of IL-32, a unified view has not been agreed. In the present study, through the establishment of highly invasive cells from pancreatic cancer and a comprehensive gene analysis, it is suggested that IL-32 may serve an important role as a molecule involved in the invasiveness of this neoplasm.

Relevance of the hippocampal endoplasmic reticulum stress response in a mouse model of chronic kidney disease.

It has been shown that the incidence of cognitive impairment increases with the severity of chronic kidney disease (CKD). A previous study has demonstrated that hippocampal oxidative stress contributes to cognitive dysfunction in CKD model mice. Endoplasmic reticulum (ER) stress is thought to contribute significantly to neuronal dysfunction, but its role in the hippocampal dysfunction seen in CKD still remains unclear. The present study examined whether the ER stress response as well as oxidative stress was activated in the hippocampus of CKD model mice. Western blotting revealed that the expression level of 4-hydroxy-2-nonenal (HNE)-protein adducts, a marker of oxidative stress, was increased in the hippocampus 8 weeks after 5/6 nephrectomy. In these mice, the expression level of glucose-regulated protein 78 (GRP78), a typical ER stress marker, also showed a pronounced increase in the hippocampus. Correlation analyses showed that the levels of these two marker proteins in the hippocampus are positively correlated with the serum concentrations of BUN and creatinine. These results suggest that ER stress as well as oxidative stress are induced in the hippocampus of CKD mice and that the levels of these stress markers in the hippocampus are correlated with the renal impairment caused by CKD.

Endoglin (CD105) and SMAD4 regulate spheroid formation and the suppression of the invasive ability of human pancreatic cancer cells.

In this study, we investigated the ability of pancreatic cancer cell lines to form spheroids with the aim of identifying factors involved in cell invasiveness, a property that leads to a poor prognosis in pancreatic cancer. For this purpose, 8 cell lines derived from human pancreatic cancer tissues were cultured in non-adherent culture conditions to form spheroids, as well as normal monolayers. The morphology of the cells was observed and spheroid diameters measured. mRNA expression was compared between cells cultured under both culture conditions. The gene knockdown of endoglin (ENG) and SMAD4, components of the transforming growth factor-ß (TGF-ß) signaling system, using siRNAs was conducted in spheroids in order to identify affected protein signaling factors, determine the morphological changes occurring over time and to measure the invasive capacity of the cells constituting spheroids. The cell lines exhibited differences in their spheroid-forming abilities. The expression of SMAD4 and ENG concomitantly increased in the cells that formed spheroids. SMAD4 was transported into the nucleus when spheroids were formed. The expression of ENG was decreased in the cells in which SMAD4 was knocked down; by contrast, the expression of BMP and activin membrane-bound inhibitor (BAMBI) and noggin (NOG), further components of the TGF-ß signaling system, increased. In the cells in which ENG was knocked down, the decreased mRNA expression of TGF-ß receptor type 2 (TGFBR2) and SMAD9 was observed, as well as a change in the expression of pSMAD1/5/9, and a tendency of spheroids to decrease in size. Spheroids cultured on Matrigel exhibited a tendency towards a reduction in size over time, as well as a tendency to invade into the Matrigel. In particular, the cells in which ENG was knocked down exhibited spheroids which were reduced in size, and also exhibited an increase in invasiveness, and a decrease in adhesiveness. Thus, our data indicate that in pancreatic cancer cells, the expression of ENG may be controlled by a pathway mediated by SMAD4. In addition, ENG was found to be related to the spheroid-forming ability of cells and to be involved in the invasive capacity of pancreatic cancer cells.

Insight into the molecular mechanism of heme oxygenase-1 induction by docosahexaenoic acid in U937 cells.

