Chih-Ching Wu

Subcellular localization of Photofrin® determines the death phenotype of human epidermoid carcinoma A431 cells triggered by photodynamic therapy: When plasma membranes are the main targets

Photodynamic therapy (PDT) is a kind of photochemo‐therapeutic treatment that exerts its effect mainly through the induction of cell death. Distinct types of cell death may be elicited by different PDT regimes. In this study, the mechanisms involved in the death of human epidermoid carcinoma A431 cells triggered by PDT with Photofrin® (a clinically approved photosensitizer) were characterized. Photofrin distributes dynamically in A431 cells; the plasma membranes and Golgi complex are the main target sites of Photofrin after a brief (3 h) and prolonged (24 h) incubation, respectively. Cells with differentially localized Photofrin displayed distinct death phenotypes in response to PDT. The effects of PDT on cells with plasma membrane‐localized Photofrin were further studied in details. Cells stopped proliferating post PDT at Photofrin dose >7 μg/ml, and at higher dose (28 μg/ml) plasma membrane disruption and cell swelling were observed immediately after PDT. Dramatic alterations of several important signaling events were detected in A431 cells post Photofrin®‐PDT, including (i) immediate formation of reactive oxygen species (ROS), (ii) rapid activation of c‐Jun N‐terminal kinase, (iii) delayed activation of caspase‐3 and cleavage of polyADP‐ribose polymerase and p21‐activated kinase 2, and (iv) loss of mitochondrial membrane potential. Intriguingly, the characteristics of typical apoptosis such as phosphatidylserine externalization and DNA fragmentation were not detected in the cell death process caused by this PDT regime. In conclusion, our results show that when plasma membranes are the main targets, Photofrin‐PDT can lead to instant ROS formation and subsequent activation of downstream signaling events similar to those elicited by many apoptotic stimuli, but the damage of plasma membranes renders the death phenotype more necrosis like.

Candidate Serological Biomarkers for Cancer Identified from the Secretomes of 23 Cancer Cell Lines and the Human Protein Atlas

Although cancer cell secretome profiling is a promising strategy used to identify potential body fluid-accessible cancer biomarkers, questions remain regarding the depth to which the cancer cell secretome can be mined and the efficiency with which researchers can select useful candidates from the growing list of identified proteins. Therefore, we analyzed the secretomes of 23 human cancer cell lines derived from 11 cancer types using one-dimensional SDS-PAGE and nano-LC-MS/MS performed on an LTQ-Orbitrap mass spectrometer to generate a more comprehensive cancer cell secretome. A total of 31,180 proteins was detected, accounting for 4,584 non-redundant proteins, with an average of 1,300 proteins identified per cell line. Using protein secretion-predictive algorithms, 55.8% of the proteins appeared to be released or shed from cells. The identified proteins were selected as potential marker candidates according to three strategies: (i) proteins apparently secreted by one cancer type but not by others (cancer type-specific marker candidates), (ii) proteins released by most cancer cell lines (pan-cancer marker candidates), and (iii) proteins putatively linked to cancer-relevant pathways. We then examined protein expression profiles in the Human Protein Atlas to identify biomarker candidates that were simultaneously detected in the secretomes and highly expressed in cancer tissues. This analysis yielded 6–137 marker candidates selective for each tumor type and 94 potential pan-cancer markers. Among these, we selectively validated monocyte differentiation antigen CD14 (for liver cancer), stromal cell-derived factor 1 (for lung cancer), and cathepsin L1 and interferon-induced 17-kDa protein (for nasopharyngeal carcinoma) as potential serological cancer markers. In summary, the proteins identified from the secretomes of 23 cancer cell lines and the Human Protein Atlas represent a focused reservoir of potential cancer biomarkers.

Curcumin inhibits UV irradiation-induced oxidative stress and apoptotic biochemical changes in human epidermoid carcinoma A431 cells.

