Nuclear survivin expression: a prognostic factor for the response to taxane-platinum chemotherapy in patients with advanced non-small cell lung cancer.

Survivin, a structurally unique protein expressed in most common human neoplasms, is thought to support cell cycle progression and suppress apoptosis. Survivin expression is highly correlated with advanced non-small cell lung cancer (NSCLC) and poor prognosis. In this retrospective study of banked pathology tissue of patients with advanced NSCLC, we tested for correlations of N-survivin expression in tumor tissues and responsiveness to treatment with platinum-based regimens containing paclitaxel or docetaxel. The 48 patients with NSCLC included 32 (66.7 %) males and 16 (33.3 %) females. Mean age at diagnosis was 59.4 years (range 36-83 years), and median follow-up time was 20.4 months (range 3.4-59.0 months). Patients with high tumor N-survivin expression had significantly better responses to taxane-platinum chemotherapy than those with low tumor N-survivin expression (P < 0.001). Adjusted multivariate modeling found high tumor N-survivin expression to be an independent prognostic factor for a clinical response to chemotherapy (high vs. low, OR 6.14, 95 % CI 1.62-23.29; P = 0.008). Median overall survival differed significantly between those with high tumor N-survivin expression who did/did not respond to chemotherapy and between those with low tumor N-survivin expression who did/did not respond to chemotherapy (P < 0.05). Tumor N-survivin expression shows promise as a predictive biomarker in the chemotherapy setting as a surrogate marker of high proliferation status.

Blockade of phospholipid scramblase 1 with its N-terminal domain antibody reduces tumorigenesis of colorectal carcinomas in vitro and in vivo.

BACKGROUND: Membrane-bound phospholipid scramblase 1 (PLSCR1) is involved in both lipid trafficking and cell signaling. Previously, we showed that PLSCR1 is overexpressed in many colorectal carcinomas (CRCs). In the present study, we investigated the tumorigenic role of PLSCR1 in CRC and suggest that it is a potential therapeutic target. METHODS: To identify PLSCR1 as a therapeutic target, we studied the tumorigenic properties of CRC cell lines treated with a monoclonal antibody (NP1) against the N-terminus of PLSCR1 in vitro and in vivo. We also investigated cell cycle status and epidermal growth factor receptor-related pathways and downstream effectors of PLSCR1 after blocking its function with NP1. RESULTS: Treating CRC cells with NP1 in vitro and in vivo decreased cell proliferation, anchorage-independent growth, migration, and invasion. Adding NP1 to the CRC cell line HT29 caused arrest at G1/S. Treating HT29 cells with NP1 significantly decreased the expression of cyclin D1 and phosphorylation levels of Src, the adaptor protein Shc, and Erks. The reduced level of cyclin D1 led to an increase in the activated form of the tumor suppressor retinoblastoma protein via dephosphorylation. These actions led to attenuation of tumorigenesis. CONCLUSIONS: Therefore, PLSCR1 may serve as a potential therapeutic target for CRC.

Detection of autoantibodies against Rabphilin-3A-like protein as a potential biomarker in patient's sera of colorectal cancer.

BACKGROUND: Rabphilin-3A-like (RPH3AL) protein functions in the regulation of hormone exocytosis, and mutations in the RPHA3L gene have been associated with tumorigenesis in colorectal cancer (CRC). We evaluated the potential use of anti-RPH3AL autoantibodies as a marker for CRC detection. METHODS: Sera from 84 patients with CRC and 63 healthy controls were analysed for the presence of RPH3AL autoantibodies with a Western blotting assay. RESULTS: The frequencies of RPH3AL autoantibodies in the early stage, advanced stage and all CRC patients were 64.7%, 78.0% and 72.6%, respectively. These values are significantly higher than the frequency of RPH3AL autoantibodies in healthy controls (15.9%, P<0.001). Although the presence of RPH3AL autoantibodies did not correlate with clinical parameters, RPH3AL autoantibodies were found in 69.4% (34/49) of CRC patients who were negative for carcinoembryonic antigen. The value of the area under the receiver operating characteristic curve of RPH3AL autoantibody was 0.84, which suggests that screening for these autoantibodies could potentially be used for CRC diagnosis. CONCLUSION: Circulating RPH3AL autoantibodies are prevalent in patients with CRC, and detection of these autoantibodies might provide a novel non-invasive approach for CRC diagnosis.

