Knockdown of Annexin A1 Enhances Radioresistance and Inhibits Apoptosis in Nasopharyngeal Carcinoma.

Radiotherapy is the primary treatment for nasopharyngeal carcinoma while radioresistance can hinder efficient treatment. To explore the role of annexin A1 and its potential mechanisms in radioresistance of nasopharyngeal carcinoma, human nasopharyngeal carcinoma cell line CNE2-sh annexin A1 (knockdown of annexin A1) and the control cell line CNE2-pLKO.1 were constituted and CNE2-sh annexin A1 xenograft mouse model was generated. The effect of annexin A1 knockdown on the growth of xenograft tumor after irradiation and radiation-induced DNA damage and repair was analyzed. The results of immunohistochemistry assays and Western blotting showed that the level of annexin A1 was significantly downregulated in the radioresistant nasopharyngeal carcinoma tissues or cell line compared to the radiosensitive nasopharyngeal carcinoma tissues or cell line. Knockdown of annexin A1 significantly promoted CNE2-sh annexin A1 xenograft tumor growth compared to the control groups after irradiation. Moreover, the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assays revealed that knockdown of annexin A1 significantly inhibited apoptosis in vivo compared to the control groups. We assessed the intracellular reactive oxygen species levels and the extent of radiation-induced DNA damage and repair using reactive oxygen species assay, comet assays, and immunohistochemistry assay. The results showed that knockdown of annexin A1 remarkedly reduced the intracellular reactive oxygen species levels, level of DNA double-strand breaks, and the phosphorylation level of H2AX and increased the accumulation of DNA-dependent protein kinase in nasopharyngeal carcinoma cells after irradiation. The findings suggest that knockdown of annexin A1 inhibits DNA damage via decreasing the generation of intracellular reactive oxygen species and the formation of γ-H2AX and promotes DNA repair via increasing DNA-dependent protein kinase activity and therefore improves the radioresistance in nasopharyngeal carcinoma cells. Together, our findings suggest that knockdown of annexin A1 promotes radioresistance in nasopharyngeal carcinoma and provides insights into therapeutic targets for nasopharyngeal carcinoma radiotherapy.

Dissecting characteristics and dynamics of differentially expressed proteins during multistage carcinogenesis of human colorectal cancer.

AIM: To discover novel biomarkers for early diagnosis, prognosis or treatment of human colorectal cancer. METHODS: iTRAQ 2D LC-MS/MS analysis was used to identify differentially expressed proteins (DEPs) in the human colonic epithelial carcinogenic process using laser capture microdissection-purified colonic epithelial cells from normal colon, adenoma, carcinoma in situ and invasive carcinoma tissues. RESULTS: A total of 326 DEPs were identified, and four DEPs (DMBT1, S100A9, Galectin-10, and S100A8) with progressive alteration in the carcinogenic process were further validated by immunohistochemistry. The DEPs were involved in multiple biological processes including cell cycle, cell adhesion, translation, mRNA processing, and protein synthesis. Some of the DEPs involved in cellular process such as "translation" and "mRNA splicing" were progressively up-regulated, while some DEPs involved in other processes such as "metabolism" and "cell response to stress" was progressively down-regulated. Other proteins with up- or down-regulation at certain stages of carcinogenesis may play various roles at different stages of the colorectal carcinogenic process. CONCLUSION: These findings give insights into our understanding of the mechanisms of colorectal carcinogenesis and provide clues for further investigation of carcinogenesis and identification of biomarkers.

The function and significance of SELENBP1 downregulation in human bronchial epithelial carcinogenic process.

