Simone Venz

Purification and characterization of AKR1B10 from human liver: role in carbonyl reduction of xenobiotics

Members of the aldo-keto reductase (AKR) superfamily have a broad substrate specificity in catalyzing the reduction of carbonyl group-containing xenobiotics. In the present investigation, a member of the aldose reductase subfamily, AKR1B10, was purified from human liver cytosol. This is the first time AKR1B10 has been purified in its native form. AKR1B10 showed a molecular mass of 35 kDa upon gel filtration and SDS-polyacrylamide gel electrophoresis. Kinetic parameters for the NADPH-dependent reduction of the antiemetic 5-HT3 receptor antagonist dolasetron, the antitumor drugs daunorubicin and oracin, and the carcinogen 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK) to the corresponding alcohols have been determined by HPLC. Km values ranged between 0.06 mM for dolasetron and 1.1 mM for daunorubicin. Enzymatic efficiencies calculated as kcat/Km were more than 100 mM–1 min–1 for dolasetron and 1.3, 0.43, and 0.47 mM–1 min–1 for daunorubicin, oracin, and NNK, respectively. Thus, AKR1B10 is one of the most significant reductases in the activation of dolasetron. In addition to its reducing activity, AKR1B10 catalyzed the NADP+-dependent oxidation of the secondary alcohol (S)-1-indanol to 1-indanone with high enzymatic efficiency (kcat/Km = 112 mM–1 min–1). The gene encoding AKR1B10 was cloned from a human liver cDNA library and the recombinant enzyme was purified. Kinetic studies revealed lower activity of the recombinant compared with the native form. Immunoblot studies indicated large interindividual variations in the expression of AKR1B10 in human liver. Since carbonyl reduction of xenobiotics often leads to their inactivation, AKR1B10 may play a role in the occurrence of chemoresistance of tumors toward carbonyl group-bearing cytostatic drugs.

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.

Identification of Clinically Relevant Protein Targets in Prostate Cancer with 2D-DIGE Coupled Mass Spectrometry and Systems Biology Network Platform

Prostate cancer (PCa) is the most common type of cancer found in men and among the leading causes of cancer death in the western world. In the present study, we compared the individual protein expression patterns from histologically characterized PCa and the surrounding benign tissue obtained by manual micro dissection using highly sensitive two-dimensional differential gel electrophoresis (2D-DIGE) coupled with mass spectrometry. Proteomic data revealed 118 protein spots to be differentially expressed in cancer (n = 24) compared to benign (n = 21) prostate tissue. These spots were analysed by MALDI-TOF-MS/MS and 79 different proteins were identified. Using principal component analysis we could clearly separate tumor and normal tissue and two distinct tumor groups based on the protein expression pattern. By using a systems biology approach, we could map many of these proteins both into major pathways involved in PCa progression as well as into a group of potential diagnostic and/or prognostic markers. Due to complexity of the highly interconnected shortest pathway network, the functional sub networks revealed some of the potential candidate biomarker proteins for further validation. By using a systems biology approach, our study revealed novel proteins and molecular networks with altered expression in PCa. Further functional validation of individual proteins is ongoing and might provide new insights in PCa progression potentially leading to the design of novel diagnostic and therapeutic strategies.

Altered expression of tumor protein D52 regulates apoptosis and migration of prostate cancer cells.

Tumor protein D52 (TPD52) is a protein found to be overexpressed in prostate and breast cancer due to gene amplification. However, its physiological function remains under investigation. In the present study, we investigated the response of the LNCaP human prostate carcinoma cell line to deregulation of TPD52 expression. Proteomic analysis of prostate biopsies showed TPD52 overexpression at the protein level, whereas its transcriptional upregulation was demonstrated by real‐time PCR. Transfection of LNCaP cells with a specific small hairpin RNA giving efficient knockdown of TPD52 resulted in significant cell death of the carcinoma LNCaP cells. As demonstrated by activation of caspases (caspase‐3 and ‐9), and by the loss of mitochondrial membrane potential, cell death occurs due to apoptosis. The disruption of the mitochondrial membrane potential indicates that TPD52 acts upstream of the mitochondrial apoptotic reaction. To study the effect of TPD52 expression on cell proliferation, LNCaP cells were either transfected with enhanced green fluorescence protein‐TPD52 or a specific small hairpin RNA. Enhanced green fluorescence protein‐TPD52 overexpressing cells showed an increased proliferation rate, whereas TPD52‐depleted cells showed the reverse effect. Additionally, we demonstrate that exogenous expression of TPD52 promotes cell migration via αvβ3 integrin in prostate cancer cells through activation of the protein kinase B/Akt signaling pathway. From these results, we conclude that TPD52 plays an important role in various molecular events, particularly in the morphological diversification and dissemination of prostate carcinoma cells, and may be a promising target with respect to developing new therapeutic strategies to treat prostate cancer.