Ultraviolet (UV) light is a strong apoptotic trigger that induces caspase‐dependent biochemical changes in cells. Previously we showed that UV irradiation can activate caspase‐3, and the subsequent cleavage and activation of p21Cdc42/Rac‐activated kinase 2 (PAK2) in human epidermoid carcinoma A431 cells. In this study we demonstrate that curcumin (Cur), the yellow pigment of Curcuma longa with known anti‐oxidant and anti‐inflammatory properties, can prevent UV irradiation‐induced apoptotic changes, including c‐Jun N‐terminal kinase (JNK) activation, loss of mitochondrial membrane potential (MMP), mitochondrial release of cytochrome C, caspase‐3 activation, and cleavage/activation of PAK2 in A431 cells. Flow cytometric analysis using the cell permeable dye 2′,7′‐dichlorofluorescin diacetate (DCF‐DA) as an indicator of reactive oxygen species (ROS) generation revealed that the increase in intracellular oxidative stress caused by UV irradiation could be abolished by Cur. In addition, we found that SP600125, a JNK‐specific inhibitor, reduced UV irradiation‐induced JNK activation as well as caspase‐3 activation, indicating that JNK activity is required for UV irradiation‐induced caspase activation. Collectively, our results demonstrate that Cur significantly attenuates UV irradiation‐induced ROS formation, and suggest that ROS triggers JNK activation, which in turn causes MMP change, cytochrome C release, caspase activation, and subsequent apoptotic biochemical changes. J. Cell. Biochem. 90: 327–338, 2003.

Multiplexed quantification of 63 proteins in human urine by multiple reaction monitoring-based mass spectrometry for discovery of potential bladder cancer biomarkers.

Three common urological diseases are bladder cancer, urinary tract infection, and hematuria. Seventeen bladder cancer biomarkers were previously discovered using iTRAQ - these findings were verified by MRM-MS in this current study. Urine samples from 156 patients with hernia (n=57, control), bladder cancer (n=76), or urinary tract infection/hematuria (n=23) were collected and subjected to multiplexed LC-MRM/MS to determine the concentrations of 63 proteins that are normally considered to be plasma proteins, but which include proteins found in our earlier iTRAQ study. Sixty-five stable isotope-labeled standard proteotypic peptides were used as internal standards for 63 targeted proteins. Twelve proteins showed higher concentrations in the bladder cancer group than in the hernia and the urinary tract infection/hematuria groups, and thus represent potential urinary biomarkers for detection of bladder cancer. Prothrombin had the highest AUC (0.796), with 71.1% sensitivity and 75.0% specificity for differentiating bladder cancer (n=76) from non-cancerous (n=80) patients. The multiplexed MRM-MS data was used to generate a six-peptide marker panel. This six-peptide panel (afamin, adiponectin, complement C4 gamma chain, apolipoprotein A-II precursor, ceruloplasmin, and prothrombin) can discriminate bladder cancer subjects from non-cancerous subjects with an AUC of 0.814, with a 76.3% positive predictive value, and a 77.5% negative predictive value. This article is part of a Special Section entitled: Understanding genome regulation and genetic diversity by mass spectrometry.

Identification of collapsin response mediator protein‐2 as a potential marker of colorectal carcinoma by comparative analysis of cancer cell secretomes

The cancer cell secretome may contain many potentially useful biomarkers. We therefore sought to identify proteins in the conditioned media of colorectal carcinoma (CRC) cell lines but not in those from other cancer cell lines. The secretomes of 21 cancer cell lines derived from 12 cancer types were analyzed by SDS‐PAGE combined with MALDI‐TOF MS. Among the 325 proteins identified, collapsin response mediator protein‐2 (CRMP‐2) was chosen for evaluation as a potential CRC biomarker, since it was selectively detected in the CRC cell line secretome and has never been reported as a cancer biomarker. Immunohistochemical analysis of 169 CRC specimens showed that CRMP‐2 was positively detected in 58.6% of the tumors, but weakly or not detected in >90% of the adjacent nontumor epithelial cells. Moreover, the CRMP‐2‐positive rate was significantly increased in earlier stage tumors and lymph node metastasis. Plasma CRMP‐2 levels were significantly higher in CRC patients (N = 201) versus healthy controls (N = 201) (61.3 ± 34.6 vs. 40.2 ± 24.3 ng/mL, p = 0.001). Our results indicate that comparative analysis of cancer cell secretome is a feasible strategy for identifying potential cancer biomarkers, and that CRMP‐2 may be a novel CRC biomarker.