BACKGROUND: Our quantitative proteomic study showed that selenium-binding protein 1 (SELENBP1) was progressively decreased in human bronchial epithelial carcinogenic process. However, there is little information on expression and function of SELENBP1 during human lung squamous cell cancer (LSCC) carcinogenesis. METHODS: iTRAQ-tagging combined with 2D LC-MS/MS analysis was used to identify differentially expressed proteins in the human bronchial epithelial carcinogenic process. SELENBP1, member of selenoproteins family and progressively downregulated in this process, was selected to further study. Both Western blotting and immunohistochemistry were performed to detect SELENBP1 expression in independent sets of tissues of bronchial epithelial carcinogenesis, and ability of SELENBP1 for discriminating NBE (normal bronchial epithelium) from preneoplastic lesions from invasive LSCC was evaluated. The effects of SELENBP1 downregulation on the susceptibility of benzo(a)pyrene (B[a]P)-induced human bronchial epithelial cell transformation were determined. RESULTS: 102 differentially expressed proteins were identified by quantitative proteomics, and SELENBP1 was found and confirmed being progressively decreased in the human bronchial epithelial carcinogenic process. The sensitivity and specificity of SELENBP1 were 80% and 79% in discriminating NBE from preneoplastic lesions, 79% and 82% in discriminating NBE from invasive LSCC, and 77% and 71% in discriminating preneoplastic lesions from invasive LSCC, respectively. Furthermore, knockdown of SELENBP1 in immortalized human bronchial epithelial cell line 16HBE cells significantly increased the efficiency of B[a]P-induced cell transformation. CONCLUSIONS: The present data shows for the first time that decreased SELENBP1 is an early event in LSCC, increases B[a]P-induced human bronchial epithelial cell transformation, and might serve as a novel potential biomarker for early detection of LSCC.

Identification of flotillin-1 as a novel biomarker for lymph node metastasis and prognosis of lung adenocarcinoma by quantitative plasma membrane proteome analysis.

To identify a novel lung adenocarcinoma (AdC) biomarker, iTRAQ-tagging combined with 2D LC-MS/MS analysis was used to identify differentially expressed plasma membrane (PM) proteins in primary lung AdCs and paraneoplastic normal lung tissues (PNLTs). As a result, 36 differentially expressed membrane proteins were identified. Two differential PM proteins flotillin-1 and caveolin-1 were selectively validated by Western blotting. As there has been no report on the association of flotillin-1 with lung AdC, immunohistochemistry was further performed to detect the expression of flotillin-1 in the archival tissue specimens including 42 cases of PNLTs, 62 cases of primary lung AdCs with lymph node metastasis (LNM AdCs), and 46 cases of primary lung AdCs without lymph node metastasis (non-LNM AdCs), and the correlation of flotillin-1 expression levels in lung AdCs with clinicopathological features and clinical outcomes were evaluated. The results showed that up-regulation of flotillin-1 expression in lung AdCs was significantly correlated with advanced clinical stage, lymph node metastasis, increased postoperative relapse and decreased overall survival. Cox regression analysis revealed that the expressional level of flotillin-1 was an independent prognostic factor. The data suggest that flotillin-1 is a potential novel biomarker for lymph node metastasis and prognosis of lung AdC, and flotillin-1 up-regulation might play an important role in the pathogenesis of lung AdC.

Identification of heat shock protein 27 as a radioresistance-related protein in nasopharyngeal carcinoma cells.

PURPOSE: To identify the proteins involved in radioresistance in nasopharyngeal cancer (NPC) cells. METHODS: Sublethal ionizing radiation was applied to establish a radioresistant NPC cell line from its parental NPC cell line CNE1. Clonogenic survival assay, cell growth assay and flow cytometry analysis were used to examine the difference of radiosensitivity in the radioresistant CNE1 cells (CNE1-IR) and control CNE1 cells. Comparative proteomics was performed to identify the differential proteins in the two cell lines. Association of HSP27, one of upregulated proteins in CNE1-IR cells, with NPC cell radioresistance was selected for further investigation using antisense oligonucleotides (ASOs), clonogenic survival assay, Hoechst 33258 staining of apoptotic cells and MTT assay of cell viability. RESULTS: Radioresistant NPC cell line CNE1-IR derived from its parental cell line CNE1 was established. Thirteen differential proteins in the CNE1-IR and CNE1 cells were identified by proteomics, and differential expression of HSP27, one of identified proteins, was selectively confirmed by western blot. Inhibition of HSP27 expression by HSP27 ASOs decreased clonogenic survival and cell viability and increased cell apoptosis of CNE1-IR cells after irradiation, that is, enhanced radiosensitivity of CNE1-IR cells. CONCLUSION: The data suggest that HSP27 is a radioresistant protein in NPC cells, and its upregulation may be involved in the NPC radioresistance.

