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.     

Macrophage-capping protein as a tissue biomarker for prediction of response to gemcitabine treatment and prognosis in cholangiocarcinoma

Cholangiocarcinoma is one of the deadliest malignancies worldwide. Recent studies reported that treatment with gemcitabine was effective in prolonging survival. However, as the treatment only benefited a limited subset of patients, selection of patients before treatment is required. To discover biomarkers predictive of the response to gemcitabine treatment in cholangiocarcinoma, we examined the proteome of three types of material resource; ten cell lines, nine xenografts and nine surgically resected primary tumors from patients who exhibited different response to gemcitabine treatment. Two-dimensional difference gel electrophoresis generated quantitative protein expression profiles including 3571 protein spots. We detected 172 protein spots with significant correlation with response to gemcitabine treatment. All proteins corresponding to these 172 protein spots were identified by mass spectrometry. We found that the macrophage-capping protein (CapG) was associated with response to gemcitabin treatment in all three types of material source. Immunohistochemical validation in an additional set of 196 cholangiocarcinoma cases revealed that CapG expression was associated with lymphatic invasion status and overall survival. Multivariate analysis showed that CapG protein expression was an independent prognostic factor for overall survival. In conclusion, CapG was identified as a novel candidate biomarker to predict response to gemcitabine treatment and survival in cholangiocarcinoma.     

Proteomic-based identification of the APC-binding protein EB1 as a candidate of novel tissue biomarker and therapeutic target for colorectal cancer

Novel candidates of biomarker and therapeutic target in colorectal cancer (CRC) were investigated using a proteomic approach. The proteome of normal colorectal epithelial tissues was compared with that of the tumor ones in 59 CRC patients using two-dimensional difference gel electrophoresis. Of 3458 protein spots, 110 exhibited statistically significant (p<0.01) differences in intensity (more than 2.5-folds) between the normal and tumor tissue groups. Of 67 unique gene products that were identified for 105 of the 110 protein spots, we focused on the higher expression of the adenoma polyposis coli-binding protein EB1 (EB1). EB1 was originally discovered as a binding protein of APC, which is a tumor suppressor gene product, and the expression of EB1 has been associated with poor prognosis in several malignancies but not in CRC. Immunohistochemical analysis of the 132 CRC cases revealed that EB1 was overexpressed in tumor cells in correlation with poor prognosis. Suppression of EB1 by RNAi inhibited CRC cell proliferation and invasion. In this study, the overexpression of EB1 in CRC tissues correlating with prognosis, and its functional contribution to the malignant phenotypes of CRC cells are described. The present findings indicate that EB1 is a potential biomarker and therapeutic target in CRC.     

Proteomic search for potential diagnostic markers and therapeutic targets for ovarian clear cell adenocarcinoma

Clear cell adenocarcinoma (CCA) has a highly malignant potential in human epithelial ovarian cancer. The serum CA-125 is widely used as a marker for ovarian cancer, but the level is relatively low in CCA. Therefore, new sensitive biomarkers are required. In this report, we describe a promising proteomic analysis that is differentially expressed in CCA when compared to mucinous adenocarcinoma, using the ovarian cultured cell lines OVISE, OVTOKO, and MCAS. The disease-associated proteins were identified by 2-D differential gel electrophoresis (2-D DIGE) and MS. In this analysis, 18 up-regulated and 31 down-regulated spots were observed that had at least two-fold differences in the two CCA cell lines than in MCAS as control cells. Some of the proteins differentially expressed in CCA were previously observed as alternative expression levels in ovarian and/or other cancers in clinical samples. In a subsequent preliminary differential study using surgical specimens from patients with CCA, it was demonstrated that the identified proteins were expressed differentially in actual tissues, as well as in the CCA culture cells. The results from this investigation show the potentiality of a proteomic approach for identifying disease-associated proteins, which may eventually serve as diagnostic markers or therapeutic targets in CCA.     

Trimodality treatment in malignant pleural mesothelioma – Ordeal or real deal?