In vitro and in vivo analysis of a biodegradable poly(lactide-co-glycolide) copolymer capsule and collagen composite system for antibiotics and bone cells delivery.

The authors investigated poly(lactide-co-glycolide) (PLGA) capsule and collagen composite system for antibiotics and bone cells delivery to treat infected bone defects. Poly(lactide-co-glycolide) was mixed with vancomycin and hot compressing molded to form an antibiotic capsule. Rabbit mesenchymal stem cells (MSCs) were entrapped in collagen gel phase and dispersed throughout the void volume of capsule. In vitro study, the composite systems were cultured in complete or osteogenic medium for 21 days. The profiles of vancomycin released from the systems were evaluated using the high-performance liquid chromatography method. Relative activity of vancomycin against Staphylococcus aureus was determined by an antibiotic disk diffusion method. The expression of osteogenic gene was determined by reverse-transcription polymerase chain reaction. The alkaline phosphatase activity and calcium level of the MSCs were assessed. Analytical results demonstrated that the concentrations of vancomycin eluted from the composite system were above the minimal inhibitory concentration for 21 days. Sample inhibition zone was 10 to 24 mm, and the relative activity was 17.6% to 100%. mRNA of Cbfa1 and osteocalcin were detected, and increased alkaline phosphatase activity and calcium levels were noted. In in vivo investigation, the PKH 26-labeled MSCs and composite systems were implanted in the distal femoral cavities of four rabbits. The local concentration of vancomycin was above the minimal inhibitory concentration for 56 days. Sample inhibition zone was 9 mm to 24 mm, and the relative activity was 11.8% to 100%. Implanted PKH 26-labeled MSCs were identified in the newly formed bony trabeculae in specimens at 2 and 4 months after implantation. The results offer a potential approach to meet clinical requirements in the treatment of infected bone defects.

Identification of SEC61ß and its autoantibody as biomarkers for colorectal cancer.

BACKGROUND: To identify novel serological biomarkers for human colorectal cancer (CRC), we analyzed CRC tissues using gel-assisted digestion and isobaric tags with related and absolute quantitation (iTRAQ) labeling mass spectrometry (MS). By comparing pairs of tumor tissues and matched normal tissues, we discovered the SEC61ß with expression changes 3.3-fold and a marginal statistical significance (p=0.052) previously. METHODS: SEC61ß expression in CRC tissues was further analyzed by western blotting and immunohistochemistry. We next assessed the putative diagnostic value of the SEC61ß autoantibody as a serum marker. RESULTS: Using western blotting analysis, SEC61ß expression was increased 1.9-fold in tumor tissues. Immunohistochemical analysis of 64 CRC specimens showed that SEC61ß was positively detected in 64% of the tumors, but weakly or not detected in >80% of the adjacent nontumor epithelial cells. Western blot analysis with plasma samples showed that the sensitivity and specificity of the SEC61ß autoantibody from patients with CRC were 79% and 75%, respectively. Importantly, the results of the SEC61ß autoantibody for early detection of colorectal cancer revealed a higher sensitivity of 77% than the carcinoembryonic antigen (CEA) assay. CONCLUSIONS: Measurement of SEC61ß autoantibody levels may provide an alternative detection indicator for CRC, particularly among early-stage patients.

An informatics-assisted label-free approach for personalized tissue membrane proteomics: case study on colorectal cancer.