Proteomic and bioinformatics analyses of mouse liver microsomes.

Microsomes are derived mostly from endoplasmic reticulum and are an ideal target to investigate compound metabolism, membrane-bound enzyme functions, lipid-protein interactions, and drug-drug interactions. To better understand the molecular mechanisms of the liver and its diseases, mouse liver microsomes were isolated and enriched with differential centrifugation and sucrose gradient centrifugation, and microsome membrane proteins were further extracted from isolated microsomal fractions by the carbonate method. The enriched microsome proteins were arrayed with two-dimensional gel electrophoresis (2DE) and carbonate-extracted microsome membrane proteins with one-dimensional gel electrophoresis (1DE). A total of 183 2DE-arrayed proteins and 99 1DE-separated proteins were identified with tandem mass spectrometry. A total of 259 nonredundant microsomal proteins were obtained and represent the proteomic profile of mouse liver microsomes, including 62 definite microsome membrane proteins. The comprehensive bioinformatics analyses revealed the functional categories of those microsome proteins and provided clues into biological functions of the liver. The systematic analyses of the proteomic profile of mouse liver microsomes not only reveal essential, valuable information about the biological function of the liver, but they also provide important reference data to analyze liver disease-related microsome proteins for biomarker discovery and mechanism clarification of liver disease.

Comparative proteome analysis of human lung squamous carcinoma using two different methods: two-dimensional gel electrophoresis and iTRAQ analysis.

Discovery of early-diagnosis biomarkers is the key to improve the early-diagnosis and prognosis of human lung squamous carcinoma (hLSC). In order to identify more exhaustive and systematic protein biomarkers for early-diagnosis of hLSC, we chose LCM purifed cells from hLSC tissues and paired normal bronchial epithelia(NBE) tissues and used two methods, the classical 2-DE/MS approach and the new iTRAQ analysis. We found a total of 63 differential proteins, 22 proteins in 2-DE and 59 proteins in iTRAQ analysis, between hLSC and NBE tissues. Among them, 18 proteins were quantified using both methods. The expression level of 15 proteins (68.2%) in 2-DE was consistent with that in iTRAQ analysis. Series of proteins involved in cytoskeleton, chaperone, GTP binding, metabolic process, cell apoptosis, cell proliferation and differentiation, signal transduction, transcription and translation were identified, suggesting their possible role in the emergence of oncogenic pathways leading to carcinogenesis of hLSC. These proteins may make as potential biomarkers for diagnosis of hLSC. The two methods gave us closely related but different information about proteins, suggesting they are complementary or at least supplementary methods at present. Our results show both the usefulness of iTRAQ reagent technology for identification of further potential marker proteins as well as for prevalidation of biomarker.

Identification of candidate biomarkers for early detection of human lung squamous cell cancer by quantitative proteomics.