Proteomic Profiling of Germ Cell Cancer Cells Treated with Aaptamine, a Marine Alkaloid with Antiproliferative Activity

Aaptamine is a marine compound isolated from the sponge Aaptos aaptos showing antiproliferative properties via an undefined mode of action. We analyzed the effects of aaptamine treatment on the proliferation and protein expression of the pluripotent human embryonal carcinoma cell line NT2. Effects on proliferation, cell cycle distribution, and induction of apoptosis were analyzed. At lower concentrations, including the IC50 of 50 μM, aaptamine treatment resulted in a G2/M phase cell cycle arrest, whereas at higher concentrations, induction of apoptosis was seen. Differentially expressed proteins were assessed by 2D-PAGE and mass spectrometry, followed by verification and analysis of protein modifications of the most significantly up- and down-regulated proteins. Aaptamine treatment at the IC50 for 48 h resulted in alteration of 10 proteins, of which five each showed up- and down-regulation. Changes in the 2D map were frequently noticed as a result of post-transcriptional modifications, e.g., of the hypusine modification of the eukaryotic initiation factor 5A (eIF5A). Observed alterations such as increased expression of CRABP2 and hypusination of eIF5A have previously been identified during differentiation of pluripotent cells. For the first time, we describe changes in protein expression caused by aaptamine, providing valuable information regarding the mode of action of this compound.

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.

Activated Microglia Modulate Astroglial Enzymes Involved in Oxidative and Inflammatory Stress and Increase the Resistance of Astrocytes to Oxidative Stress In Vitro

Neuropathological processes in the central nervous system are commonly accompanied by an activation of microglia and astrocytes. The involvement of both cell populations in the onset and progress of neurological disorders has been widely documented, implicating both beneficial and detrimental influences on the neural tissue. Nevertheless, little is known about the interplay of these glial cell populations, especially under diseased conditions. To examine the effects of activated microglia on astrocytes purified rat astroglial cell cultures were treated with medium conditioned by purified quiescent (MCM[−]) or lipopolysaccharide (LPS)‐activated rat microglia (MCM[+]) and subjected to a comparative proteome analysis based on two‐dimensional gel electrophoresis. No significant down regulation of proteins was observed. The majority of the 19 proteins identified by means of nano HPLC/ESI‐MS/MS in the 12 most prominent protein spots significantly overexpressed (≥2‐fold) in MCM[+] treated astrocytes are involved in inflammatory processes and oxidative stress response: superoxide dismutases (Sod), peroxiredoxins, glutathione S‐transferases (Gst), nucleoside diphosphate kinase B, argininosuccinate synthase (Ass), and cellular retinol‐binding protein I (Rbp1). Sod2, Rbp1, Gstp1, and Ass were also significantly increased on the mRNA level determined by quantitative RT‐PCR. The upregulation of antioxidative enzymes in astrocytes was accompanied by a higher resistance to oxidative stress induced by H2O2. These results show that activated microglia change the expression of antioxidative proteins in astrocytes and protect them against oxidative stress, which might be an effective way to increase the neuroprotective potential of astrocytes under pathological conditions associated with oxidative stress and inflammation.