We developed a multiplexed label-free quantification strategy, which integrates an efficient gel-assisted digestion protocol, high-performance liquid chromatography tandem MS analysis, and a bioinformatics alignment method to determine personalized proteomic profiles for membrane proteins in human tissues. This strategy provided accurate (6% error) and reproducible (34% relative S.D.) quantification of three independently purified membrane fractions from the same human colorectal cancer (CRC) tissue. Using CRC as a model, we constructed the personalized membrane protein atlas of paired tumor and adjacent normal tissues from 28 patients with different stages of CRC. Without fractionation, this strategy confidently quantified 856 proteins (≥2 unique peptides) across different patients, including the first and robust detection (Mascot score: 22,074) of the well-documented CRC marker, carcinoembryonic antigen 5 by a discovery-type proteomics approach. Further validation of a panel of proteins, annexin A4, neutrophils defensin A1, and claudin 3, confirmed differential expression levels and high occurrences (48-70%) in 60 CRC patients. The most significant discovery is the overexpression of stomatin-like 2 (STOML2) for early diagnostic and prognostic potential. Increased expression of STOML2 was associated with decreased CRC-related survival; the mean survival period was 34.77 ± 2.03 months in patients with high STOML2 expression, whereas 53.67 ± 3.46 months was obtained for patients with low STOML2 expression. Further analysis by ELISA verified that plasma concentrations of STOML2 in early-stage CRC patients were elevated as compared with those of healthy individuals (p < 0.001), suggesting that STOML2 may be a noninvasive serological biomarker for early CRC diagnosis. The overall sensitivity of STOML2 for CRC detection was 71%, which increased to 87% when combined with CEA measurements. This study demonstrated a sensitive, label-free strategy for differential analysis of tissue membrane proteome, which may provide a roadmap for the subsequent identification of molecular target candidates of multiple cancer types.

Detection of annexin A autoantibodies in sera from colorectal cancer patients.

BACKGROUND: Annexins (ANXAs) belong to a superfamily of closely related calcium and membrane-binding proteins and are overexpressed in some carcinomas. The overexpression of ANXAs presumably induces an autoantibody response, but this has not been demonstrated for sera from colorectal cancer (CRC) patients. STUDY: We examined serum samples from 220 CRC patients and 216 healthy volunteers to evaluate the ANXA autoantibody response in patients by using an enzyme-linked immunosorbent assay with recombinant ANXA A4 as the antigen. RESULTS: The sensitivity of the anti-ANXA response from CRC patient sera was about 85% and the specificity was about 61.6%. When a cut-off value of 5.0 ng/mL was chosen for carcinoembryonic antigen (CEA) in the same sera samples, the sensitivity and specificity values were 43.2% and 85.2%, respectively. Combined detection using ANXA autoantibodies and CEA produced better sensitivity (71.8%) and specificity (79.2%) compared with CEA sensitivity (43.2%) and anti-ANXA specificity (61.6%). The area under a receiver operating characteristic curve was 0.794 for ANXA autoantibodies, 0.666 for CEA, and 0.84 for both markers together. Importantly, in comparison to CEA (27.5% seropositivity), ANXA autoantibody showed a remarkable change (81.3% seropositivity) at the early stage of CRC. CONCLUSIONS: Measurement of ANXA autoantibody levels may provide an alternative detection indicator for CRC, particularly among early-stage patients.

Identification of phospholipid scramblase 1 as a biomarker and determination of its prognostic value for colorectal cancer.

The purpose of this study was to examine the expression of phospholipid scramblase 1 (PLSCR1) in tumor tissues and plasma specimens of patients with colorectal cancer (CRC), as well as analyze its association with clinical parameters. The expression levels of PLSCR1 protein in 104 matched CRC and adjacent normal tissue sections and 50 pairs of CRC tissue blocks were determined by use of immunohistochemical and Western blot analyses, respectively. To evaluate the diagnostic potential of PLSCR1, the plasma levels of PLSCR1 were investigated in 111 additional subjects (59 CRC patients and 52 healthy controls) by Western blot. PLSCR1 was overexpressed in malignant adenocarcinoma tissues compared with normal colorectal mucosa (P < 0.001). In addition, the plasma level of PLSCR1 was not only significantly elevated in CRC patients compared with healthy individuals (P < 0.001), but it was also substantially increased in early stage CRC (P < 0.001). Importantly, the overall sensitivity and specificity of PLSCR1 for CRC detection were 80% and 59.6%, respectively. The area under the ROC curve of PLSCR1 for CRC diagnosis is 0.75, which increases to 0.8 if combined with the measurement of carcinoembryonic antigen. Univariate analysis with the Cox regression model revealed that elevated PLSCR1 expression indicated a poor prognosis for CRC. This study showed that PLSCR1 protein levels were significantly elevated in both the cancer tissue and plasma of CRC patients. Moreover, the plasma levels of PLSCR1 were significantly elevated in patients with early stage CRC compared with healthy individuals, suggesting that PLSCR1 might be used as a noninvasive serological diagnostic and prognostic biomarker for CRC.