BackgroundManagement of MPM is complex and controversial as there is a paucity of good quality evidence. We report the toxicity and outcomes in patients who received trimodality treatment for non-metastatic MPM at our institution.Methods & materialsWe reviewed the electronic medical records of surgically managed MPM patients at our institution in the last decade. Dosimetric parameters of target volume and organs at risk were documented by the treatment planning workstation. SPSS was used for statistical analysis.ResultsBetween January 2008 and October 2018, 21 patients underwent surgery for MPM – all but 2 patients underwent extra-pleural pneumonectomy (EPP); epithelioid MPM was the most common histology. All patients, except 2, received neoadjuvant Pemetrexed/platinum doublet chemotherapy. Fourteen patients received adjuvant hemithoracic RT; ten patients were treated with a conformal technique at our institute and dosimetric data was available for analysis. Average time to start RT after surgery was 51 days (range 32–82 days). All patients were treated with a conformal technique using IMRT/VMAT to a dose of 45Gy in 25 fractions. Mean overall RT duration was 35 days (range 30–42 days). Grade I/II Pneumonitis was seen in 4 patients. One patient developed grade III acute lung toxicity unrelated to RT. At a median follow up of 25 months, 8 patients had died, of whom six died due to the disease and two died in the immediate post op period. Two-year DFS and OS were 58% and 73%, respectively.ConclusionIn spite of the extensive surgery and complex hemithoracic RT, we demonstrated excellent dosimetry, toxicity profile and favorable outcomes in non-metastatic MPM.     

Dickkopf-1 antagonizes Wnt signaling independent of beta-catenin in human mesothelioma

Dickkopf-1 (Dkk-1) is a secreted protein that acts as a potent inhibitor of the Wnt signal transduction pathway. It is thought that the antagonistic effect of Dkk-1 is specific to the canonical (Wnt/beta-catenin) pathway. In this study, we demonstrate that restoration of Dkk-1 expression suppresses cell growth and induces apoptotic cell death in beta-catenin-deficient mesothelioma cell lines H28 and MS-1. Furthermore, we found that a small-molecule inhibitor of JNK inhibited the apoptosis induced by Dkk-1 overexpression in these cells. Together, our data suggest that Dkk-1 may be able to antagonize Wnt signaling and exert its tumor suppressive effects through beta-catenin-independent non-canonical pathways (i.e., the Wnt/JNK pathway).     

Comparative proteomic and transcriptomic profiling of the human hepatocellular carcinoma

Proteome analysis of human hepatocellular carcinoma (HCC) was done using two-dimensional difference gel electrophoresis. To gain an understanding of the molecular events accompanying HCC development, we compared the protein expression profiles of HCC and non-HCC tissue from 14 patients to the mRNA expression profiles of the same samples made from a cDNA microarray. A total of 125 proteins were identified, and the expression profiles of 93 proteins (149 spots) were compared to the mRNA expression profiles. The overall protein expression ratios correlated well with the mRNA ratios between HCC and non-HCC (Pearson’s correlation coefficient: r = 0.73). Particularly, the HCC/non-HCC expression ratios of proteins involved in metabolic processes showed significant correlation to those of mRNA (r = 0.9). A considerable number of proteins were expressed as multiple spots. Among them, several proteins showed spot-to-spot differences in expression level and their expression ratios between HCC and non-HCC poorly correlated to mRNA ratios. Such multi-spotted proteins might arise as a consequence of post-translational modifications.     