To discover novel biomarkers for early detection of human lung squamous cell cancer (LSCC) and explore possible mechanisms of LSCC carcinogenesis, iTRAQ-tagging combined with two dimensional liquid chromatography tandem MS analysis was used to identify differentially expressed proteins in human bronchial epithelial carcinogenic process using laser capture microdissection-purified normal bronchial epithelium (NBE), squamous metaplasia (SM), atypical hyperplasia (AH), carcinoma in situ (CIS) and invasive LSCC. As a result, 102 differentially expressed proteins were identified, and three differential proteins (GSTP1, HSPB1 and CKB) showing progressively expressional changes in the carcinogenic process were selectively validated by Western blotting. Immunohistochemistry was performed to detect the expression of the three proteins in an independent set of paraffin-embedded archival specimens including various stage tissues of bronchial epithelial carcinogenesis, and their ability for early detection of LSCC was evaluated by receiver operating characteristic analysis. The results showed that the combination of the three proteins could perfectly discriminate NBE from preneoplastic lesions (SM, AH and CIS) from invasive LSCC, achieving a sensitivity of 96% and a specificity of 92% in discriminating NBE from preneoplatic lesions, a sensitivity of 100% and a specificity of 98% in discriminating NBE from invasive LSCC, and a sensitivity of 92% and a specificity of 91% in discriminating preneoplastic lesions from invasive LSCC, respectively. Furthermore, we knocked down GSTP1 in immortalized human bronchial epithelial cell line 16HBE cells, and then measured their susceptibility to carcinogen benzo(a)pyrene-induced cell transformation. The results showed that GSTP1 knockdown significantly increased the efficiency of benzo(a)pyrene-induced 16HBE cell transformation. The present data first time show that GSTP1, HSPB1 and CKB are novel potential biomarkers for early detection of LSCC, and GSTP1 down-regulation is involved in human bronchial epithelial carcinogenesis.

Quantitative proteomic analysis identifying three annexins as lymph node metastasis-related proteins in lung adenocarcinoma.

Lymph node status is a strong predictor of outcome for lung adenocarcinoma (AdC) patients. To explore novel potential protein markers for predicting lymph node metastasis of lung AdC, differential proteomic analysis on microdissected cancer cells from primary lung AdC and matched lymph node (LN) metastatic tissues by laser capture microdissection (LCM) was conducted using two-dimensional differential in-gel electrophoresis (2D-DIGE) coupled with mass spectrometry (MS). Annexins including annexin-1, annexin-2 and annexin-3 were identified and found to be overexpressed in matched LN metastatic tissues compared to primary lung AdC. Furthermore, differential expression levels of the three annexins were evaluated in paraffin-embedded 188 primary lung AdC tissues and 65 matched positive lymph node specimens using immunohistochemistry. High expression of annexin-1, annexin-2, and annexin-3 was all frequently observed in matched positive lymph node tissues compared to primary lung AdC. In primary lung AdC, expression levels of the three annexins in primary lymph node-positive AdC tissues were higher than primary lymph node-negative AdC tissues. Multivariate logistic regression analysis indicated annexin-1, annexin-2, and annexin-3 were all significant risk factors for lymph node metastasis. Furthermore, statistical analysis indicated that the concomitant expression of annexin-1/annexin-2, annexin-1/annexin-3, or annexin-2/annexin-3 and combined expression of all three markers had stronger correlation with lymph node metastasis. Our results suggest that annexin-1, annexin-2, and annexin-3 are identified as potential biomarkers associated with lymph node metastasis in lung AdC.

Identification of GLIPR1 tumor suppressor as methylation-silenced gene in acute myeloid leukemia by microarray analysis.

PURPOSE: To identify methylation-silenced genes in acute myeloid leukemia (AML). METHODS: Microarray analyses were performed in AML cell line HL-60 cells exposed to the demethylating agent 5-aza-2dC. The methylation status and expression of glioma pathogenesis-related protein 1 (GLIPR1), one of highly induced genes by demethylation, were further detected in six hematopoietic malignancy cell lines and 260 bone marrow samples from leukemia patients and nonmalignant diseases as control, as well as pre-treated and post-treated bone marrow samples from 24 complete remission AML patients received chemotherapy using MS-PCR, bisulfite DNA sequencing, RT-PCR, and Western blotting. RESULTS: One hundred and nine genes were significantly induced by demethylation in HL-60 cells, 12 genes of which were confirmed by RT-PCR. GLIPR1, a tumor suppressor gene, was frequently methylation-silenced in AML cell lines and AML patients, but not in the other hematopoietic malignancy cell lines and patients. The frequencies of methylation-silenced GLIPR1 in the pre-treatment were significantly higher than those in the post-treatment in complete remission AML patients. CONCLUSION: We identify 109 genes induced by demethylation in HL-60 cells, and demonstrate that GLIPR1 is a methylation-silenced gene in the AML patients, and may serve as a marker for monitoring disease activity during therapy in the AML patients. The data provide the important information for studying the pathogenesis of AML and discovering the target genes of methylating agents.

