Global Protein Profiling in Processed Immunohistochemistry Tissue Sections.

Tissue sections, which are widely used in research and diagnostic laboratories and have already been examined by immunohistochemistry (IHC), may subsequently provide a resource for proteomic studies, even though only small amount of protein is available. Therefore, we established a workflow for tandem mass spectrometry-based protein profiling of IHC specimens and characterized defined brain area sections. We investigated the CA1 region of the hippocampus dissected from brain slices of adult C57BL/6J mice. The workflow contains detailed information on sample preparation from brain slices, including removal of antibodies and cover matrices, dissection of region(s) of interest, protein extraction and digestion, mass spectrometry measurement, and data analysis. The Gene Ontology (GO) knowledge base was used for further annotation. Literature searches and Gene Ontology annotation of the detected proteins verify the applicability of this method for global protein profiling using formalin-fixed and embedded material and previously used IHC slides.

Identification of the Regulatory Targets of miR-3687 and miR-4417 in Prostate Cancer Cells Using a Proteomics Approach.

MicroRNAs (miRNA) are ubiquitous non-coding RNAs that have a prominent role in cellular regulation. The expression of many miRNAs is often found deregulated in prostate cancer (PCa) and castration-resistant prostate cancer (CRPC). Although their expression can be associated with PCa and CRPC, their functions and regulatory activity in cancer development are poorly understood. In this study, we used different proteomics tools to analyze the activity of hsa-miR-3687-3p (miR-3687) and hsa-miR-4417-3p (miR-4417), two miRNAs upregulated in CRPC. PCa and CRPC cell lines were transfected with miR-3687 or miR-4417 to overexpress the miRNAs. Cell lysates were analyzed using 2D gel electrophoresis and proteins were subsequently identified using mass spectrometry (Maldi-MS/MS). A whole cell lysate, without 2D-gel separation, was analyzed by ESI-MS/MS. The expression of deregulated proteins found across both methods was further investigated using Western blotting. Gene ontology and cellular process network analysis determined that miR-3687 and miR-4417 are involved in diverse regulatory mechanisms that support the CRPC phenotype, including metabolism and inflammation. Moreover, both miRNAs are associated with extracellular vesicles, which point toward a secretory mechanism. The tumor protein D52 isoform 1 (TD52-IF1), which regulates neuroendocrine trans-differentiation, was found to be substantially deregulated in androgen-insensitive cells by both miR-3687 and miR-4417. These findings show that these miRNAs potentially support the CRPC by truncating the TD52-IF1 expression after the onset of androgen resistance.

Immunoproteasome impairment via ß5i/LMP7-deletion leads to sustained pancreatic injury from experimental pancreatitis.

Uncovering potential new targets involved in pancreatitis may permit the development of new therapies and improvement of patient's outcome. Acute pancreatitis is a primarily sterile disease characterized by a severe systemic inflammatory response associated with extensive necrosis and a mortality rate of up to 24%. Considering that one of the reported disease mechanisms comprises the endoplasmic reticulum (ER) stress response and that the immunoproteasome is a key regulator to prevent proteotoxic stress in an inflammatory context, we investigated its role in acute pancreatitis. In this study, we demonstrate that immunoproteasome deficiency by deletion of the ß5i/LMP7-subunit leads to persistent pancreatic damage. Interestingly, immunoproteasome-deficient mice unveil increased activity of pancreatic enzymes in the acute disease phase as well as higher secretion of Interleukin-6 and transcript expression of the Interleukin IL-1ß, IFN-ß cytokines and the CXCL-10 chemokine. Cell death was increased in immunoproteasome-deficient mice, which appears to be due to the increased accumulation of ubiquitin-protein conjugates and prolonged unfolded protein response. Accordingly, our findings suggest that the immunoproteasome plays a protective role in acute pancreatitis via its role in the clearance of damaged proteins and the balance of ER stress responses in pancreatic acini and in macrophages cytokine production.

Proteomic-based investigations on the mode of action of the marine anticancer compound rhizochalinin.