Heme oxygenase-1 (HO-1) has anti-inflammatory effects on myeloid cells in response to various stimuli. To date, little is known about whether fatty acids can affect HO-1 induction. Here, we report the induction of HO-1 by docosahexaenoic acid (DHA) and the associated molecular mechanisms in human myelomonocytic lymphoma U937 cells. When U937 cells were treated with DHA, eicosapentaenoic acid, palmitic acid or oleic acid, DHA was the most effective inducer of HO-1. The activation of AKT and glycogen synthase kinase-3ß did not significantly change after DHA treatment. However, DHA increased the activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), but not of other mitogen-activated protein kinases such as p38 and JNK. The increase in HO-1 expression was significantly inhibited by U0126, an ERK1/2 inhibitor. Nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf-2) and its binding to the HO-1 promoter significantly increased upon DHA treatment. An increase in intracellular reactive oxygen species was detected by dichlorofluorescein diacetate, but not by hydroethidium or 2-[6-(4-hydroxy)phenoxy-3H-xanthen-3-on-9-yl] benzoic acid after DHA treatment. Pretreatment with NAC dramatically inhibited the ERK1/2 activation, binding of Nrf-2 to antioxidant response elements (AREs) located in the HO-1 promoter and the induction of HO-1 by DHA. In conclusion, DHA increased HO-1 expression in U937 cells via activation of ERK1/2 and increased Nrf-2 binding to ARE in the HO-1 promoter. These findings will help develop better strategies for treating inflammatory disorders with DHA.

Toxicity monitoring with primary cultured hepatocytes underestimates the acetaminophen-induced inflammatory responses of the mouse liver.

In vitro gene expression profiling with isolated hepatocytes has been used to assess the hepatotoxicity of certain chemicals because of animal welfare issues. However, whether an in vitro system can completely replace the in vivo system has yet to be elucidated in detail. Using a focused microarray established in our laboratory, we examined gene expression profiles in the mouse liver and primary cultured hepatocytes after treatment with different doses of acetaminophen, a widely used analgesic that frequently causes liver injury. The acute hepatotoxicity of acetaminophen was confirmed by showing the induction of an oxidative stress marker, heme oxygenase-1, elevated levels of serum transaminase, and histopathological findings. In vivo microarray and network analysis showed that acetaminophen treatment provoked alterations in relation to the inflammatory response, and that tumor necrosis factor-α plays a central role in related pathway alterations. By contrast, pathway analyses in in vitro isolated hepatocytes did not find such prominent changes in the inflammation-related networks compared with the in vivo situation. Thus, although in vitro gene expression profiles are useful for evaluating the direct toxicity of chemicals, indirect toxicities including inflammatory responses mediated by cell-cell interactions or secondary toxicity due to pathophysiological changes in the whole body may be overlooked. Our results indicate that the in vitro hepatotoxicity prediction system using isolated hepatocytes does not fully reflect the in vivo cellular response. An in vitro system may be appropriate, therefore, for high throughput screening to detect the direct hepatotoxicity of a test compound.

Molecular mechanisms involved in the adaptive response to cadmium-induced apoptosis in human myelomonocytic lymphoma U937 cells.

We examined the molecular mechanisms involved in the adaptive response to cadmium (Cd)-induced apoptosis in human myelomonocytic lymphoma U937 cells. When U937 cells were treated with 50 µM cadmium chloride (CdCl2) for 12 h, significant apoptosis occurred. This was associated with an increase in intracellular reactive oxygen species (ROS), sustained phosphorylation of JNK, activation of caspase-3, a decrease in Mcl-1 (anti-apoptotic Bcl-2 proteins), and increases in Bim, Noxa and tBid (a pro-apoptotic protein under the Bcl-2 family). No apoptosis occurred when the cells were treated with 1 µM CdCl2 for 72 h. However, pretreatment with low-dose CdCl2 dramatically altered the sensitivity of the cells to 50 µM CdCl2 with inhibition of apoptosis. Concomitantly, there were significant decreases in the generation of intracellular ROS and the activation of JNK. Pretreatment with 1 µM CdCl2 also attenuated the decrease in Mcl-1 and the increases in Bim, Noxa and tBid induced by 50 µM CdCl2. In conclusion, pretreatment with low-dose Cd inhibited apoptosis induced by high-dose Cd. The mechanism involves inhibition of intracellular ROS generation and JNK activation, and modulating the balance between the expression of Mcl-1 and its binding partners, Bim, Noxa and tBid.