Analysis of EGFR signaling pathway in nasopharyngeal carcinoma cells by quantitative phosphoproteomics.

BACKGROUND: The epidermal growth factor receptor (EGFR) is usually overexpressed in nasopharyngeal carcinoma (NPC) and is associated with pathogenesis of NPC. However, the downstream signaling proteins of EGFR in NPC have not yet been completely understood at the system level. The aim of this study was identify novel downstream proteins of EGFR signaling pathway in NPC cells. RESULTS: We analyzed EGFR-regulated phosphoproteome in NPC CNE2 cells using 2D-DIGE and mass spectrometry analysis after phosphoprotein enrichment. As a result, 33 nonredundant phosphoproteins including five known EGFR-regulated proteins and twenty-eight novel EGFR-regulated proteins in CNE2 were identified, three differential phosphoproteins were selectively validated, and two differential phosphoproteins (GSTP1 and GRB2) were showed interacted with phospho-EGFR. Bioinformatics analysis showed that 32 of 33 identified proteins contain phosphorylation modification sites, and 17 identified proteins are signaling proteins. GSTP1, one of the EGFR-regulated proteins, associated with chemoresistance was analyzed. The results showed that GSTP1 could contribute to paclitaxel resistance in EGF-stimulated CNE2 cells. Furthermore, an EGFR signaling network based on the identified EGFR-regulated phosphoproteins were constructed using Pathway Studio 5.0 software, which includes canonical and novel EGFR-regulated proteins and implicates the possible biological roles for those proteins. CONCLUSION: The data not only can extend our knowledge of canonical EGFR signaling, but also will be useful to understand the molecular mechanisms of EGFR in NPC pathogenesis and search therapeutic targets for NPC.

Activation of EGFR promotes squamous carcinoma SCC10A cell migration and invasion via inducing EMT-like phenotype change and MMP-9-mediated degradation of E-cadherin.

EGFR is a potent stimulator of invasion and metastasis in head and neck squamous cell carcinomas (HNSCC). However, the mechanism by which EGFR may stimulate tumor cell invasion and metastasis still need to be elucidated. In this study, we showed that activation of EGFR by EGF in HNSCC cell line SCC10A enhanced cell migration and invasion, and induced loss of epitheloid phenotype in parallel with downregulation of E-cadherin and upregulation of N-cadherin and vimentin, indicating that EGFR promoted SCC10A cell migration and invasion possibly by an epithelial to mesenchymal transition (EMT)-like phenotype change. Interestingly, activation of EGFR by EGF induced production of matrix metalloproteinase-9 (MMP-9) and soluble E-cadherin (sE-cad), and knockdown of MMP-9 by siRNA inhibited sE-cad production induced by EGF in SCC10A. Moreover, both MMP-9 knockdown and E-cadherin overexpression inhibited cell migration and invasion induced by EGF in SCC10A. The results indicate that EGFR activation promoted cell migration and invasion through inducing MMP-9-mediated degradation of E-cadherin into sE-cad. Pharmacologic inhibition of EGFR, MEK, and PI3K kinase activity in SCC10A reduced phosphorylated levels of ERK-1/2 and AKT, production of MMP-9 and sE-cad, cell migration and invasion, and expressional changes of EMT markers (E-cadherin and N-cadherin) induced by EGF, indicating that EGFR activation promotes cell migration and invasion via ERK-1/2 and PI3K-regulated MMP-9/E-cadherin signaling pathways. Taken together, the data suggest that EGFR activation promotes HNSCC SCC10A cell migration and invasion by inducing EMT-like phenotype change and MMP-9-mediated degradation of E-cadherin into sE-cad related to activation of ERK-1/2 and PI3K signaling pathways.

Identification of annexin A1 as a proinvasive and prognostic factor for lung adenocarcinoma.

