Cathepsin D is secreted from M-BE cells: its potential role as a biomarker of lung cancer

The early diagnosis of lung cancer is an effective approach to reduce the mortality caused by malignancy. To explore serum biomarkers of lung cancer at early stage, M-BE, a SV40T-transformed human bronchial epithelial cell line with the phenotypic features of early tumorigenesis at high passage, was cultured in the conditioned media to collect its secretory proteins. The proteins secreted from different passage M-BE cells were extracted and then separated by two-dimensional electrophoresis (2-DE). MALDI-TOF/TOF mass spectrometry was adopted to identify the passage-dependent 2-DE spots. Totally, 47 proteins were identified, including 23 that were up-regulated and 24 that were down-regulated. Of these proteins, cathepsin D was a typical secretory protein that exhibited the increased abundance either in culture media or in cells during passaging. Furthermore, the proteomic conclusions were validated in the clinical samples of lung cancer patients. When sandwich ELISA was used, the concentrations of cathepsin D in plasma showed significant differences between lung squamous cell carcinomas (SCC, 104 cases) and normal donors (36 cases, p 相似文献   

Proteomic signatures for histological types of lung cancer

We performed proteomic studies on lung cancer cells to elucidate the mechanisms that determine histological phenotype. Thirty lung cancer cell lines with three different histological backgrounds (squamous cell carcinoma, small cell lung carcinoma and adenocarcinoma) were subjected to two-dimensional difference gel electrophoresis (2-D DIGE) and grouped by multivariate analyses on the basis of their protein expression profiles. 2-D DIGE achieves more accurate quantification of protein expression by using highly sensitive fluorescence dyes to label the cysteine residues of proteins prior to two-dimensional polyacrylamide gel electrophoresis. We found that hierarchical clustering analysis and principal component analysis divided the cell lines according to their original histology. Spot ranking analysis using a support vector machine algorithm and unsupervised classification methods identified 32 protein spots essential for the classification. The proteins corresponding to the spots were identified by mass spectrometry. Next, lung cancer cells isolated from tumor tissue by laser microdissection were classified on the basis of the expression pattern of these 32 protein spots. Based on the expression profile of the 32 spots, the isolated cancer cells were categorized into three histological groups: the squamous cell carcinoma group, the adenocarcinoma group, and a group of carcinomas with other histological types. In conclusion, our results demonstrate the utility of quantitative proteomic analysis for molecular diagnosis and classification of lung cancer cells.     

Identificating cathepsin D as a biomarker for differentiation and prognosis of nasopharyngeal carcinoma by laser capture microdissection and proteomic analysis

In this study, we applied laser capture microdissection and a proteomic approach to identify novel nasopharyngeal carcinoma (NPC) biomarkers. Proteins from pooled microdissected NPC and normal nasopharyngeal epithelial tissues (NNET) were separated by two-dimensional gel electrophoresis, and differential proteins were identified by mass spectrometry. Expression of the differential protein cathepsin D in the above two tissues as well as four NPC cell lines was determined by Western blotting. Next, siRNA was used to inhibit the expression of cathepsin D in highly metastatic NPC cell line 5-8F to examine whether it associates with NPC metastasis. Immunohistochemistry was also performed to detect the expression of cathepsin D in 72 cases of primary NPC, 28 cases of NNET, and 20 cases of cervical lymph node metastases, and the correlation of its expression level with clinicopathologic features and clinical outcomes were evaluated. Thirty-six differential proteins between the NPC and NNET were identified. The expression level of cathepsin D in the two types of tissues was confirmed by Western blotting and related to differentiation degree and metastatic potential of the NPC cell lines. Down-regulated cathepsin D expression by siRNA significantly decreased in vitro invasive ability of 5-8F cells. Significant cathepsin D down-regulation was observed in NPC versus NNET, whereas significant cathepsin D up-regulation was observed in lymph node metastasis versus primary NPC. In addition, cathepsin D down-regulation was significantly correlated with poor histological differentiation, whereas cathepsin D up-regulation was significantly correlated with advanced clinical stage, recurrence, and lymph node and distant metastasis. Furthermore, survival curves showed that patients with cathepsin D up-regulation had a poor prognosis. Multivariate analysis confirmed that cathepsin D expression was an independent prognostic indicator. The data suggest that cathepsin D is a potential biomarker for the differentiation and prognosis of NPC, and its dysregulation might play an important role in the pathogenesis of NPC.     