Rhizochalinin (Rhiz) is a novel marine natural sphingolipid-like compound, which shows promising in vitro and in vivo activity in human castration-resistant prostate cancer. In the present study, a global proteome screening approach was applied to investigate molecular targets and biological processes affected by Rhiz in castration-resistant prostate cancer. Bioinformatical analysis of the data predicted an antimigratory effect of Rhiz on cancer cells. Validation of proteins involved in the cancer-associated processes, including cell migration and invasion, revealed downregulation of specific isoforms of stathmin and LASP1, as well as upregulation of Grp75, keratin 81, and precursor IL-1ß by Rhiz. Functional analyses confirmed an antimigratory effect of Rhiz in PC-3 cells. Additionally, predicted ERK1/2 activation was confirmed by Western blotting analysis, and revealed prosurvival effects in Rhiz-treated prostate cancer cells indicating a potential mechanism of resistance. A combination of Rhiz with MEK/ERK inhibitors PD98059 (non-ATP competitive MEK1 inhibitor) and FR180204 (ATP-competitive ERK1/2 inhibitor) resulted in synergistic effects. This work provides further insights into the molecular mechanisms underlying Rhiz bioactivity. Furthermore, our research is exemplary for the ability of proteomics to predict drug targets and mode of action of natural anticancer agents.

Isoform 1 of TPD52 (PC-1) promotes neuroendocrine transdifferentiation in prostate cancer cells.

The tumour protein D52 isoform 1 (PC-1), a member of the tumour protein D52 (TPD52) protein family, is androgen-regulated and prostate-specific expressed. Previous studies confirmed that PC-1 contributes to malignant progression in prostate cancer with an important role in castration-resistant stage. In the present work, we identified its impact in mechanisms leading to neuroendocrine (NE) transdifferentiation. We established for long-term PC-1 overexpression an inducible expression system derived from the prostate carcinoma cell line LNCaP. We observed that PC-1 overexpression itself initiates characteristics of neuroendocrine cells, but the effect was much more pronounced in the presence of the cytokine interleukin-6 (IL-6). Moreover, to our knowledge, this is the first report that treatment with IL-6 leads to a significant upregulation of PC-1 in LNCaP cells. Other TPD52 isoforms were not affected. Proceeding from this result, we conclude that PC-1 overexpression enhances the IL-6-mediated differentiation of LNCaP cells into a NE-like phenotype, noticeable by morphological changes and increased expression of typical NE markers, like chromogranin A, synaptophysin or beta-3 tubulin. Immunofluorescent staining of IL-6-treated PC-1-overexpressing LNCaP cells indicates a considerable PC-1 accumulation at the end of the long-branched neuron-like cell processes, which are typically formed by NE cells. Additionally, the experimentally initiated NE transdifferentiation correlates with the androgen receptor status, which was upregulated additively. In summary, our data provide evidence for an involvement of PC-1 in NE transdifferentiation, frequently associated with castration resistance, which is a major therapeutic challenge in the treatment of advanced prostate cancer.

Immunoproteasomes control activation of innate immune signaling and microglial function.

Microglia are the resident immune cells of the central nervous system (CNS) and play a major role in the regulation of brain homeostasis. To maintain their cellular protein homeostasis, microglia express standard proteasomes and immunoproteasomes (IP), a proteasome isoform that preserves protein homeostasis also in non-immune cells under challenging conditions. The impact of IP on microglia function in innate immunity of the CNS is however not well described. Here, we establish that IP impairment leads to proteotoxic stress and triggers the unfolded and integrated stress responses in mouse and human microglia models. Using proteomic analysis, we demonstrate that IP deficiency in microglia results in profound alterations of the ubiquitin-modified proteome among which proteins involved in the regulation of stress and immune responses. In line with this, molecular analysis revealed chronic activation of NF-κB signaling in IP-deficient microglia without further stimulus. In addition, we show that IP impairment alters microglial function based on markers for phagocytosis and motility. At the molecular level IP impairment activates interferon signaling promoted by the activation of the cytosolic stress response protein kinase R. The presented data highlight the importance of IP function for the proteostatic potential as well as for precision proteolysis to control stress and immune signaling in microglia function.

Deficiency in FTSJ1 Affects Neuronal Plasticity in the Hippocampal Formation of Mice.