Metastasis is the most common cause of death in lung cancer patients and is a major obstacle to the successful treatment. To discover novel metastasis-related proteins in lung adenorcinoma (AdC), quantitative proteomic analysis was performed between primary lung AdC tissues with (LNM AdC) and without lymph node metastasis (non-LNM AdC). In this study, annexin A1 was identified to be significantly up-regulated in LNM AdC compared with non-LNM AdC. Immunohistochemistry showed that annexin A1 over-expression was frequently observed in LNM AdCs and matched lymph node metastases compared with non-LNM AdCs. Annexin A1 over-expression was significantly associated with advanced clinical stage (P < 0.05) and lymph node metastasis (P < 0.05) and increased relapse rate (P < 0.05) and decreased overall survival (P < 0.05) in lung AdCs. Cox regression analysis indicated annexin A1 over-expression was an independent prognostic factor. Furthermore, suppression of annexin A1 expression by siRNA interference significantly inhibited the invasion ability of lung adenocarcinoma cell A549 in vitro. In conclusion, annexin A1 expression correlated with tumor stage, lymph node metastasis, relapse, and patient survival. Annexin A1 is proposed to function importantly in the progression of lung AdC.

Identification of the proteins related to p53-mediated radioresponse in nasopharyngeal carcinoma by proteomic analysis.

Radiotherapy is the primary treatment for nasopharyngeal cancer (NPC), and p53 is closely associated with the radiosensitivity of cancer, but the molecular mechanisms of p53-mediated radioresponse in NPC remains unclear. We previously established NPC CNE2sip53 cell line with p53 knockdown and paired control cell line CNE2/pSUPER, which provides a cell model system to investigate mechanisms of p53-mediated radioresponse in NPC. In this study, we first compared the radiosensitivity of CNE2sip53 and CNE2/pSUPER by a clonogenic survival assay, cell growth assay, and Hoechst 33258 staining and flow cytometry analysis of apoptotic cells. The results showed that the radiosensitivity of CNE2sip53 was significantly lower than that of CNE2/pSUPER, indicating that p53 plays a role in mediating NPC radiosensitivity. To search for the proteins associated with the p53-mediated radioresponse in NPC, a proteomic approach was performed to identify the radioresponsive proteins in CNE2sip53 and CNE2p/SUPER, respectively, and then the difference of radioresponsive proteins in CNE2sip53 and CNE2p/SUPER was compared. As a result, 14 differential radioresponsive proteins were identified in the two cell lines, 4 proteins of which were conformed by Western blot. Among them, 9 and 5 proteins were identified solely from CNE2p/SUPER and CNE2sip53, respectively. Furthermore, protein-protein interaction analysis showed that 7 differential radioresponsive proteins identified only in CNE2p/SUPER were related to p53 protein. Our results suggest that the differential radioresponsive proteins unique to CNE2p/SUPER may be involved in p53-mediated radioresponse in NPC, which will be helpful for elucidating the mechanisms of p53-mediated NPC cellular response to radiotherapy.

Enhancing the stability of ¹8O-labeled peptides through removal of immobilized trypsin by ZipTips.

Trypsin-catalyzed ¹8O labeling is increasingly used in shotgun proteomics for relative peptide/protein quantitation. However, precise quantitative measurements are often complicated by the instability of ¹8O-labeled peptides caused mainly by oxygen back-exchange. Although a number of attempts have been made to reduce or prevent oxygen back-exchange, there is still room for improvement. Here we demonstrate that the removal of immobilized trypsin by filtration using ZipTips can efficiently minimize oxygen back-exchange and enhance the stability of ¹8O-labeled peptides under various pH conditions. The ¹8O-labeled peptides processed by the approach were successfully separated by immobilized pH gradient-isoelectric focusing (IPG-IEF), and no marked decrease in the extent of labeling was observed. The results also demonstrated that there was no correlation between the extent of ¹8O labeling and molecular weight or isoelectric point (pI). The approach presented here is especially applicable to microscale samples. Its ability to generate stably ¹8O-labeled samples without back-exchange should expand the application scope of the ¹8O-labeling technique.