Cholesterol enhances amyloid β deposition in mouse retina by modulating the activities of Aβ-regulating enzymes in retinal pigment epithelial cells

Cancer patients frequently develop autoantibodies. To test the hypothesis that the appearance of autoantibodies precedes the clinical diagnosis of cancer, we applied an immunoproteomic approach to compare autoantibody profiles before and after appearance of malignances. Proteins from A549 cells, a lung adenocarcinoma cell line, were separated by two dimensional electrophoresis and then immunoblotted with serum samples from 8 individuals who were eventually diagnosed with lung cancer. Compared with autoantibody profiles from 3 years prior to the appearance of malignances, 21 immunoreactive spots newly appeared or presented with stronger staining intensity when clinical diagnoses were made. Among them, 10 matched spots on 2-DE gels were identified by mass spectrometry analysis as 5 proteins. With immunoprecipitation analysis, the antigenicity of protein cathepsin D was confirmed, and notably, in lung cancer sera, the occurrences of autoantibodies against the specific forms of cathepsin D differed significantly from the control groups (p<0.05). Our findings suggest that harnessing immunity may have utility for early cancer marker discovery, and that comparing autoantibodies to specific forms of cathepsin D may be a promising early marker of lung cancer.     

Proteomic signature of human cancer cells

We assessed proteomic profiles as biomarkers for monitoring cell phenotypes. Protein expression profiles were obtained by fluorescence two-dimensional difference gel electrophoresis (2-D-DIGE), in which quantitative ability is improved by labeling proteins with fluorescent dyes prior to electrophoresis. Integrated protein spot intensities were analyzed by a statistical approach. The proteomic data of two groups of cell lines: (1) adenocarcinoma (AC) cell lines derived from lung, pancreas and colon tissues and (2) lung cancer cell lines with different histological backgrounds, including AC, squamous cell carcinoma and small cell carcinoma, were assessed on the basis of prior biological information. Hierarchical clustering analysis and principal component analysis were used to divide the cell lines into subgroups on the basis of similarities between their protein expression profiles. The majority of cell lines were grouped according to their organ of origin or histological background. A machine-learning algorithm selected 32 protein spots that were responsible for the classification. The results indicate that proteomic data generated by 2-D-DIGE can provide a signature of essential cell phenotypes, suggesting that it might be possible to apply this technique to developing tumor markers that could identify the organ of origin of metastatic tumors and contribute to the differential diagnosis of lung cancer.     

Comparative proteomic analysis of hearts of adult SCNT Bama miniature pigs (Sus scrofa)

This study aims to determine the effects of SCNT on cardiac development of SCNT pigs through proteomic methods. Heart proteins from three adult SCNTs and two normal reproductive Bama miniature pigs were extracted, separated, and identified via comparative proteomic methods, including two-dimensional gel electrophoresis, mass spectrometry, and Western blot. Eleven differentially expressed spots were identified as differentially expressed proteins, of which five spots were upregulated proteins such as cardiac myosin heavy chain, cathepsin D, and heat shock protein beta-1 (HSP27). By contrast, six spots were downregulated proteins such as alpha skeletal muscle and actin. The results also demonstrated that nuclear transfer might result in abnormal expression of some important proteins in hearts from SCNT pigs, and affect the cardiac development in SCNT pigs' survival.     