Hyperglycemia perturbs biochemical networks in human trophoblast BeWo cells.

Determining the effects of hyperglycemia on gene expression in placental trophoblast is important to gain a better understanding of how diabetes adversely affects pregnancy. In this study, we examined whether exposure to high glucose during forskolin-induced differentiation affects gene expression in differentiated trophoblasts. Human trophoblast BeWo cells were differentiated under low glucose (LG: 11 mM) or high glucose (HG: 25 mM) conditions. Gene expression was analyzed using a GeneChip system and the obtained data were analyzed using Ingenuity Pathways Analysis. In HG conditions, there were marked alterations in gene expression in differentiated BeWo cells compared with LG conditions. In particular, BeWo cells responded to HG with major changes in the expression levels of cell cycle- and metabolism-related genes. We selected the aromatase gene for further investigation of the molecular mechanisms. Mannitol or 3-O-methylglucose did not mimic the expression changes caused by HG, indicating that the effect of glucose was not due to a difference in osmotic pressure, and that glucose metabolism plays an essential role in inducing the HG effects. Cotreatment with N-acetylcysteine reduced the effect of HG on aromatase gene expression, suggesting that hyperglycemia may perturb biochemical networks because of the elevation of oxidative stress. Overall, our results will aid further understanding of the effect of diabetes on the regulation of trophoblast differentiation and function.

Carbon tetrachloride affects inflammation-related biochemical networks in the mouse liver as identified by a customized cDNA microarray system.

OBJECTIVES: We have attempted to upgrade and validate an in-house cDNA microarray system developed by our group for the evaluation of chemical toxicity. METHODS: To establish an in-house microarray, we selected genes that play pivotal roles in detoxifying exogenous substances and maintaining homeostasis in the liver. To validate the system, we examined gene expression profiles in mouse liver following treatment with different doses of carbon tetrachloride (CCl(4)). The data were also analyzed by pathway analysis tools. RESULTS: We upgraded our array system by collecting genes that are responsive to xenobiotic receptors, apoptosis-related genes, and stress-responsive genes. The acute toxicity of CCl(4) was confirmed by elevated levels of serum transaminase and histopathological findings. The microarray data showed the CCl(4) treatment induced significant changes in gene expression in the mouse liver, and the ingenuity pathways analysis revealed alterations in gene expression in inflammation-related networks. CONCLUSIONS: We have established a focused microarray system that may be useful for use in toxicogenomics studies. Using this array system, we gained insight into the mechanisms by which CCl(4) exerts its toxic effects. The results of our study also indicate that the combination of focused arrays and bioinformatics tools is helpful in the mechanistic analysis of chemical toxicity.

Effect of Wnt-1 inducible signaling pathway protein-2 (WISP-2/CCN5), a downstream protein of Wnt signaling, on adipocyte differentiation.

Wnt signaling negatively regulates adipocyte differentiation, and ectopic expression of Wnt-1 in 3T3-L1 cells induces several downstream molecules of Wnt signaling, including Wnt-1 inducible signaling pathway protein (WISP)-2. In this study, we examined the role of WISP-2 in the process of adipocyte differentiation using an in vitro cell culture system. In the differentiation of 3T3-L1 cells, WISP-2 expression was observed in growing cells and declined thereafter. In the mitotic clonal expansion phase of adipocyte differentiation, WISP-2 expression was transiently down-regulated concurrently with up-regulation of CCAAT/enhancer-binding protein delta expression. Treatment of 3T3-L1 cells in the differentiation medium with lithium, an activator of Wnt signaling, inhibited the differentiation process with concomitant induction of WISP-2. Treatment of differentiated cells with lithium induced de-differentiation as evidenced by profound reduction of peroxisome proliferator-activator receptor gamma expression and concomitant induction of WISP-2. However, de-differentiation of differentiated cells induced by tumor necrosis factor-alpha did not induce WISP-2 expression. To directly examine the effect of WISP-2 on adipocyte differentiation, 3T3-L1 cells were infected with a retrovirus carrying WISP-2. Although forced expression of WISP-2 inhibited preadipocyte proliferation, it had no effect on adipocyte differentiation. Thus, although WISP-2 is a downstream protein of Wnt signaling, the role of WISP-2 on adipocyte differentiation may be marginal, at least in this in vitro culture model.