Changes in protein expression in testes of L2 strain Taiwan country chickens in response to acute heat stress

Heat stress causes a decrease of fertility in roosters. Yet, the way acute heat stress affects protein expression remains poorly understood. This study investigated differential protein expression in testes of the L2 strain of Taiwan country chickens following acute heat stress. Twelve 45-week-old roosters were allocated into four groups, including control roosters kept at 25 °C, roosters subjected to 38 °C acute heat stress for 4 hours without recovery, with 2 hours of recovery, and with 6 hours of recovery. Testis samples were collected for morphologic assay and protein analysis. Some of the differentially expressed proteins were validated by Western blot and immunohistochemistry. Abnormal and apoptotic spermatogenic cells were observed at 2 hours of recovery after acute heat stress, especially among the spermatocytes. Two-dimensional difference gel electrophoresis revealed that 119 protein spots were differentially expressed in chicken testes following heat stress, and peptide mass fingerprinting revealed that these spots contained 92 distinct proteins. In the heat-stressed samples, the heat shock proteins, chaperonin containing t-complex, and proteasome subunits were downregulated, and glutathione S-transferase, transgelin, and DJ-1 were upregulated. Our results demonstrate that acute heat stress impairs the processes of translation, protein folding, and protein degradation, and thus results in apoptosis and interferes with spermatogenesis. On the other hand, the increased expression of antioxidant enzymes, including glutathione S-transferase and DJ-1, may attenuate heat-induced damage. These findings may have implications for breeding chickens that can tolerate more extreme conditions.     

Situs inversus in the developing mouse: proteins affected by the iv mutation (genocopy) and the teratogen retinoic acid (phenocopy)

To decipher genes that are important in the determination of laterality, we compared two-dimensional protein gels from wild-type C57BL/6J mice and C57BL/6J mice that carried the iv mutation, which confers random determination of visceral situs. To span the time period(s) during which laterality determination occurs, we compared computer-analyzed two-dimensional protein gels from wild-type mouse embryos and iv/iv mouse embryos at 7.5, 8.0, and 8.5 days post-coitum. One polypeptide that was expressed only on day 8.0 of development and only in wild-type embryos represents a particular candidate for determination of laterality. Day 8.5 postcoitum represents the earliest time in murine development that laterality is manifest. Two-dimensional gels were compared from 8.5 day embryos that were C57BL/6J wild-type, C57BL/6J iv/iv, or C57BL/6J wild-type and exposed to the teratogen retinoic acid late on day 7. Reproducible alterations of protein synthesis were observed in both the iv genocopy and retinoic acid phenocopy, yielding abnormal laterality determination. The intersection of these peptide changes identifies a protein likely to play a role in the determination of laterality.     

Optimal isolation of mitochondria for proteomic analyses

Considering the key role of mitochondria in cellular (dys)functions, we compared a standard isolation protocol, followed by lysis in urea/detergent buffer, with a commercially available isolation buffer that rapidly yields a mitochondrial protein fraction. The standard protocol yielded significantly better overall resolution and coverage of both the soluble and membrane mitochondrial proteomes; although the kit was faster, it resulted in recovery of only approximately 56% of the detectable proteome. The quality of “omic” analysis depends on sample handling; for large-scale protein studies, well-resolved proteomes are highly dependent on the purity of starting material and the rigor of the extraction protocol.     

Analysis of proteins expressed at the time of murine organogenesis

Two-dimensional electrophoretograms were prepared from wild-type C57BL/6J embryos from day 7.5 through day 9.0 of development. This time period encompasses a critical window of development as the embryo traverses from an egg cylinder through major organogenesis. Consequently, we term this resource MOPED (for mouse organogenesis protein electrophoresis database). By resolving and analyzing the behavior of approximately 1,000 polypeptides per time point, we were able to track many of these polypeptides through this time period in development. Of special note was a burst of induced protein synthesis that was observed in mouse embryos development. Polypeptides observed in mouse embryos that match those identified previously in mouse fibroblasts were noted. Two of them (the intermediate filament-associated protein and tropomyosin-4) were significantly altered in 8.5 day embryos. As more polypeptides are designated, it will be possible to expand the known proteins in the database. MOPED establishes the patterns of synthesis of a large number of polypeptides during a crucial period of development. Thus MOPED is designed to analyze proteins relevant to mouse embryogenesis in the future.     