Cathepsin D expression in colorectal adenocarcinomas and adenomas

The aim of this study was to investigate the role of cathepsin D in colorectal cancer. For this purpose cathepsin D expression was evaluated by means of immunohistochemistry in stromal and tumor cells of 31 colorectal carcinomas and 29 adenomas. Cytoplasmic cathepsin D expression of tumor cells was present in 90.3% of the carcinoma cases and various degrees of stromal cell cathepsin D expression were present in all cases. In the adenomas, the epithelial cells and stromal cells expressed cathepsin D in 68.96% and 96.55% of cases, respectively. The staining intensity was always weaker in the adenomas. When the stromal and tumor cell cathepsin D expression in the adenocarcinoma and adenoma cases were compared, a statistically significant difference was observed in the staining of stromal cells. Furthermore, stromal cathepsin D expression in the adenocarcinomas was related to tumor stage when the carcinomas were divided into low and high stage. Cathepsin D expression in stromal cells may be an important indicator of poor prognosis in colorectal adenocarcinomas.     

Comparative analysis of the human urinary proteome by 1D SDS-PAGE and chip-HPLC-MS/MS identification of the AACT putative urinary biomarker

Urine is one of the most attractive analyte used for clinical diagnosis. NSCLC (non-small cell lung carcinoma), which includes adenocarcinoma, squamous cell carcinoma and large-cell carcinoma, is a leading cause of cancer-related deaths. In the present study, urinary proteomes of normal individuals and NSCLC patients were compared using 1D SDS-PAGE. From the distinctly differentially expressed bands in SDS-PAGE gel, 40 proteins were identified by chip-HPLC-MS/MS, including five proteins relevant to NSCLC. One of the selected proteins, alpha-1-antichymotrypsin (AACT), was further validated in urine by western blot and in lung tissue by immunohistochemistry staining. Higher expression level of AACT in NSCLC patients was observed by western blot when compared with normal urine samples. Significantly, the NSCLC tumor tissue (18 out of 20 cases, 90%) showed a significantly higher expression level of AACT compared to adjacent non-tumor lung tissue (3 out of 20 cases, 15%). These results establish AACT as a potential biomarker for objective and non-invasive diagnosis of NSCLC in urine and the other four NSCLC-related proteins were also listed.     

Comparative proteomic approach identifies PKM2 and cofilin-1 as potential diagnostic, prognostic and therapeutic targets for pulmonary adenocarcinoma

Lung cancer is the leading cause of cancer-related death in the world. Non-small cell lung carcinomas (Non-SCLC) account for almost 80% of lung cancers, of which 40% were adenocarcinomas. For a better understanding of the molecular mechanisms behind the development and progression of lung cancer, particularly lung adenocarcinoma, we have used proteomics technology to search for candidate prognostic and therapeutic targets in pulmonary adenocarcinoma. The protein profile changes between human pulmonary adenocarcinoma tissue and paired surrounding normal tissue were analyzed using two-dimensional polyacrylamide gel electrophoresis (2-DE) based approach. Differentially expressed protein-spots were identified with ESI-Q-TOF MS/MS instruments. As a result, thirty two differentially expressed proteins (over 2-fold, p<0.05) were identified in pulmonary adenocarcinoma compared to normal tissues. Among them, two proteins (PKM2 and cofilin-1), significantly up-regulated in adenocarcinoma, were selected for detailed analysis. Immunohistochemical examination indicated that enhanced expression of PKM2 and cofilin-1 were correlated with the severity of epithelial dysplasia, as well as a relatively poor prognosis. Knockdown of PKM2 expression by RNA interference led to a significant suppression of cell growth and induction of apoptosis in pulmonary adenocarcinoma SPC-A1 cells in vitro, and tumor growth inhibition in vivo xenograft model (P<0.05). In addition, the shRNA expressing plasmid targeting cofilin-1 significantly inhibited tumor metastases and prolonged survival in LL/2 metastatic model. While additional works are needed to elucidate the biological significance and molecular mechanisms of these altered proteins identified in this study, PKM2 and cofilin-1 may serve as potential diagnostic and prognostic biomarkers, as well as therapeutic targets for pulmonary adenocarcinoma.     

