Clarice Izumi

Proteomic analysis of low‐ to high‐grade astrocytomas reveals an alteration of the expression level of raf kinase inhibitor protein and nucleophosmin

Proteomic approaches have been useful for the identification of aberrantly expressed proteins in complex diseases such as cancer. These proteins are not only potential disease biomarkers, but also targets for therapy. The aim of this study was to identify differentially expressed proteins in diffuse astrocytoma grade II, anaplastic astrocytoma grade III and glioblastoma multiforme grade IV in human tumor samples and in non‐neoplastic brain tissue as control using 2‐DE and MS. Tumor and control brain tissue dissection was guided by histological hematoxylin/eosin tissue sections to provide more than 90% of tumor cells and astrocytes. Six proteins were detected as up‐regulated in higher grade astrocytomas and the most important finding was nucleophosmin (NPM) (p<0.05), whereas four proteins were down‐regulated, among them raf kinase inhibitor protein (RKIP) (p<0.05). We report here for the first time the alteration of NPM and RKIP expression in brain cancer. Our focus on these proteins was due to the fact that they are involved in the PI3K/AKT/mTOR and RAS/RAF/MAPK pathways, known for their contribution to the development and progression of gliomas. The proteomic data for NPM and RKIP were confirmed by Western blot, quantitative real‐time PCR and immunohistochemistry. Due to the participation of NPM and RKIP in uncontrolled proliferation and evasion of apoptosis, these proteins are likely targets for drug development.

Changes in the expression of proteins associated with aerobic glycolysis and cell migration are involved in tumorigenic ability of two glioma cell lines

BackgroundThe most frequent and malignant brain cancer is glioblastoma multiforme (GBM). In gliomas, tumor progression and poor prognosis are associated with the tumorigenic ability of the cells. U87MG cells (wild-type p53) are known to be tumorigenic in nude mice, but T98G cells (mutant p53) are not tumorigenic. We investigated the proteomic profiling of these two cell lines in order to gain new insights into the mechanisms that may be involved in tumorigenesis.ResultsWe found 24 differentially expressed proteins between T98G and U87MG cells. Gene Ontology supports the notion that over-representation of differentially expressed proteins is involved in glycolysis, cell migration and stress oxidative response. Among those associated with the glycolysis pathway, TPIS and LDHB are up-regulated in U87MG cells. Measurement of glucose consumption and lactate production suggests that glycolysis is more effective in U87MG cells. On the other hand, G6PD expression was 3-fold higher in T98G cells and this may indicate a shift to the pentose-phosphate pathway. Moreover, GRP78 expression was also three-fold higher in T98G than in U87MG cells. Under thapsigargin treatment both cell lines showed increased GRP78 expression and the effect of this agent was inversely correlated to cell migration. Quantitative RT-PCR and immunohistochemistry of GRP78 in patient samples indicated a higher level of expression of GRP78 in grade IV tumors compared to grade I and non-neoplastic tissues, respectively.ConclusionsTaken together, these results suggest an important role of proteins involved in key functions such as glycolysis and cell migration that may explain the difference in tumorigenic ability between these two glioma cell lines and that may be extrapolated to the differential aggressiveness of glioma tumors.

Linker for Activation of T-cell Family Member2 (LAT2) a Lipid Raft Adaptor Protein for AKT Signaling, Is an Early Mediator of Alkylphospholipid Anti-leukemic Activity

Lipid rafts are highly ordered membrane domains rich in cholesterol and sphingolipids that provide a scaffold for signal transduction proteins; altered raft structure has also been implicated in cancer progression. We have shown that 25 μm 10-(octyloxy) decyl-2-(trimethylammonium) ethyl phosphate (ODPC), an alkylphospholipid, targets high cholesterol domains in model membranes and induces apoptosis in leukemia cells but spares normal hematopoietic and epithelial cells under the same conditions. We performed a quantitative (SILAC) proteomic screening of ODPC targets in a lipid-raft-enriched fraction of leukemic cells to identify early events prior to the initiation of apoptosis. Six proteins, three with demonstrated palmitoylation sites, were reduced in abundance. One, the linker for activation of T-cell family member 2 (LAT2), is an adaptor protein associated with lipid rafts in its palmitoylated form and is specifically expressed in B lymphocytes and myeloid cells. Interestingly, LAT2 is not expressed in K562, a cell line more resistant to ODPC-induced apoptosis. There was an early loss of LAT2 in the lipid-raft-enriched fraction of NB4 cells within 3 h following treatment with 25 μm ODPC. Subsequent degradation of LAT2 by proteasomes was observed. Twenty-five μm ODPC inhibited AKT activation via myeloid growth factors, and LAT2 knockdown in NB4 cells by shRNA reproduced this effect. LAT2 knockdown in NB4 cells also decreased cell proliferation and increased cell sensitivity to ODPC (7.5×), perifosine (3×), and arsenic trioxide (8.5×). Taken together, these data indicate that LAT2 is an early mediator of the anti-leukemic activity of alkylphospholipids and arsenic trioxide. Thus, LAT2 may be used as a target for the design of drugs for cancer therapy.