The role of the tRNA methyltransferase FTSJ1 in the brain is largely unknown. We analyzed whether FTSJ1-deficient mice (KO) displayed altered neuronal plasticity. We explored open field behavior (10 KO mice (aged 22-25 weeks)) and 11 age-matched control littermates (WT) and examined mean layer thickness (7 KO; 6 WT) and dendritic spines (5 KO; 5 WT) in the hippocampal area CA1 and the dentate gyrus. Furthermore, long-term potentiation (LTP) within area CA1 was investigated (5 KO; 5 WT), and mass spectrometry (MS) using CA1 tissue (2 each) was performed. Compared to controls, KO mice showed a significant reduction in the mean thickness of apical CA1 layers. Dendritic spine densities were also altered in KO mice. Stable LTP could be induced in the CA1 area of KO mice and remained stable at for at least 1 h, although at a lower level as compared to WTs, while MS data indicated differential abundance of several proteins, which play a role in neuronal plasticity. FTSJ1 has an impact on neuronal plasticity in the murine hippocampal area CA1 at the morphological and physiological levels, which, in conjunction with comparable changes in other cortical areas, might accumulate in disturbed learning and memory functions.

Gelsolin Governs the Neuroendocrine Transdifferentiation of Prostate Cancer Cells and Suppresses the Apoptotic Machinery.

BACKGROUND/AIM: Interleukin 6 (IL6) is increased in patients with progressive prostate cancer and induces its transdifferentiation to neuroendocrine prostate cancer. Neuroendocrine prostate cancer has become one of the greatest challenges in treating castration-resistant disease and is linked to poor prognosis. It is necessary to understand better the cellular events associated with IL6-mediated neuroendocrine differentiation to prevent it and identify potential new therapeutic targets. MATERIALS AND METHODS: In the present study, an IL6-inducible neuroendocrine differentiation model established specifically for this purpose was applied using LNCaP cells. Proteomics and western blot analyses were used to identify proteins involved in neuroendocrine differentiation. Subsequently, the role of gelsolin (GSN) in the neuroendocrine differentiation model was characterized (knock-down analyses, microscopic co-localization analyses, apoptosis assay) and GSN expression levels in patient material were investigated. RESULTS: This study revealed that GSN is a crucial factor in the neuroendocrine differentiation process. CONCLUSION: It was shown that siRNA-mediated knock-down of GSN can inhibit neuroendocrine differentiation, making it a valid target for preventing IL6-mediated neuroendocrine differentiation.

Nicotinamide phosphoribosyltransferase and prostaglandin H2 synthase 2 are up-regulated in human pancreatic adenocarcinoma cells after stimulation with interleukin-1.

Human pancreatic cancer is today an almost incurable disease with a 5-year survival rate of <5%. Chronic inflammation in the tumor region is often associated with cancer progression. In pancreatic tumors, the pro-inflammatory cytokine IL-1 has been found to affect the development of chemoresistance in this cancer type. In a search for new therapeutic targets we investigated the effect of this pro-inflammatory mediator on pancreatic cancer protein expression. Therefore, the human pancreatic adenocarcinoma cell line Colo357 was subjected to proteomic analysis after stimulation with IL-1 using 2D gel electrophoresis and mass spectrometry. We detected 11 spots as being differentially expressed after stimulation with IL-1 representing 11 different proteins. Among them, nicotinamide phosphoribosyltransferase (NAMPT) and prostaglandin H2 synthase 2 (PGHS-2) are crucial proteins whose expression in Colo357 is increased by IL-1. This study is the first one demonstrating an up-regulation of NAMPT in a tumor model for human pancreatic cancer. Several clinical trials using selective PGHS-2 or NAMPT inhibitors alone did not lead to an improvement in clinical outcome. Against the background of a high cardiovascular risk associated with PGHS-2-specific pharmacological inhibitors, we recommend a combinatory treatment with selective NAMPT and PGHS-2 inhibitors. This might overcome the limitations associated with PGHS-2 inhibitors since agents at low doses and with complementary mechanisms will be used. Such combined administration should positively affect the balance between risk and benefit in fighting the interplay of tumor-associated pancreatic inflammation and carcinogenesis in high-risk patients with pancreatic neoplasia.