Comparison of membrane fraction proteomic profiles of normal and cancerous human colorectal tissues with gel-assisted digestion and iTRAQ labeling mass spectrometry.

The aim of this study was to uncover the membrane protein profile differences between colorectal carcinoma and neighboring normal mucosa from colorectal cancer patients. Information from cellular membrane proteomes can be used not only to study the roles of membrane proteins in fundamental biological processes, but also to discover novel targets for improving the management of colorectal cancer patients. We used solvent extraction and a gel-assisted digestion method, together with isobaric tags with related and absolute quantitation (iTRAQ) reagents to label tumoral and adjacent normal tissues in a pairwise manner (n = 8). For high-throughput quantification, these digested labeled peptides were combined and simultaneously analyzed using LC-MS/MS. Using the shotgun approach, we identified a total of 438 distinct proteins from membrane fractions of all eight patients. After comparing protein expression between cancerous and corresponding normal tissue, we identified 34 upregulated and eight downregulated proteins with expression changes greater than twofold (Student's t-test, P < 0.05). Among these, the overexpression of well-established biomarkers such as carcinoembryonic antigens (CEACAM5, CEACAM6), as well as claudin-3, HLA class I histocompatibility antigen A-1, tapasin and mitochondrial solute carrier family 25A4 were confirmed by western blotting. We conclude that gel-assisted digestion and iTRAQ labeling MS is a potential approach for uncovering and comparing membrane protein profiles of tissue samples that has the potential to identify novel biomarkers.

Combined analysis of survivin autoantibody and carcinoembryonic antigen biomarkers for improved detection of colorectal cancer.

BACKGROUND: Survivin is a member of the family of inhibitor of apoptosis proteins that is overexpressed in several human tumors. Previous studies have found that overexpression of survivin in cancer cells induces an antibody response. METHODS: We compared 232 serum samples from colorectal cancer (CRC) patients and 365 samples from healthy volunteers using an in vitro enzyme-linked immunosorbent assay to evaluate the survivin autoantibody response in patients. RESULTS: The sensitivity of the anti-survivin response from patients with CRC was 56.9%, and the specificity was 64.1%. When a cut-off value of 5.0 ng/mL was chosen for carcinoembryonic antigen (CEA) in these same serum samples, the values for sensitivity and specificity were 40.9% and 86.6%, respectively. Combined detection using survivin autoantibodies and CEA produced better sensitivity (51.3%) and specificity (89.9%) compared to the sensitivity of CEA (40.9%) and the specificities of the individual markers (64.1% and 86.6%, respectively). The area under a receiver operating characteristic curve was 0.617 for survivin autoantibodies, 0.630 for CEA and 0.694 for both markers together. CONCLUSIONS: A positive association between autoantibodies against survivin and preoperative CEA concentrations in sera of patients with CRCs was established. Our results suggest that analysis of both parameters would assist in screening patients with CRC.

Identification and clinical association of anti-cytokeratin 18 autoantibody in COPD.

The etiology of chronic obstructive pulmonary disease (COPD) remains unclear. A mechanism involving the autoimmune reaction in the pathogenesis of COPD has been proposed but not confirmed. The aim of this study was to investigate whether serum autoantibodies against pulmonary cellular proteins are present in COPD patients and to identify their autoantigens if possible. Samples from 50 COPD patients and 42 control subjects were studied. Circulating autoantibodies were detected by Western blot. Immunoprecipitation and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry were used to identify the autoantigens. Autoantibodies against pulmonary cellular antigens were found in the sera of COPD patients. Specifically, an autoantibody against the 45-kDa human cytokeratin 18 protein was found in 76.0% of COPD patients and 23.8% of control subjects (p<0.001). Furthermore, the cytokeratin 18 autoantibody level was positively correlated with the FEV(1) (L) (p=0.013) and FEV(1) (%pred.) (p=0.043) values observed in COPD patients. This study identified the pulmonary epithelial cytokeratin 18 protein as a COPD-associated autoantigen and found that anti-cytokeratin 18 autoantibodies were prevalent in COPD patients. Our results support the hypothesis that humoral autoimmunity may be involved in the pathogenesis of COPD.

Multiple serological biomarkers for colorectal cancer detection.