Proteomics as a tool for assessment of therapeutics in transfusion medicine: evaluation of prothrombin complex concentrates

BACKGROUND:  Proteomic technologies are evolving tools to analyze complex protein patterns that to date have been rarely applied to transfusion medicine. The analysis of prothrombin complex concentrates (PCCs) was used as a model to evaluate to what extent these technologies can detect differences in blood‐derived therapeutics beyond that of standard quality control.

Activity of aaptamine and two derivatives, demethyloxyaaptamine and isoaaptamine, in cisplatin-resistant germ cell cancer

UNLABELLED We analyzed the effects of all three marine alkaloids aaptamine, demethyloxyaaptamine and isoaaptamine in NT2-R, a cisplatin-resistant subline of the human embryonal carcinoma cell line NT2. All aaptamines were found to be equally effective in both cell lines, excluding cross-resistance between aaptamines and cisplatin in vitro. At the inhibitory concentration (IC50), aaptamine exerted an antiproliferative effect, whereas demethyloxyaaptamine and isoaaptamine were strong inducers of apoptosis. We analyzed the changes in the proteome of NT2-R cells treated with these compounds. 16-22 proteins were found to be significantly altered, of which several were validated by Western blotting and two-dimensional Western blotting analysis. Changes in the proteome pattern frequently resulted from post-transcriptional protein modifications, i.e. phosphorylation or hypusination in the case of eIF5A. Although the lists of altered proteins were heterogeneous and compound-specific, gene ontology analyses identified rather similar profiles regarding the affected molecular functions. Ingenuity pathway analysis by IPA put the following factors in a central position of the hypothetical networks: myc and p53 for aaptamine; tumor necrosis factor (TNF) for demethyloxyaaptamine; and all three, myc, p53, and TNF for isoaaptamine. Our results represent an important step towards a better understanding of the molecular basis underlying the observed bioactivity of these promising marine compounds. BIOLOGICAL SIGNIFICANCE We characterized the mode of action of three aaptamines, marine natural compound with anti-tumor activity, using a functional proteomics approach and the cisplatin-resistant pluripotent human embryonal carcinoma cell line NT2-R. The manuscript is of particular scientific interest, as we could reveal the similarities and differences of the modes of action. Furthermore, we were able to identify several new targets of these promising compounds. We found hypusination of eIF5A to be a prominent feature exclusively of aaptamine treatment, as this was not observed upon treatment with demethyloxyaaptamine or isoaaptamine. Our results are a step towards unraveling the mode of action of these interesting compounds.

Nephrotic syndrome and subepithelial deposits in a mouse model of immune-mediated anti-podocyte glomerulonephritis.

Subepithelial immune complex deposition in glomerular disease causes local inflammation and proteinuria by podocyte disruption. A rat model of membranous nephropathy, the passive Heymann nephritis, suggests that Abs against specific podocyte Ags cause subepithelial deposit formation and podocyte foot process disruption. In this study, we present a mouse model in which a polyclonal sheep anti-mouse podocyte Ab caused subepithelial immune complex formation. Mice developed a nephrotic syndrome with severe edema, proteinuria, hypoalbuminemia, and elevated cholesterol and triglycerides. Development of proteinuria was biphasic: an initial protein loss was followed by a second massive increase of protein loss beginning at approximately day 10. By histology, podocytes were swollen. Electron microscopy revealed 60–80% podocyte foot process effacement and subepithelial deposits, but no disruption of the glomerular basement membrane. Nephrin and synaptopodin staining was severely disrupted, and podocyte number was reduced in anti-podocyte serum-treated mice, indicating severe podocyte damage. Immunohistochemistry detected the injected anti-podocyte Ab exclusively along the glomerular filtration barrier. Immunoelectron microscopy localized the Ab to podocyte foot processes and the glomerular basement membrane. Similarly, immunohistochemistry localized mouse IgG to the subepithelial space. The third complement component (C3) was detected in a linear staining pattern along the glomerular basement membrane and in the mesangial hinge region. However, C3-deficient mice were not protected from podocyte damage, indicating a complement-independent mechanism. Twenty proteins were identified as possible Ags to the sheep anti-podocyte serum by mass spectrometry. Together, these data establish a reproducible model of immune-mediated podocyte injury in mice with subepithelial immune complex formation.