Identification of molecular targets for selective elimination of TRAIL‐resistant leukemia cells. From spots to in vitro assays using TOP15 charts

The resistance of malignant cells to chemotherapy calls for the development of novel anti‐cancer drugs. TNF‐related apoptosis‐inducing ligand (TRAIL) is a pro‐apoptotic cytokine, which selectively induces apoptosis in malignant cells. We derived two TRAIL‐resistant HL‐60 subclones, HL‐60/P1 and HL‐60/P2, from a TRAIL‐sensitive HL‐60 acute promyelocytic leukemia cell line. To identify therapeutically exploitable “weaknesses” of the TRAIL‐resistant leukemia cells that could be used as molecular targets for their elimination, we performed proteomic (2‐DE) analysis and compared both TRAIL‐resistant subclones with the original TRAIL‐sensitive HL‐60 cells. We identified over 40 differentially expressed proteins. To significantly narrow the lists of candidate proteins, we excluded proteins that are known to be often differentially expressed, regardless of experiment type and tissue (the so‐called “TOP15” proteins). Decreased expression of DNA replication and maintenance proteins MCM7 and RPA32 in HL‐60/P1 cells, and the marked down‐regulation of enzyme adenosine deaminase in HL‐60/P2 cells, suggests increased sensitivity of these cells to DNA‐interfering drugs, and adenosine and its homologues, respectively. In a series of in vitro assays, we confirmed the increased toxicity of etoposide and cisplatin to TRAIL resistant HL‐60/P1 cells, and adenosine and vidarabine to HL‐60/P2, compared with TRAIL‐sensitive HL‐60 cells.     

Proteomic characterization of Her2/neu-overexpressing breast cancer cells

The receptor tyrosine kinase HER2 is an oncogene amplified in invasive breast cancer and its overexpression in mammary epithelial cell lines is a strong determinant of a tumorigenic phenotype. Accordingly, HER2-overexpressing mammary tumors are commonly indicative of a poor prognosis in patients. Several quantitative proteomic studies have employed two-dimensional gel electrophoresis in combination with MS/MS, which provides only limited information about the molecular mechanisms underlying HER2/neu signaling. In the present study, we used a SILAC-based approach to compare the proteomic profile of normal breast epithelial cells with that of Her2/neu-overexpressing mammary epithelial cells, isolated from primary mammary tumors arising in mouse mammary tumor virus-Her2/neu transgenic mice. We identified 23 proteins with relevant annotated functions in breast cancer, showing a substantial differential expression. This included overexpression of creatine kinase, retinol-binding protein 1, thymosin 4 and tumor protein D52, which correlated with the tumorigenic phenotype of Her2-overexpressing cells. The differential expression pattern of two genes, gelsolin and retinol binding protein 1, was further validated in normal and tumor tissues. Finally, an in silico analysis of published cancer microarray data sets revealed a 23-gene signature, which can be used to predict the probability of metastasis-free survival in breast cancer patients.     

Differential expression of protease activity in serum samples of prostate carcinoma patients with metastases

We present here the results from MS peptide profiling experiments of prostate carcinoma patients and controls with a specific focus on protease activity‐related protein fragments. After purification with surface‐active magnetic beads, MALDI‐TOF profiling experiments were performed on tryptic digests of serum samples of prostate cancer patients with metastases (n=27) and controls (n=30). This resulted in the reproducible detection of eight differentially expressed peptides, which were then identified by nanoLC‐MALDI‐TOF/TOF and confirmed by MALDI‐FTMS exact mass measurements. All differentially expressed peptides are derived from two homologous parts of human serum albumin; two of the eight peptides were tryptic and six were nontryptic. The presence of the nontryptic fragments indicates that a proteolysis process occurs which is not mediated by trypsin. Since the nontryptic fragments were found at significantly higher levels in control samples compared with metastases samples, it is proposed that a specific proteolytic inhibition process is in effect in the serum of prostate cancer patients. Experiments using synthetic peptides showed that this proteolytic activity occurs ex vivo and is sequence specific. Importantly, the observed prostate carcinoma‐related inhibition of the proteolysis was reproduced ex vivo using synthetic peptides.     