The aim of this study was to initiate a survey of human autoantibody responses to a panel of select colorectal tumor-associated antigens identified by previous serological analysis of a cDNA expression library and to subsequently identify multiple serological biomarkers for the detection of colorectal cancer. For screening of autoantibodies against colorectal tumor-associated antigens, sera from 94 colorectal cancer patients and 54 normal controls were analyzed by enzyme-linked immunosorbent assay using recombinant rCCCAP, rHDAC5, rP53, rNMDAR and rNY-CO-16 proteins as coating antigens. Seropositivity among colorectal cancer patients to the 5 individual coating antigens varied from 18.1% to 35.1%. Seropositivity to any of the 5 coating antigens was 58.5% and combining this analysis with evaluation of serum carcinoembryonic antigen (> or =5 ng/ml) significantly increased the seropositivity to 77.6%. Seropositivity of early-stage (Dukes' Stages A and B) colorectal cancer patients to CEA was 21.9%, and seropositivity to any of the 5 colorectal cancer-associated antigens was 53.7%, and the combination of these 2 measurements resulted in a higher diagnostic capacity (65.9%) than either marker alone. In conclusion, these results collectively indicated that combined detection of serum autoantibody profiles against our panel of colorectal tumor-associated antigens and the analysis of carcinoembryonic antigen provides a promising diagnostic biomarker for colorectal cancer, particularly among early-stage patients.

Cytotoxic effect of recombinant Mycobacterium tuberculosis CFP-10/ESAT-6 protein on the crucial pathways of WI-38 cells.

To unravel the cytotoxic effect of the recombinant CFP-10/ESAT-6 protein (rCFES) on WI-38 cells, an integrative analysis approach, combining time-course microarray data and annotated pathway databases, was proposed with the emphasis on identifying the potentially crucial pathways. The potentially crucial pathways were selected based on a composite criterion characterizing the average significance and topological properties of important genes. The analysis results suggested that the regulatory effect of rCFES was at least involved in cell proliferation, cell motility, cell survival, and metabolisms of WI-38 cells. The survivability of WI-38 cells, in particular, was significantly decreased to 62% with 12.5 microM rCFES. Furthermore, the focal adhesion pathway was identified as the potentially most-crucial pathway and 58 of 65 important genes in this pathway were downregulated by rCFES treatment. Using qRT-PCR, we have confirmed the changes in the expression levels of LAMA4, PIK3R3, BIRC3, and NFKBIA, suggesting that these proteins may play an essential role in the cytotoxic process in the rCFES-treated WI-38 cells.

Quantitative detection of survivin in malignant pleural effusion for the diagnosis and prognosis of lung cancer.

In the present study, pleural effusions are the first time to be used as the specimens for detection of survivin expression in lung cancer patients. We demonstrated that by quantifying survivin expression with enzyme-linked immunosorbent assay (ELISA) in the 80 effusion samples exhibited a diagnostic power of 85% and 75% in sensitivity and specificity, respectively. A multivariate analysis with the Cox regression model revealed that both high survivin expression and cancer cells of stage IV were the indicators for poor prognosis of lung cancer. In conclusion, quantitative assay of survivin in pleural effusion could be useful both in diagnosis and prognosis for lung cancer.

An aberrant autocrine activation of the platelet-derived growth factor alpha-receptor in follicular and papillary thyroid carcinoma cell lines.

Platelet-derived growth factor receptor (PDGFR) can bind to its ligand and consequently possess a kinase activity, and which is associated with the carcinogenesis of different cell types, including astrocytomas, oligodendrogliomas, and glioblastoma. In a cDNA microarray analysis, we observe the over-expressed mRNA of both PDGF-A and PDGF-alpha receptor in thyroid carcinoma cells. And the elevated protein expressions of PDGF-A and PDGF-alpha receptor in thyroid carcinoma cells were also confirmed by a Western blot analysis. The phosphorylation of PDGF-alpha receptor evaluated by an antibody against Tyr 720-phosphate was found in thyroid carcinoma cells. The tyrosine kinase activity of PDGF-alpha receptor was inhibited by tyrphostin AG1295 and showed a dose-dependent inhibition for the proliferation of thyroid carcinoma cells. These findings imply that autocrine activation of PDGF-alpha receptor plays a crucial role in the carcinogenesis of thyroid cells.