Nicotinamide N-Methyltransferase and Its Precursor Substrate Methionine Directly and Indirectly Control Malignant Metabolism During Progression of Renal Cell Carcinoma.

BACKGROUND/AIM: Renal cell carcinoma (RCC) is one of the most common tumor diseases in adults, and new specific biomarkers are urgently needed to define diagnosis and prognosis of patients with RCC as well as monitor the outcome of therapeutic interventions. The enzyme nicotinamide N-methyltransferase (NNMT) is believed to represent such a marker molecule in RCC therapy. MATERIALS AND METHODS: NNMT expression was examined by western blotting in samples from patients with RCC and in RCC cell lines. Effects of NNMT on cell growth and metabolism were assessed using the Hoechst 33342 reagent assay and Vita-Orange cell viability assay. Incubation experiments were performed to study the influence of methionine and interleukin-6 (IL6) on expression of NNMT. RESULTS: In patient samples, NNMT was up-regulated depending on the stage of progression. Investigations in an RCC cell culture model showed that after modulation of NNMT expression, cellular metabolism, but not cell growth was affected. This regulatory function was also dependent on the presence of the NNMT precursor substrate methionine and IL6. CONCLUSION: The metabolism-regulatory activity of NNMT depends on the precursor substrate methionine and the presence of IL6. The function of methionine appears to be dependent on the stage of progression, since in individual RCC cell lines, opposing effects on metabolism were demonstrated. This, in turn, reflects the thoroughly complex situation in the clinic.

Molecular Insight Into the IRE1α-Mediated Type I Interferon Response Induced by Proteasome Impairment in Myeloid Cells of the Brain.

Proteostasis is critical for cells to maintain the balance between protein synthesis, quality control, and degradation. This is particularly important for myeloid cells of the central nervous system as their immunological function relies on proper intracellular protein turnover by the ubiquitin-proteasome system. Accordingly, disruption of proteasome activity due to, e.g., loss-of-function mutations within genes encoding proteasome subunits, results in systemic autoinflammation. On the molecular level, pharmacological inhibition of proteasome results in endoplasmic reticulum (ER) stress-activated unfolded protein response (UPR) as well as an induction of type I interferons (IFN). Nevertheless, our understanding as to whether and to which extent UPR signaling regulates type I IFN response is limited. To address this issue, we have tested the effects of proteasome dysfunction upon treatment with proteasome inhibitors in primary murine microglia and microglia-like cell line BV-2. Our data show that proteasome impairment by bortezomib is a stimulus that activates all three intracellular ER-stress transducers activation transcription factor 6, protein kinase R-like endoplasmic reticulum kinase and inositol-requiring protein 1 alpha (IRE1α), causing a full activation of the UPR. We further demonstrate that impaired proteasome activity in microglia cells triggers an induction of IFNß1 in an IRE1-dependent manner. An inhibition of the IRE1 endoribonuclease activity significantly attenuates TANK-binding kinase 1-mediated activation of type I IFN. Moreover, interfering with TANK-binding kinase 1 activity also compromised the expression of C/EBP homologous protein 10, thereby emphasizing a multilayered interplay between UPR and type IFN response pathway. Interestingly, the induced protein kinase R-like endoplasmic reticulum kinase-activation transcription factor 4-C/EBP homologous protein 10 and IRE1-X-box-binding protein 1 axes caused a significant upregulation of proinflammatory cytokine interleukin 6 expression that exacerbates STAT1/STAT3 signaling in cells with dysfunctional proteasomes. Altogether, these findings indicate that proteasome impairment disrupts ER homeostasis and triggers a complex interchange between ER-stress sensors and type I IFN signaling, thus inducing in myeloid cells a state of chronic inflammation.

Prohibitin identified by proteomic analysis of prostate biopsies distinguishes hyperplasia and cancer.