Comparative and Targeted Proteomic Analyses of Urinary Microparticles from Bladder Cancer and Hernia Patients

Bladder cancer is a common urologic cancer whose incidence continues to rise annually. Urinary microparticles are an attractive material for noninvasive bladder cancer biomarker discovery. In this study, we applied isotopic dimethylation labeling coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to discover bladder cancer biomarkers in urinary microparticles isolated from hernia (control) and bladder cancer patients. This approach identified 2964 proteins based on more than two distinct peptides, of which 2058 had not previously been reported as constituents of human urine exosomes/microparticles. A total of 107 differentially expressed proteins were identified as candidate biomarkers. Differences in the concentrations of 29 proteins (41 signature peptides) were precisely quantified by LC-MRM/MS in 48 urine samples of bladder cancer, hernia, and urinary tract infection/hematuria. Concentrations of 24 proteins changed significantly (p<0.05) between bladder cancer (n=28) and hernia (n=12), with area-under-the-curve values ranging from 0.702 to 0.896. Finally, we quantified tumor-associated calcium-signal transducer 2 (TACSTD2) in raw urine specimens (n=221) using a commercial ELISA and confirmed its potential value for diagnosis of bladder cancer. Our study reveals a strong association of TACSTD2 with bladder cancer and highlights the potential of human urinary microparticles in the noninvasive diagnosis of bladder cancer.

Discovery of novel bladder cancer biomarkers by comparative urine proteomics using iTRAQ technology.

A urine sample preparation workflow for the iTRAQ (isobaric tag for relative and absolute quantitation) technique was established. The reproducibility of this platform was evaluated and applied to discover proteins with differential levels between pooled urine samples from nontumor controls and three bladder cancer patient subgroups with different grades/stages (a total of 14 controls and 23 cancer cases in two multiplex iTRAQ runs). Combining the results of two independent clinical sample sets, a total of 638 urine proteins were identified. Among them, 55 proteins consistently showed >2-fold differences in both sample sets. Western blot analyses of individual urine samples confirmed that the levels of apolipoprotein A-I (APOA1), apolipoprotein A-II, heparin cofactor 2 precursor and peroxiredoxin-2 were significantly elevated in bladder cancer urine specimens (n = 25-74). Finally, we quantified APOA1 in a number of urine samples using a commercial ELISA and confirmed again its potential value for diagnosis (n = 126, 94.6% sensitivity and 92.0% specificity at a cutoff value of 11.16 ng/mL) and early detection (n = 71, 83.8% sensitivity and 94.0% specificity). Collectively, our results provide the first iTRAQ-based quantitative profile of bladder cancer urine proteins and represent a valuable resource for the discovery of bladder cancer markers.

Importin subunit alpha-2 is identified as a potential biomarker for non-small cell lung cancer by integration of the cancer cell secretome and tissue transcriptome

The cancer cell secretome may contain potentially useful biomarkers. In this study, we integrated the profiles of secreted proteins in lung cancer cell lines with mRNA expression levels from pulmonary adenocarcinoma tissue, with a view to identify effective biomarkers for non‐small cell lung cancer (NSCLC). Among the novel candidates isolated, importin subunit alpha‐2 (also known as karyopherin subunit alpha [KPNA]‐2), was selected for further validation. Immunohistochemical staining revealed overexpression of KPNA2 in the nuclei of tumor cells, compared with adjacent normal cells. A sandwich ELISA assay developed to detect KPNA2 levels in serum samples showed significantly higher serum KPNA2 in NSCLC patients than in healthy controls. A combination of serum KPNA2 and carcinoembryonic antigen displayed higher diagnostic capacity than either marker alone. Importantly, protein levels of KPNA2 in pleural effusion from NSCLC patients were also significantly higher than those from non‐lung cancer. Moreover, knockdown of KPNA2 inhibited the migration ability and viability of lung cancer cells. Our results collectively suggest that integration of the cancer cell secretome and transcriptome datasets provides an efficient means of identifying novel biomarkers for NSCLC, such as KPNA2.