Upregulation of plasma C9 protein in gastric cancer patients

Gastric cancer is one of the leading causes of cancer‐related deaths worldwide. Current biomarkers used in the clinic do not have sufficient sensitivity for gastric cancer detection. To discover new and better biomarkers, protein profiling on plasma samples from 25 normal, 15 early‐stage and 21 late‐stage cancer was performed using an iTRAQ‐LC‐MS/MS approach. The level of C9 protein was found to be significantly higher in gastric cancer compared with normal subjects. Immunoblotting data revealed a congruent trend with iTRAQ results. The discriminatory power of C9 between normal and cancer states was not due to inter‐patient variations and was independent from gastritis and Helicobacter pylori status of the patients. C9 overexpression could also be detected in a panel of gastric cancer cell lines and their conditioned media compared with normal cells, implying that higher C9 levels in plasma of cancer patients could be attributed to the presence of gastric tumor. A subsequent blind test study on a total of 119 plasma samples showed that the sensitivity of C9 could be as high as 90% at a specificity of 74%. Hence, C9 is a potentially useful biomarker for gastric cancer detection.     

对生物医药产业可持续发展战略的思考

全球生物药品销售额以年均约30%的速度增长,大大高于全医药行业年均不到10%的增长速度,生物技术产业已不是几个国家的产业,而是一个全球化发展的产业,由于全球生态环境日益恶化,严重影响了各国发展的后劲,于是它们纷纷将发展生物技术产业作为可持续发展战略的一个重要内容。生物医药产业可持续发展是环境与发展战略的需要,其核心是技术创新,提出了生物医药产业可持续发展链和区域经济竞争力是促进生物医药产业可持续发展的基石,阐述了生物医药产业发展只有立足现实、谋划长远,紧抓发展优势,才能实现可持续发展战略目标。     

Integrated and convenient procedure for protein extraction from formalin‐fixed,paraffin‐embedded tissues for LC‐MS/MS analysis

Because fresh‐frozen tissue samples associated with long‐term clinical data and of rare diseases are often unobtainable at the present time, formalin‐fixed paraffin‐embedded (FFPE) tissue samples are considered a highly valuable resource for researchers. However, protein extraction from FFPE tissues faces challenges of deparaffinization and cross‐link reversion. Current procedures for protein extraction from FFPE tissue require separate steps and toxic solvents, resulting in inconvenience in protein extraction. To overcome these limitations, an integrated method was developed using nontoxic solvents in four types of FFPE tissues. The average amount of proteins from three replicates of bladder, kidney, liver, and lung FFPE tissues were 442.6, 728.9, 736.4, and 694.7 μg with CVs of 7.5, 5.8, 2.4, and 4.5%, respectively. Proteomic analysis showed that 348, 417, 607, and 304 unique proteins were identified and quantified without specification of isoform by a least two peptides from bladder, kidney, liver, and lung FFPE tissue samples, respectively. The analysis of individual protein CV demonstrated that 97–99% of the proteins were quantified with a CV ≤ 30%, verifying the reproducibility of the integrated protein extraction method. In summary, the developed method is high‐yield, reproducible, convenient, simple, low cost, nonvolatile, nonflammable, and nontoxic.     

Proteomic characteristics of human sperm cryopreservation

Human sperm cryopreservation in assisted reproductive technology is the only proven method that enables infertile men to father their own children. However, freezing and thawing reduces spermatozoon motility, viability, and fertilizing ability. An association between dysfunctional spermatozoa due to cryoinjury and protein changes has not been established. We investigated through proteomic analysis the differential protein characteristics between freeze‐thawed and fresh sperm samples obtained from nine normozoospermic donors. Twenty‐seven proteins differed in abundance between the two groups, and results were verified for four proteins via Western blot and immunofluorescent staining. These proteins are putatively involved in sperm motility, viability, acrosomal integrity, ATP and isocitrate content, mitochondrial membrane potential, capacitation, acrosome reaction, and intracellular calcium concentration. These marked differences suggest that dysfunctional spermatozoon after cryopreservation may be due to protein degradation and protein phosphorylation.     

