RESUMEN
BACKGROUND: Proteomics-based approaches for biomarker discovery are promising strategies used in cancer research. We present state-of-art label-free quantitative proteomics method to assess proteome of renal cell carcinoma (RCC) compared with noncancer renal tissues. METHODS: Fresh frozen tissue samples from eight primary RCC lesions and autologous adjacent normal renal tissues were obtained from surgically resected tumour-bearing kidneys. Proteins were extracted by complete solubilisation of tissues using filter-aided sample preparation (FASP) method. Trypsin digested proteins were analysed using quantitative label-free proteomics approach followed by data interpretation and pathways analysis. RESULTS: A total of 1761 proteins were identified and quantified with high confidence (MASCOT ion score threshold of 35 and P-value <0.05). Of these, 596 proteins were identified as differentially expressed between cancer and noncancer tissues. Two upregulated proteins in tumour samples (adipose differentiation-related protein and Coronin 1A) were further validated by immunohistochemistry. Pathway analysis using IPA, KOBAS 2.0, DAVID functional annotation and FLink tools showed enrichment of many cancer-related biological processes and pathways such as oxidative phosphorylation, glycolysis and amino acid synthetic pathways. CONCLUSIONS: Our study identified a number of differentially expressed proteins and pathways using label-free proteomics approach in RCC compared with normal tissue samples. Two proteins validated in this study are the focus of on-going research in a large cohort of patients.
Asunto(s)
Biomarcadores de Tumor/análisis , Carcinoma de Células Renales/química , Neoplasias Renales/química , Proteómica/métodos , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/biosíntesis , Biomarcadores de Tumor/orina , Carcinoma de Células Renales/metabolismo , Carcinoma de Células Renales/patología , Carcinoma de Células Renales/orina , Femenino , Humanos , Neoplasias Renales/metabolismo , Neoplasias Renales/patología , Neoplasias Renales/orina , Masculino , Espectrometría de Masas , Persona de Mediana Edad , Proteínas de Neoplasias/análisis , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/orina , Transducción de SeñalRESUMEN
Bipolar affective disorder is a severe and debilitating psychiatric condition characterized by the alternating mood states of mania and depression. Both the molecular pathophysiology of the disorder and the mechanism of action of the mainstays of its treatment remain largely unknown. Here, (1)H NMR spectroscopy-based metabonomic analysis was performed to identify molecular changes in post-mortem brain tissue (dorsolateral prefrontal cortex) of patients with a history of bipolar disorder. The observed changes were then compared to metabolic alterations identified in rat brain following chronic oral treatment with either lithium or valproate. This is the first study to use (1)H NMR spectroscopy to study post-mortem bipolar human brain tissue, and it is the first to compare changes in disease brain with changes induced in rat brain following mood stabilizer treatment. Several metabolites were found to be concordantly altered in both the animal and human tissues. Glutamate levels were increased in post-mortem bipolar brain, while the glutamate/glutamine ratio was decreased following valproate treatment, and gamma-aminobutyric acid levels were increased after lithium treatment, suggesting that the balance of excitatory/inhibitory neurotransmission is central to the disorder. Both creatine and myo-inositol were increased in the post-mortem brain but depleted with the medications. Lastly, the level of N-acetyl aspartate, a clinically important metabolic marker of neuronal viability, was found to be unchanged following chronic mood stabilizer treatment. These findings promise to provide new insight into the pathophysiology of bipolar disorder and may be used to direct research into novel therapeutic strategies.
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Trastorno Bipolar/metabolismo , Corteza Prefrontal/metabolismo , Adulto , Análisis de Varianza , Animales , Antimaníacos/uso terapéutico , Ácido Aspártico/análogos & derivados , Ácido Aspártico/efectos de los fármacos , Ácido Aspártico/metabolismo , Trastorno Bipolar/tratamiento farmacológico , Trastorno Bipolar/patología , Estudios de Casos y Controles , Creatina/efectos de los fármacos , Creatina/metabolismo , Modelos Animales de Enfermedad , Femenino , Ácido Glutámico/efectos de los fármacos , Ácido Glutámico/metabolismo , Glutamina/efectos de los fármacos , Glutamina/metabolismo , Humanos , Inositol/metabolismo , Espectroscopía de Resonancia Magnética , Masculino , Análisis por Apareamiento , Metabolómica , Persona de Mediana Edad , Corteza Prefrontal/efectos de los fármacos , Corteza Prefrontal/patología , Ratas , Ratas Endogámicas WKY , Valores de Referencia , Ácido gamma-Aminobutírico/efectos de los fármacos , Ácido gamma-Aminobutírico/metabolismoRESUMEN
Although some insights into the etiology of schizophrenia have been gained, an understanding of the illness at the molecular level remains elusive. Recent advances in proteomic profiling offer great promise for the discovery of markers underlying pathophysiology of diseases. In the present study, we employed two high-throughput proteomic techniques together with traditional methods to investigate cerebrospinal fluid (CSF), brain and peripheral tissues (liver, red blood cells and serum) of schizophrenia patients in an attempt to identify peripheral/surrogate disease markers. The cohorts used to investigate each tissue were largely independent, although some CSF and serum samples were collected from the same patient. To address the major confounding factor of antipsychotic drug treatment, we also included a large cohort of first-onset drug-naive patients. Apolipoprotein A1 (apoA1) showed a significant decrease in expression in schizophrenia patients compared to controls in all five tissues examined. Specifically, using SELDI-TOF mass spectrometry, apoA1 was found decreased in CSF from schizophrenia patients (-35%, P=0.00001) and, using 2D-DIGE, apoA1 was also found downregulated in liver (-30%, P=0.02) and RBCs (-60%, P=0.003). Furthermore, we found a significant reduction of apoA1 in sera of first-onset drug-naive schizophrenia patients using enzyme-linked immunosorbent assay (-18%, P=0.00008) and in two investigations of post-mortem brain tissue using western blot analysis (-35%, P=0.05; -51%, P=0.05). These results show that apoA1 is consistently downregulated in the central nervous system as well as peripheral tissues of schizophrenia patients and may be linked to the underlying disease mechanism.
Asunto(s)
Apolipoproteína A-I/metabolismo , Encéfalo/metabolismo , Regulación hacia Abajo/fisiología , Proteoma/metabolismo , Esquizofrenia , Adulto , Electroforesis en Gel Bidimensional , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Masculino , Espectrometría de Masas , Análisis por Matrices de Proteínas/métodos , Proteómica/métodos , Esquizofrenia/sangre , Esquizofrenia/líquido cefalorraquídeo , Esquizofrenia/patología , Adulto JovenRESUMEN
The etiology and pathophysiology of schizophrenia remain unknown. A parallel transcriptomics, proteomics and metabolomics approach was employed on human brain tissue to explore the molecular disease signatures. Almost half the altered proteins identified by proteomics were associated with mitochondrial function and oxidative stress responses. This was mirrored by transcriptional and metabolite perturbations. Cluster analysis of transcriptional alterations showed that genes related to energy metabolism and oxidative stress differentiated almost 90% of schizophrenia patients from controls, while confounding drug effects could be ruled out. We propose that oxidative stress and the ensuing cellular adaptations are linked to the schizophrenia disease process and hope that this new disease concept may advance the approach to treatment, diagnosis and disease prevention of schizophrenia and related syndromes.