Expression profile of liver genes in response to hepatotoxicants identified using a SAGE-based customized DNA microarray system.

Gene expression analysis using customized or focused DNA microarrays is favorable because of a reduction in the cost and time needed for the analysis. To examine the effect of chemicals on the liver, we developed an in-house cDNA microarray system, mouse Liver Stress Array ver. 1.0, containing 355 unique genes involved in drug metabolism, inflammation and liver-related proteins. These genes were selected for sensing the homeostasis of the liver and based on the information of liver transcriptome revealed by serial analysis of gene expression. By using this customized microarray, we analyzed gene expression changes in the mouse liver treated by 11 known hepatotoxicants. Gene expression measurements corresponding to the in vivo response to known hepatotoxicants revealed that profiles of chemicals with similar mechanisms clustered together. For each of the chemicals tested, several genes that were induced or repressed were common in each chemical exposure, whereas other genes were unique for the specific class compound. Ingenuity pathways analysis revealed that significant alterations in gene expression occurred in a number of biological networks by these treatments. Although the genes spotted on our array was limited to a highly focused set for toxicity classification, this work provides proof of concept that patterns of gene regulation assessed by a focused array system are useful to classify unknown chemicals.

[Advances in "omics" technologies for toxicological research].

Toxicology research can be applied to evaluate potential human health risks resulting from exposure to chemicals and other factors in the environment. The tremendous advances that have been made in high-throughput "omics" technologies (e.g., genomics, transcriptomics, proteomics and metabolomics) are providing good tools for toxicological research. Toxicogenomics is the study of changes in gene expression, protein and metabolite profiles, and combines the tools of traditional toxicology with those of genomics and bioinformatics. In particular, identification of changes in gene expression using DNA microarrays is an important method for understanding toxicological processes and obtaining an informative biomarker. Although these technologies have emerged as a powerful tool for clarifying hazard mechanisms, there are some concerns for the application of these technologies to toxicological research. This review summarizes the impact of "omics" technologies in toxicological study, followed by a brief discussion of future research.

Environmental chemical tributyltin augments adipocyte differentiation.

Scientific attention has been drawn to environmental factors that affect obesity and type II diabetes. Previously, acute organotin toxicosis was reported to induce hyperglycemia without morphological abnormalities in islet tissue, suggesting that these compounds have a direct effect on adipose tissue. Therefore, we investigated the effect of tributyltin (TBT) on adipocyte differentiation. When confluent 3T3-L1 cells were incubated with TBT for 2 days in the presence or absence of isobutyl methylxanthine, dexamethasone and insulin (MDI), the lipid accumulation in adipocytes was greatly enhanced. These morphological changes induced by TBT were accompanied by the expression of a differentiation marker for adipocytes in a dose-dependent manner. Co-treatment with the peroxisome proliferator-activated receptor (PPAR)gamma antagonist GW9662 did not inhibit the effect of TBT, suggesting that the observed effect of TBT may not be PPARgamma-dependent. Although TBT was reported to exert androgenic effects and inhibit the activity of aromatase, treatments with dihydrotestosterone or 17beta-estradiol did not influence the aP2 expression in 3T3-L1 cells, suggesting that the TBT effect does not occur via sex-steroids. These findings indicate that TBT may be one of the environmental chemicals that lead to excessive accumulation of adipose tissue, which can result in obesity.