Proteomic profiles of human lung adeno and squamous cell carcinoma using super‐SILAC and label‐free quantification approaches

Nonsmall cell lung cancer (NSCLC) accounts for 85% of lung cancers, and is subdivided into two major histological subtypes: adenocarcinoma (ADC) and squamous cell carcinoma (SCC). There is an unmet need to further subdivide NSCLC according to distinctive molecular features that may be associated with responsiveness to therapies. Four primary tumor‐derived xenograft proteomes (two‐each ADC and SCC) were quantitatively compared by using a super‐SILAC labeling approach together with ultrahigh‐resolution MS. Proteins highly differentially expressed in the two subtypes were identified, including 30 that were validated in an independent cohort of 12 NSCLC primary tumor‐derived xenograft tumors whose proteomes were quantified by an alternative, label‐free shotgun MS methodology. The 30‐protein signature contains metabolism enzymes including phosphoglycerate dehydrogenase, which is more highly expressed in SCC, as well as a comprehensive set of cytokeratins and other components of the epithelial barrier, which is therefore distinctly different between ADC and SCC. These results demonstrate the utility of the super‐SILAC method for the characterization of primary tissues, and compatibility with datasets derived from different MS‐based platforms. The validation of proteome signatures of NSCLC subtypes supports the further development and application of MS‐based quantitative proteomics as a basis for precision classifications and treatments of tumors. All MS data have been deposited in the ProteomeXchange with identifier PXD000438 ( http://proteomecentral.proteomexchange.org/dataset/PXD000438 ).     

Spike‐in SILAC proteomic approach reveals the vitronectin as an early molecular signature of liver fibrosis in hepatitis C infections with hepatic iron overload

Hepatitis C virus (HCV)‐induced iron overload has been shown to promote liver fibrosis, steatosis, and hepatocellular carcinoma. The zonal‐restricted histological distribution of pathological iron deposits has hampered the attempt to perform large‐scale in vivo molecular investigations on the comorbidity between iron and HCV. Diagnostic and prognostic markers are not yet available to assess iron overload‐induced liver fibrogenesis and progression in HCV infections. Here, by means of Spike‐in SILAC proteomic approach, we first unveiled a specific membrane protein expression signature of HCV cell cultures in the presence of iron overload. Computational analysis of proteomic dataset highlighted the hepatocytic vitronectin expression as the most promising specific biomarker for iron‐associated fibrogenesis in HCV infections. Next, the robustness of our in vitro findings was challenged in human liver biopsies by immunohistochemistry and yielded two major results: (i) hepatocytic vitronectin expression is associated to liver fibrogenesis in HCV‐infected patients with iron overload; (ii) hepatic vitronectin expression was found to discriminate also the transition between mild to moderate fibrosis in HCV‐infected patients without iron overload.     

Discovery and identification of serum potential biomarkers for pulmonary tuberculosis using iTRAQ‐coupled two‐dimensional LC‐MS/MS

Pulmonary tuberculosis (TB) caused by Mycobacterium tuberculosis is a chronic disease. Currently, there are no sufficiently validated biomarkers for early diagnosis of TB infection. In this study, a panel of potential serum biomarkers was identified between patients with pulmonary TB and healthy controls by using iTRAQ‐coupled 2D LC‐MS/MS technique. Among 100 differentially expressed proteins screened, 45 proteins were upregulated (>1.25‐fold at p < 0.05) and 55 proteins were downregulated (<0.8‐fold at p < 0.05) in the TB serum. Bioinformatics analysis revealed that the differentially expressed proteins were related to the response to stimulus, the metabolic and immune system processes. The significantly differential expression of apolipoprotein CII (APOCII), CD5 antigen‐like (CD5L), hyaluronan‐binding protein 2 (HABP2), and retinol‐binding protein 4 (RBP4) was further confirmed using immunoblotting and ELISA analysis. By forward stepwise multivariate regression analysis, a panel of serum biomarkers including APOCII, CD5L, and RBP4 was obtained to form the disease diagnostic model. The receiver operation characteristic curve of the diagnostic model was 0.98 (sensitivity = 93.42%, specificity = 92.86%). In conclusion, APOCII, CD5L, HABP2, and RBP4 may be potential protein biomarkers of pulmonary TB. Our research provides useful data for early diagnosis of TB.