The effects of photodynamic treatment with new methylene blue N on the Candida albicans proteome

Candida albicans is a human pathogenic fungus mainly affecting immunocompromised patients. Resistance to the commonly used fungicides can lead to poor treatment of mucosal infections which, in turn, can result in life-threatening systemic candidiasis. In this scenario, antimicrobial photodynamic treatment (PDT) has emerged as an effective alternative to treat superficial and localized fungal infections. Microbial death in PDT is a consequence of the oxidation of many cellular biomolecules, including proteins. Here, we report a combination of two-dimensional electrophoresis and tandem mass spectrometry to study the protein damage resulting from treating C. albicans with PDT with new methylene blue N and red light. Two-dimensional gels of treated cells showed an increase in acidic spots in a fluence-dependent manner. Amino acid analysis revealed a decrease in the histidine content after PDT, which is one plausible explanation for the observed acidic shift. However, some protein spots remained unchanged. Protein identification by mass spectrometry revealed that both modified and unmodified proteins could be localized to the cytoplasm, ruling out subcellular location as the only explanation for damage selectivity. Therefore, we hypothesize that protein modification by PDT is a consequence of both photosensitizer binding affinity and the degree of exposure of the photooxidizable residues on the protein surface.

Ontogenesis of prolyl endopeptidase in the chick retina

The time course of prolyl endopeptidase (PE) activity, measured using 7-(N-succinyl-Gly-Pro)-4-methyl-coumarinamide as substrate, was determined in the developing chick retina, and in monolayer and aggregate culture of embryonic retinal cells. PE activity/retina increased 12.5-fold between embryonic days 7 and 12 and remained constant from the 12th embryonic day until the 3rd post-hatched day. PE activity/retina decreased 2.3-fold from the 3rd to the 9th post-hatched day. The levels of PE specific activity in aggregates and in retina were similar, whereas they were 44-81% higher in monolayer than in aggregate cultures between 3 and 13 days in culture. The data suggest that the development of PE activity and of plexiform layers occurs in parallel during chick retina ontogenesis, and that the chick retina can be an adequate in vivo and in vitro model to study PE development.

Quantitative proteomic analysis and functional studies reveal that nucleophosmin is involved in cell death in glioblastoma cell line transfected with siRNA

Previously, we reported that nucleophosmin (NPM) was increased in glioblastoma multiforme (GBM). NPM is a phosphoprotein related to apoptosis, ribosome biogenesis, mitosis, and DNA repair, but details about its function remain unclear. We treated U87MG and A172 cells with small interference RNA (siRNA) and obtained a reduction of 80% in NPM1 expression. Knockdown at the protein level was evident after the 4th day and was maintained until the 7th day of transfection that was investigated by quantitative proteomic analysis using isobaric tags. The comparison of proteomic analysis of NPM1‐siRNA against controls allowed the identification of 14 proteins, two proteins showed increase and 12 presented a reduction of expression levels. Gene ontology assigned most of the hypoexpressed proteins to apoptosis regulation, including GRP78. NPM1 silencing did not impair cell proliferation until the 7th day after transfection, but sensitized U87MG cells to temozolomide (TMZ), culminating with an increase in cell death and provoking at a later period a reduction of colony formation. In a large data set of GBM patients, both GRP78 and NPM1 genes were upregulated and presented a tendency to shorter overall survival time. In conclusion, NPM proved to participate in the apoptotic process, sensitizing TMZ‐treated U87MG and A172 cells to cell death, and in association with upregulation of GRP78 may be helpful as a predictive factor of poor prognosis in GBM patients.