Prostate cancer (PCA) is the most common type of cancer found in men of western countries and is the leading cancer death next to lung cancer and colorectal cancer. Prostate-specific antigen (PSA) test is an established diagnostic tool for PCA detection, but confirmation of diagnosis by histopathological evaluation of prostate needle biopsies is performed. To define protein expression pattern of prostate biopsies, in the present study we investigated biopsy samples from benign prostate hyperplasia (BPH, n=11) and prostate cancer (PCA, n=12) patients by two-dimensional gel electrophoresis (2-DE) and mass spectrometry to identify potential biomarkers which might distinguish the two clinical situations. 2-DE results revealed 88 protein spots expressed differentially among hyperplasia and cancer groups with statistical significance. Interesting spots were analyzed by MALDI-TOF-MS-MS and 79 different proteins were identified. The important proteins identified included prostatic acid phosphatase precursor, a significant overexpressed protein in PCA, prohibitin, NDRG1 tumor suppressor proteins, heat shock proteins, cytoskeletal proteins, enzymes like DDAH1 and ALDH2. Prohibitin was investigated in detail at mRNA level and protein level using immunohistochemistry on prostatectomized specimens. We found that the level of mRNA for prohibitin correlates with the increased amount of protein indicating involvement of changes at transcriptional level. Furthermore, immunohistochemistry revealed no staining in BPH (n=13), moderate staining in prostate intra-epithelial neoplasia (PIN, n=5) but strong staining in PCA (n=18). Our results demonstrate that protein profiling and mRNA studies can be performed on the same prostate biopsy. Moreover, our study revealed a significant up-regulation of prohibitin in prostate cancer compared to BPH which may be a potential marker to distinguish PCA and BPH. Some of the interesting proteins identified in this approach may serve to develop new targets for PCA diagnosis and treatment.

Annexin A3 expression after stroke in the aged rat brain.

In an effort to identify new proteins involved in functional recovery after cerebral ischemia, young (3 months) and aged (18 months) male rats were subjected to middle cerebral artery (MCA) occlusion. Brains were harvested at 3- and 14-days post ischemia and proteins from the peri-infarcted and the corresponding contralateral area and total proteins were analyzed by two-dimensional polyacrylamide gel electrophoresis followed by mass spectrometry analysis. Annexin A3 (ANXA3) was identified as one upregulated protein in the post-ischemic rat brain. Using western blotting, real-time PCR and immunohistochemistry, we confirmed that at 3-14 days post-stroke, ANXA3 expression in the peri-infarct area was consistently increased over the corresponding area of control rats. Double staining revealed that ANXA3 is produced by activated microglial cells. We found that aged rats also had more newly proliferating cells expressing ANXA3 than young rats do. Occasionally, ANXA3-immunopositive cells wraped around neurons, suggesting that annexin A3 may be involved in the removal of dying neurons after stroke.

Increased expression and altered location of annexin IV in renal clear cell carcinoma: a possible role in tumour dissemination.

The proteome of renal cell carcinoma and non-neoplastic kidney tissue was analysed from 12 patients by two-dimensional polyacrylamide gel electrophoresis to search for differentially expressed proteins in the tumour. Annexin IV was identified to be up-regulated in tumour cells. These patients and further 11 were characterized by RT-PCR. We found an increased amount of annexin IV mRNA. Immunohistochemical analysis revealed an altered localization of annexin IV in tumour cells. Additionally we demonstrate that over-expressed annexin IV promotes cell migration in a carcinoma model system. From these results above it seems possible that annexin IV plays an important role in the morphological diversification and dissemination of the clear cell renal cell carcinoma.

Fully automated determination of cannabinoids in hair samples using headspace solid-phase microextraction and gas chromatography-mass spectrometry.

This paper describes a fully automated procedure using alkaline hydrolysis and headspace solid-phase microextraction (HS-SPME) followed by on-fiber derivatization and gas chromatographic-mass spectrometric (GC-MS) detection of cannabinoids in human hair samples. Ten milligrams of hair was washed with deionized water, petroleum ether, and dichloromethane. After the addition of deuterated internal standards the sample was hydrolyzed with sodium hydroxide and directly submitted to HS-SPME. After absorption of analytes for an on-fiber derivatization procedure the fiber was directly placed into the headspace of a second vial containing N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA) before GC-MS analysis. The limit of detection was 0.05 ng/mg for delta9-tetrahydrocannabinol (THC), 0.08 ng/mg for cannabidiol (CBD), and 0.14 ng/mg for cannabinol (CBN). Absolute recoveries were in the range between 0.3 and 7.5%. Linearity was proved over a range from 0.1 to 20 ng/mg with coefficients of correlation from 0.998 to 0.999. Validation of the whole procedure revealed excellent results. In comparison with conventional methods of hair analysis this automated HS-SPME-GC-MS procedure is substantially faster. It is easy to perform without use of solvents and with minimal sample quantities, but with the same degree of sensitivity and reproducibility. The applicability was demonstrated by the analysis of 25 hair samples from several forensic cases. The following concentration ranges were determined: THC 0.29-2.20 (mean 1.7) ng/mg, CBN 0.55-4.54 (mean 1.2) ng/mg, and CBD 0.53-18.36 (mean 1.3) ng/mg. 11-nor-Delta9-tetrahydrocannabinol-9-carboxylic acid could not be detected with this method.