A Pilot Proteogenomic Study with Data Integration Identifies MCT1 and GLUT1 as Prognostic Markers in Lung Adenocarcinoma

We performed a pilot proteogenomic study to compare lung adenocarcinoma to lung squamous cell carcinoma using quantitative proteomics (6-plex TMT) combined with a customized Affymetrix GeneChip. Using MaxQuant software, we identified 51,001 unique peptides that mapped to 7,241 unique proteins and from these identified 6,373 genes with matching protein expression for further analysis. We found a minor correlation between gene expression and protein expression; both datasets were able to independently recapitulate known differences between the adenocarcinoma and squamous cell carcinoma subtypes. We found 565 proteins and 629 genes to be differentially expressed between adenocarcinoma and squamous cell carcinoma, with 113 of these consistently differentially expressed at both the gene and protein levels. We then compared our results to published adenocarcinoma versus squamous cell carcinoma proteomic data that we also processed with MaxQuant. We selected two proteins consistently overexpressed in squamous cell carcinoma in all studies, MCT1 (SLC16A1) and GLUT1 (SLC2A1), for further investigation. We found differential expression of these same proteins at the gene level in our study as well as in other public gene expression datasets. These findings combined with survival analysis of public datasets suggest that MCT1 and GLUT1 may be potential prognostic markers in adenocarcinoma and druggable targets in squamous cell carcinoma. Data are available via ProteomeXchange with identifier PXD002622.     

Proteomic profiling of endothelial cells in human lung cancer

Genomic and proteomic analysis of normal and diseased tissues have yielded an abundance of molecular information for diagnostic and potential therapeutic targets. Changing the target of analysis from poorly accessible cells within tissues to easily accessible vascular endothelium has theoretical advantages in tissue-specific targeting. In this study, we sought to map a large-scale proteome of microvascular endothelium in human non-small cell lung cancer (NSCLC) and normal lung tissues, and identify lung cancer-related endothelial cell (EC)-selective proteins. Endothelial cells were isolated within NSCLC tissues and adjacent-normal lung tissue of lung cancer patients by using CD31-immunomagnetic beads. The complex proteins from the ECs were separated by one-dimensional gel electrophoresis, and the proteins in each gel band were digested by trypsin. Peptides were separated by online reverse-phase liquid-chromatography and analyzed by electrospray ionization (ESI) ion trap tandem mass spectrometry. Approximately 600-1000 proteins were identified in each individual sample. Five patient cases of paired individual data, extracted from the protein identification data sets of both normal- and cancer-derived ECs, were analyzed by subtractive proteomics. An average of 300 proteins was specifically identified from each lung cancer-derived EC isolate, compared to normal lung-derived ECs. With the use of several comparative analyses, we identified among those 300 proteins, 16 common candidate proteins that were detected in at least 3 of 5 cases specific to lung cancer-derived ECs. Proteins selectively identified in cancer-derived ECs, including coatomer protein complex, subunit gamma (COPG), and peroxiredoxin 4 (PRDX4), were validated by Western blot analysis. In an additional experiment in which 16 cancer samples were analyzed by immunohistochemistry, PRDX4, thymopoietin (TMPO), and COPG were confirmed to be abundantly expressed in lung cancer-derived ECs and in cancerous lung cells. Further ongoing analysis of these 16 candidate proteins will determine their potential applicability to NSCLC-specific diagnosis and therapeutics.     

Detection of cathepsin B up-regulation in neoplastic thyroid tissues by proteomic analysis