Identification of potential bladder cancer markers in urine by abundant-protein depletion coupled with quantitative proteomics.

UNLABELLED In this study, we evaluated the reproducibility of abundant urine protein depletion by hexapeptide-based library beads and an antibody-based affinity column using the iTRAQ technique. The antibody-based affinity-depletion approach, which proved superior, was then applied in conjunction with iTRAQ to discover proteins that were differentially expressed between pooled urine samples from hernia and bladder cancer patients. Several proteins, including seven apolipoproteins, TIM, SAA4, and proEGF were further verified in 111 to 203 individual urine samples from patients with hernia, bladder cancer, or kidney cancer. Six apolipoproteins (APOA1, APOA2, APOB, APOC2, APOC3, and APOE) were able to differentiate bladder cancer from hernia. SAA4 was significantly increased in bladder cancer subgroups, whereas ProEGF was significantly decreased in bladder cancer subgroups. Additionally, the combination of SAA4 and ProEGF exhibited higher diagnostic capacity (AUC=0.80 and p<0.001) in discriminating bladder cancer from hernia than either marker alone. Using MetaCore software to interpret global changes of the urine proteome caused by bladder cancer, we found that the most notable alterations were in immune-response/alternative complement and blood-coagulation pathways. This study confirmed the clinical significance of the urine proteome in the development of non-invasive biomarkers for the detection of bladder cancer. BIOLOGICAL SIGNIFICANCE In this study, we evaluated the reproducibility of abundant urine protein depletion by hexapeptide-based library beads and an antibody-based affinity column using the iTRAQ technique. The antibody-based affinity-depletion approach, which proved superior, was then applied in conjunction with iTRAQ to discover proteins that were differentially expressed between pooled urine samples from hernia and bladder cancer patients. Several proteins, including seven apolipoproteins, TIM, SAA4, and proEGF were further verified in 111 to 203 individual urine samples from patients with hernia, bladder cancer, or kidney cancer. SAA4 was significantly increased in bladder cancer subgroups, whereas ProEGF was significantly decreased in bladder cancer subgroups. Additionally, the combination of SAA4 and ProEGF exhibited higher diagnostic capacity in discriminating bladder cancer from hernia than either marker alone. A marker panel composed by two novel biomarker candidates, SAA4 and proEGF, was first discovered and verified successfully using Western blotting. To the best of our knowledge, the associations of urinary SAA4 and proEGF with bladder tumor and kidney cancer have not been mentioned before. In the present study, we discovered and verified SAA4 and proEGF as potential bladder cancer biomarker for the first time.

Discovery of Retinoblastoma-Associated Binding Protein 46 as a Novel Prognostic Marker for Distant Metastasis in Nonsmall Cell Lung Cancer by Combined Analysis of Cancer Cell Secretome and Pleural Effusion Proteome

Nonsmall cell lung cancer (NSCLC) is the most common type of lung cancer, which is one of the most prominent causes of cancer-related mortality worldwide. Discovery of serum tumor markers could facilitate early NSCLC detection and metastatic prognosis. Here, we simultaneously analyzed the NSCLC cell secretome and proteomic profiles of pleural effusion from lung adenocarcinoma patients for NSCLC biomarker discovery. Retinoblastoma-associated binding protein 46 (RbAp46), one of the proteins detected both in NSCLC cell secretome and pleural effusion proteome, was chosen for further evaluation. Both of RbAp46 mRNA and protein levels were upregulated significantly in NSCLC cancer tissues. Serum levels of RbAp46 were markedly higher in NSCLC patients than in healthy controls, and a combination of RbAp46 and CEA could outperform CEA alone in discriminating NSCLC patients from healthy persons. Importantly, elevated serum RbAp46 level was highly correlated with NSCLC distant metastasis. Moreover, knockdown of RbAp46 inhibited the migration ability of lung cancer cells. Our data collectively suggest that RbAp46 serves as a novel biomarker and prognosticator for NSCLC, and is involved in lung cancer cell migration.