Simple determination of 22 organophosphorous pesticides in human blood using headspace solid-phase microextraction and gas chromatography with mass spectrometric detection.

A simple and rapid procedure for the determination of 22 organophosphorous pesticides (bromophos-ethyl, bromophos-methyl, chlorfenvinphos, chlorpyriphos, demethon-S-methylsulfon, diazinon, dichlorvos, dicrotophos, dimethoate, disulfoton, edifenphos, fenitrothion, fenthion, malathion, methidathion, mevinphos, monocrotophos, omethoate, parathion-ethyl, parathion-methyl, phosphamidon, and quinalphos) in human blood using headspace (HS) solid-phase microextraction (SPME) and gas chromatography (GC)-mass spectrometry (MS) is presented. The effects of various sample additions, incubation temperatures, absorption times, desorption times, and depths of fiber insertion into the injection port of the GC are optimized to enhance the sensitivity of the procedure. The recoveries of spiked blood samples are determined between 70% and 95% compared with samples prepared in water, and absolute recoveries are in the range between 0.1% and 19.6%. For quantitation in the single ion monitoring mode, linearity is established over concentration ranges from 0.025 to 5.0 microg/g with excellent coefficients of correlation (0.991-0.998). The detection limits are in the range between 0.01 and 0.3 microg/g. The time for analysis is 44 min per sample including extraction and GC-MS analysis. HS-SPME in combination with GC-MS is an effective method for the determination of organophosphorous pesticides in human blood and shows a great potential for use in rapid on-site analytical work, which is highly demanded in clinical and forensic toxicology.

Fully automated determination of amphetamines and synthetic designer drugs in hair samples using headspace solid-phase microextraction and gas chromatography-mass spectrometry.

This study describes a fully automated procedure using alkaline hydrolysis and headspace (HS) solid-phase microextraction (SPME) followed by on-fiber derivatization and gas chromatographic (GC)-mass spectrometric (MS) detection of amphetamine, methamphetamine, methylendioxyamphetamine, methylendioxymethamphetamine, methylendioxyethylamphetamine, methylendioxyphenylbutanamine, and methylmethylendioxyphenylbutanamine in human hair samples. Ten milligrams of hair is washed with deionized water, petroleum ether, and dichloromethane. After the addition of deuterated internal standards the sample is hydrolyzed with sodium hydroxide and directly submitted to HS-SPME. After the absorption of analytes for an on-fiber derivatization procedure the fiber is directly placed into the HS of a second vial containing N-methyl-bis(trifluoroacetamide) before GC-MS analysis. The limits of detection are determined between 0.01 and 0.17 ng/mg. Absolute analyte recoveries are in the range between 0.3% and 7.5%. Linearity is proven over a range from 0.1 to 50 ng/mg with coefficients of correlation from 0.998 to 1. In comparison with conventional methods of hair analysis, this fully automated HS-SPME-GC-MS procedure is substantially faster and easier to perform without using solvents. It uses minimal sample amounts and has the same degree of sensitivity and reproducibility.

Prolonged gaseous hypothermia prevents the upregulation of phagocytosis-specific protein annexin 1 and causes low-amplitude EEG activity in the aged rat brain after cerebral ischemia.