Nodular or multinodular goiter is the most common non-neoplastic thyroid disease and may be difficult to distinguish from true neoplastic thyroid diseases using microscopic criteria. We have used two-dimensional gel electrophoresis to study the protein patterns of thyroid tissues including normal thyroid, multinodular goiter, diffuse hyperplasia, follicular adenoma, follicular carcinoma and papillary carcinoma. Specific proteins, in the region of molecular mass 15-30 kDa and isoelectric point 4.5-6.5, were identified by electrospray tandem mass spectrometry and protein sequencing. The most distinctive protein found is cathepsin B, which could be detected as four spots, with differential expression in different thyroid diseases. In particular, two of these cathepsin B spots CB2 and CB3 are strongly up-regulated in neoplastic diseases, compared to non-neoplastic diseases. In addition, overexpression of ATP synthase D chain and prohibitin were observed in papillary carcinoma, which should allow it to be differentiated from follicular carcinoma. Changes in expression of other proteins were also observed in disease states compared to normal tissues, namely translationally controlled tumor protein, thioredoxin peroxidase 1, glutathione-S-transferase P, DJ-1 protein, superoxide dismutase (Cu, Zn), and heat shock protein 27, but these changes are less characteristic, so they do not allow the differentiation between neoplastic and non-neoplastic tissues. Thus, the proteomic approach is a useful diagnostic tool for studying diseases involving the thyroid nodule.     

Proteomics Approaches for Identification of Tumor Relevant Protein Targets in Pulmonary Squamous Cell Carcinoma by 2D-DIGE-MS

Potential markers for progression of pulmonary squamous cell carcinoma (SCC) were identified by examining samples of lung SCC and adjacent normal tissues using a combination of fluorescence two-dimensional difference gel electrophoresis (2D-DIGE), matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS), and electrospray ionization quadrupole-time of flight mass spectrometry (ESI-Q-TOF). The PANTHER System was used for gel image based quantification and statistical analysis. An analysis of proteomic data revealed that 323 protein spots showed significantly different levels of expression (P≤0.05) in lung SCC tissue compared to expression in normal lung tissue. A further analysis of these protein spots by MALDI-TOF-MS identified 81 different proteins. A systems biology approach was used to map these proteins to major pathways involved in numerous cellular processes, including localization, transport, cellular component organization, apoptosis, and reproduction. Additionally, the expression of several proteins in lung SCC and normal tissues was examined using immunohistochemistry and western blot. The functions of individual proteins are being further investigated and validated, and the results might provide new insights into the mechanism of lung SCC progression, potentially leading to the design of novel diagnostic and therapeutic strategies.     

Genomic and proteomic expression patterns in HPV-16 E6 gene transfected stable human carcinoma cell lines

cDNA microarray and proteomics studies were performed to analyze the genomic and proteomic expression patterns in HPV-16 E6 gene transfected stable human carcinoma cell lines. Among 1024 known genes and ESTs tested by cDNA microarray, we found 50 upregulated and 35 downregulated genes in RC10.1 HPV-16 E6 transfected human colon adenocarcinoma cells compared to RKO cells, and 27 upregulated and 43 downregulated genes in A549E6 HPV-16 E6 transfected human lung adenocarcinoma cells compared to A549 cells. Employing two dimensional gel electrophoresis and MALDI-TOF-MS, the global pattern of protein expressions in RC10.1 human colon adenocarcinoma and A549E6 human lung adenocarcinoma cell lines stably expressing the HPV 16-E6 gene were compared with those of RKO and A549 cell lines to generate a differential protein expression catalog. We found 13 upregulated and 13 downregulated proteins in RC10.1 (E6-expressing RKO) cells compared to RKO cells and 12 upregulated and 14 downregulated proteins in A549E6 (E6-expressing A549) cells compared to A549 cells. The identified genes and proteins were classified into several groups according to the subcellular function. Expressing pattern of three genes and proteins (CDK5, Bak, and I-TRAF) were matched in both analyses of cDNA microarray and proteomics. These powerful approaches using cDNA microarray and proteomics could provide in-depth information on the impact of HPV-16 E6-related genes and proteins. Differential gene and protein expression patterns by transfection of HPV-16 E6 will provide the nucleus of valuable resource for investigation of the biochemical basis of cervical carcinogenesis. Further understanding of this data base may provide valuable resources for developing novel diagnostic markers and therapeutic targets of cervical cancer.     