Preparation and scaling up of a low phenylalanine enzymatic hydrolysate of bovine whey proteins

We describe the preparation of pancreatic enzymes hydrolysate of milk whey proteins containing low levels of aromatic amino acids. Pancreatin and trypsin/chymotrypsin (6.3% w/w protein) when used to hydrolyze whey proteins for 27 h at 37±2 oC, released 74% of the Phe, 100% of the Tyr and 100% of the Trp as free amino acids. Most of the free aromatic amino acids present in 2 kg hydrolysate were separated from the remaining peptides and other amino acids by gel filtration on a 15 liter Sephadex G-25 column eluted with 5% acetic acid at 60 liters h-1 at 25oC. The product, recovered in 37% yield, contained 0.70 mmol Phe, 0.41 mmol Tyr, and <0.01 mmol Trp/100 mmol recovered amino acids. The hydrolysate had a general amino acid composition similar to the whey proteins from which it was prepared and could be used as a nitrogen source for patients with phenylketonuria or tyrosinemia after the addition of appropriate aromatic amino acids.

Biomarker verification using selected reaction monitoring and shotgun proteomics.

Shotgun proteomics (liquid chromatography-electrospray ionization-mass spectrometry, LC-ESI-MS/MS) has dominated the strategies for global protein expression in subcells, cells, tissues, and whole organisms with several types of approaches, as isobaric tags for relative and absolute quantification (iTRAQ), isotope-coded affinity tags (ICAT), or stable isotope labeling using amino acids in cell culture (SILAC) and non-labeling (label free) methods. Shotgun proteomics practically replaced the classical 2D gel electrophoresis. Selected reaction monitoring (SRM), also denominated multiple reaction monitoring (MRM), is a targeted quantitative technology that uses a complex mixture of tryptic peptides that can be selectively detected by liquid chromatography coupled to electrospray triple-quadrupole mass spectrometer; this system can select precursor ions in combination with their correspondent product ions during collision-induced dissociation to produce specific detection related to a particular protein. Here we describe protocols that are efficient to produce a complete enzymatic trypsin digestion from complex biological matrices and concomitant material to be used for LC-SRM-MS and LC-ESI-MS/MS (labeled or label free).

Iron supplementation prevents the development of iron deficiency in rats with omeprazole-induced hypochlorhydria

Abstract Gastric acidity is an important luminal factor for non-heme iron absorption. The effect of iron supplementation (1 mg Fe/kg body weight) was studied in rats submitted to hypochlorhydria by daily oral administration of omeprazole (40 μmol/kg). Forty (40) rats received omeprazole (experimental group) and 20 rats received vehicle (control group) for 4 weeks. At the end of this period, 10 animals from each group were sacrificed. The remaining rats in the control group continued receiving vehicle alone for 2 additional weeks. The experimental group was divided into three subgroups of 10 rats each. One subgroup received omeprazole alone, and the other subgroups received omeprazole plus iron supplementation with iron sulphate (Fe +2 ) or iron-peptide complex (Fe +3 ) for 2 additional weeks. After 4 weeks of treatment, the group that received omeprazole presented an increase of serum transferrin and a decrease of hepatic iron levels. However, only after 6 weeks did a decrease of haemoglobin occur in this subgroup. Supplementation started during the 5th week prevented the decrease of haemoglobin, improved the transferrin levels but did not cause hepatic iron to return to control levels. These results suggest that iron deficiency due to hypochlorhydria could be prevented by iron supplementation and that the two iron sources were equally efficient in this respect.

Omega-3 fatty acids induce biochemical changes in the small bowel of rats before and after resection

No presente estudo, foi avaliado o efeito da dieta com oleo de peixe, uma fonte de acidos graxos altamente insaturados, sobre a mucosa intestinal antes e apos reseccao do intestino delgado proximal em ratos. Quarenta ratos Wistar foram alimentados com dieta definida contendo oleo de peixe (grupo experimental) ou oleo de milho (grupo controle). Apos duas semanas, os animais foram submetidos a resseccao de 50% do intestino delgado proximal. Os niveis das dissacaridases, fosfatase alcalina, animopeptidade, proteina, DNA e TBARS foram avaliados, imediatamente antes e 6 e 12 dias apos a cirurgia. As atividades das dissacaridases foram reduzidas na mucosa ileal no grupo de ratos alimentados com oleo de peixe, comparado com grupo controle antes e 12 dias apos a cirurgia. Os outros parâmetros nao mostraram diferencas significativas. Este resultado indica que o oleo de peixe (acidos graxos omega-3) altera parâmetros funcionais na mucosa intestinal normal e apos reseccao, comparado ao acido graxo omega-6, sugerindo cautela no uso de acidos graxos omega-3 como adjuvante no processo adaptativo apos reseccao.