In aged humans, stroke is a major cause of disability for which no neuroprotective measures are available. In animal studies of focal ischemia, short-term hypothermia often reduces infarct size. Nevertheless, efficient neuroprotection requires long-term, regulated lowering of whole-body temperature. Previously, we reported that post-stroke exposure to hydrogen sulfide (H(2)S) effectively lowers whole-body temperature and confers neuroprotection in aged animals. In the present study using magnetic resonance imaging, electroencephalogram recording, DNA arrays, reverse transcriptase polymerase chain reaction, western blotting and immunofluorescence, we characterized the central nervous system response to H(2)S-induced hypothermia and report, for the first time, that annexin A1, a major pro-inflammatory protein that is upregulated after stroke, was consistently downregulated in polymorphonuclear cells in the peri-lesional cortex of post-ischemic, aged rat brain after 48 hours of hypothermia induced by exposure to H(2)S. Our data suggest that long-term hypothermia may be a viable clinical approach to protecting the aged brain from cerebral injury. Our findings further suggest that, in contrast to monotherapies that have thus far uniformly failed in clinical practice, hypothermia has pleiotropic effects on brain physiology that may be necessary for effective protection of the brain after stroke.

Stage-related alterations in renal cell carcinoma--comprehensive quantitative analysis by 2D-DIGE and protein network analysis.

Renal cell carcinoma accounts for about 3% of adult malignancies and 85% of neoplasms arising from the kidney. To identify potential progression markers for kidney cancer we examined non-neoplastic and neoplastic kidney tissue from three groups of patients, which represent different tumor stages (pT1, pT2, pT3) by a fluorescence two-dimensional difference gel electrophoresis (2D-DIGE) approach combined with MALDI-ToF-MS/MS. Delta2D software package was used for gel image based quantification and statistical analysis. Thereby, a comprehensive Principal Component Analysis (PCA) could be performed and allowed a robust quality control of the experiment as well as a classification of the analyzed samples, which correlated with the predicted stages from the pathological examination. Additionally for selected candidate proteins we detected a correlation to the tumor grading as revealed by immunohistochemistry. On the 2D protein map 176 spots out of 989 were detected as at least 2-fold differentially expressed. These spots were analyzed by MALDI-ToF-MS/MS and 187 different proteins were identified. The functional clustering of the identified proteins revealed ten groups. Within these groups we found 86 enzymes, 63 proteins of unknown function, 14 transporter, 8 peptidases and 7 kinases. From the systems biology approach we could map many of these proteins in major pathways involved in remodelling of cytoskeleton, mitochondrial dysfunctions and changes in lipid metabolism. Due to complexity of the highly interconnected pathway network, further expression and functional validation of these proteins might provide new insights in kidney cancer progression to design novel diagnostic and therapeutic strategies.

EGCG downregulates IL-1RI expression and suppresses IL-1-induced tumorigenic factors in human pancreatic adenocarcinoma cells.

Human pancreatic cancer is currently one of the fifth-leading causes of cancer-related mortality with a 5-year survival rate of less than 5%. Since pancreatic carcinoma is largely refractory to conventional therapies, there is a strong medical need for the development of novel and innovative therapeutic strategies. Increasing evidence suggests an association of carcinogenesis and chronic inflammation. Because IL-1 plays a crucial role in inflammation-associated carcinogenesis, we analyzed the biological effects of IL-1 and its modulation by the chemopreventive green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) in the human pancreatic adenocarcinoma cell line Colo357. Proinflammatory IL-6 and PGHS-2 as well as proangiogenic IL-8 and VEGF were induced by IL-1, whereas the secretion of invasion-promoting MMP-2 remained unaffected. IL-1 responsiveness and constitutive MMP-2 release in Colo357 were downregulated by EGCG in a dose- and time-dependent manner. Moreover, EGCG reduced cell viability via induction of apoptosis in Colo357. Since EGCG effects on cytokine production precede reduction in cell viability, we hypothesize that these findings are not only a result of cell death but also depend on alterations in the IL-1 signaling cascade. In this context, we found for the first time an EGCG-induced downregulation of the IL-1RI expression possibly being caused by NF-κB inhibition and causative for its inhibitory action on the production of tumorigenic factors. Thus, our data might have future clinical implications with respect to the development of novel approaches as an adjuvant therapy in high-risk patients with human pancreatic carcinoma.