MALDI imaging mass spectrometry reveals COX7A2, TAGLN2 and S100-A10 as novel prognostic markers in Barrett's adenocarcinoma

To characterize proteomic changes found in Barrett's adenocarcinoma and its premalignant stages, the proteomic profiles of histologically defined precursor and invasive carcinoma lesions were analyzed by MALDI imaging MS. For a primary proteomic screening, a discovery cohort of 38 fresh frozen Barrett's adenocarcinoma patient tissue samples was used. The goal was to find proteins that might be used as markers for monitoring cancer development as well as for predicting regional lymph node metastasis and disease outcome. Using mass spectrometry for protein identification and validating the results by immunohistochemistry on an independent validation set, we could identify two of 60 differentially expressed m/z species between Barrett's adenocarcinoma and the precursor lesion: COX7A2 and S100-A10. Furthermore, among 22 m/z species that are differentially expressed in Barrett's adenocarcinoma cases with and without regional lymph node metastasis, one was identified as TAGLN2. In the validation set, we found a correlation of the expression levels of COX7A2 and TAGLN2 with a poor prognosis while S100-A10 was confirmed by multivariate analysis as a novel independent prognostic factor in Barrett's adenocarcinoma. Our results underscore the high potential of MALDI imaging for revealing new biologically significant molecular details from cancer tissues which might have potential for clinical application. This article is part of a Special Issue entitled: Translational Proteomics.     

Quantitative Proteomic Analysis of Formalin-fixed and Paraffin-embedded Nasopharyngeal Carcinoma Using iTRAQ Labeling,Two-dimensional Liquid Chromatography,and Tandem Mass Spectrometry

Formalin-fixed, paraffin-embedded (FFPE) tissue specimens represent a potentially valuable resource for protein biomarker investigations. In this study, proteins were extracted by a heat-induced antigen retrieval technique combined with a retrieval solution containing 2% SDS from FFPE tissues of normal nasopharyngeal epithelial tissues (NNET) and three histological types of nasopharyngeal carcinoma (NPC) with diverse differentiation degrees. Then two-dimensional liquid chromatography-tandem mass spectrometry coupled with isobaric tags for relative and absolute quantification (iTRAQ) labeling was employed to quantitatively identify the differentially expressed proteins among the types of NPC FFPE tissues. Our study resulted in the identification of 730 unique proteins, the distributions of subcellular localizations and molecular functions of which were similar to those of the proteomic database of human NPC and NNET that we had set up based on the frozen tissues. Additionally, the relative expression levels of cathepsin D, keratin8, SFN, and stathmin1 identified and quantified in this report were consistent with the immunohistochemistry results acquired in our previous study. In conclusion, we have developed an effective approach to identifying protein changes in FFPE NPC tissues utilizing iTRAQ technology in conjunction with an economical and easily accessible sample preparation method. (J Histochem Cytochem 58:517–527, 2010)     

Buffer optimization for high resolution of human lung cancer tissue proteins by two-dimensional gel electrophoresis

A problem in proteomic analysis of lung cancer tissue is the presence of complex components of different histological backgrounds (squamous cell carcinoma, small cell lung carcinoma, and adenocarcinoma). The efficient solubilization of protein components before two-dimensional electrophoresis (2-DE) is a very critical. Poor solubilization has been associated with a failure to detect proteins and diffuse, streaked and/or trailing protein spots. Here, we have optimized the solubilization of human lung cancer tissue to increase protein resolution. Isoelectric focusing (IEF) rehydration buffer containing a thiourea–urea mixture provided superior resolution, whereas a buffer without thiourea yielded consistently poor results. In addition, IEF rehydration buffers containing CHAPS and DTT gave superior resolution, whereas buffers containing Nonidet P-40 (NP-40) and/or Triton X-100 did not. A tributylphosphine-containing buffer gave consistently poor results. Using optimized conditions, we used 2-D gel analysis of human lung cancer tissue to identify 11 differentially-expressed protein spots by MALDI-mass spectrometry. This study provides a methodological tool to study the complex mammalian proteomes.