G-electrode-loading method for isoelectric focusing, enabling separation of low-abundance and high-molecular-mass proteins

The G-electrode-loading method (GELM) is a technique enabling a large number of proteins from rat liver to enter an immobilized pH gradient (IPG) gel strip for isoelectric focusing (IEF). In this method, three slips containing the sample solution are placed on the cathodic edge of an IPG gel strip and a slip containing Chaps solution, a filtration membrane, and an electrode slip are placed on top. Finally, a G-electrode is placed on these slips. The Chaps solution (an amphoteric compound) is supplied gently to the sample solution during IEF and helps the proteins in the sample solution to enter the IPG gel strips with a high solubilization capacity. This method was compared with traditional slip-loading and in-gel rehydration, and it showed the best results for protein separation, including high-molecular-mass proteins.     

金鱼雌核发育单倍体发育过程中的比较蛋白质组学研究

前期已有工作发现在金鱼雌核发育单倍体中一些与发育调控相关的重要蛋白质表达受阻导致单倍体的发育畸形。为了进一步阐明单倍体的发育机制,我们共收集了3个不同发育时期金鱼单倍体胚胎（HE-1、HE-2、HE-3）进行雌核发育单倍体的差异蛋白质组研究。研究采用二维凝胶电泳进行分离,利用PDQuest软件进行图谱分析,质谱分析初步鉴定到了15个差异蛋白质。这些蛋白质在金鱼雌核发育单倍体的发育中起着重要作用,为进一步阐明单倍体的发育机制奠定了良好的基础。     

Protein databases for compacted eight-cell and blastocyst-stage mouse embryos

High-resolution two-dimensional sodium dodecyl sulfate-polyacrylamide (2D-SDS) gel electrophoresis combined with computerized analysis of gel images was used to construct and analyze protein databases for two stages of preimplantation mouse embryogenesis, the compacted eight-cell stage and the fully expanded blastocyst stage. These stages were chosen for their ease in identification of multiple synchronous embryos. Synchronous cohorts of 30–50 embryos were labelled with L-[³⁵S]methionine for 2 hr. The embryos were then lysed in 30 μl hot SDS sample buffer, and the lysates were stored at ?80°C until the gels were run. Five replicates were run for eight-cell embryos, and four for blastocyst-stage embryos. The samples were processed for 2D gel electrophoresis and fluorography; multiple exposures were made. Gel images were analyzed using the PDQUEST system, and databases were constructed. Analysis of the databases for both developmental stages showed high reproducibility of protein spots in multiple gel images. Of 1,674 total spots in eight-cell embryo standards, >79% of spots had a percentage error (S.E.M./average) <50%, and >45% had a percentage error <30%. Similarly, of 1,653 total spots in blastocyst-stage embryo standards, 74% of spots had a percentage error <50%, and approximately 47% of spots had a percentage error <30%. Forty-three spots (approximately 3% of the total spots) were found to be detected only in the eight-cell stage, while 75 spots were detected solely in the blastocyst stage. Sixty-nine proteins showed a greater than threefold increase in isotope incorporation from the eight-cell to the blastocyst stage, with a percentage error <50% in both the eight-cell and the blastocyst stages. In contrast, 41 of the proteins showed a decrease during this period. Analysis of the protein databases described in this study has allowed us to document the overall quantitative changes in proteins from the compacted eight-cell stage to the blastocyst stage of mouse preimplantation development. These databases provide a valuable tool for further detailed quantitative analysis of specific proteins associated with developmental events. In addition they will permit analysis of the effects of environmental factors, such as growth factors, on early embryo development. © 1994 Wiley-Liss, Inc.     

水稻花药总蛋白质双向电泳方法的改良与优化(简报)

双向电泳作为蛋白质组学核心技术之一,目前已广泛地应用在植物领域,并且成功应用于水稻代谢和调节等方面的研究。谢锦云等利用溶液法提取温敏核不育水稻花药总蛋白质,利用pH3-10线性胶条分离,经银染显色后检测到约1,000个