Identification of the amyloid ß-protein precursor and cystatin C as novel epidermal growth factor receptor regulated secretory proteins in nasopharyngeal carcinoma by proteomics.

The epidermal growth factor receptor (EGFR) is usually overexpressed in nasopharyngeal carcinoma (NPC) and is associated with pathogenesis of NPC. However, while EGFR-modulated intracellular proteins have been extensively studied, little is known concerning their extracellular counterparts. To identify EGFR-regulated secreted proteins in NPC, we compared the secretome profiles of TGF-α-stimulated and unstimulated NPC cell line CNE-2. CNE-2 cells were cultured in the absence or presence of TGF-α for 24 h, and secreted proteins were obtained from conditioned serum-free media and enriched by ultrafiltration centrifugation. Using 2-DE and subsequent mass spectrometry, we identified 16 differential secreted proteins, among which the amyloid ß-protein precursor (APP) was up-regulated and cystatin C was down-regulated after TGF-α stimulation. We further showed that the secretory changes of APP and cystatin C in CNE-2 after TGF-α stimulation could be abrogated by pretreatment of EGFR tyrosine kinase inhibitor PD153035 and PI3 kinase inhibitor Wortmannin, validating that APP and cystatin C are EGFR-regulated secreted proteins in NPC cells. Immunohistochemistry showed that the expression level of EGFR was positively correlated with the expression level of APP and negatively correlated with the expression level of cystatin C in NPC tissues, indicating that EGFR also regulates expression of APP and cystatin C in clinical NPC tissues. Furthermore, functional analysis showed that the growth and migration of CNE-2 cells decreased after neutralization of secretory APP in the medium using the anti-APP antibody. Our data provide substantial evidence that APP and cystatin C are target secreted proteins of EGFR in NPC, and upregulation of secretory APP by EGFR may be involved in the pathogenesis of NPC.

Raf kinase inhibitor protein correlates with sensitivity of nasopharyngeal carcinoma to radiotherapy.

Raf kinase inhibitory protein (RKIP) is a metastasis suppressor whose expression is reduced in nasopharyngeal carcinoma (NPC) tissues and is absent in NPC metastases. To investigate the effect of RKIP on radiosensitivity of NPC, high metastatic 5-8F with low RKIP expression and non-metastatic 6-10B with high RKIP expression were stably transfected with plasmids that expressed sense and antisense RKIP cDNA. Overexpression of RKIP sensitized 5-8F cells to radiation-induced cell death, G(2)-M cell cycle arrest and apoptosis. In contrast, downexpression of RKIP in 6-10B cells protected cells from radiation-induced cell death, G(2)-M cell cycle arrest and apoptosis. In addition, RKIP expression altered the radiosensitivity of NPC cells through MEK and ERK phosphorylation changes of Raf-1/MEK/ERK signaling pathway. We further investigated the RKIP expression in NPC patients and its association with patients' survival after radiotherapy. Downexpression of RKIP was significantly correlated with advanced clinical stage, lymph node metastasis and radioresistance. Furthermore, survival curves showed that patients with RKIP downexpression had a poor prognosis and induced relapse. Multivariate analysis confirmed that RKIP expression was an independent prognostic indicator. The data suggested that RKIP was a potential biomarker for the radiosensitivity and prognosis of NPC, and its dysregulation might play an important role in the radioresistance of NPC.

Identification of biomarkers for predicting nasopharyngeal carcinoma response to radiotherapy by proteomics.

Radiotherapy is the primary treatment for nasopharyngeal cancer (NPC), but radioresistance remains a serious obstacle to successful treatment in many cases. To identify the proteins involved in this resistance and to evaluate their potential for predicting NPC response to radiotherapy, we first established a radioresistant subclone cell line (CNE2-IR) derived from NPC cell line CNE2 by treating the cells with five rounds of sublethal ionizing radiation. Proteomics was then performed to compare the protein profiles of CNE2-IR and CNE2, and a total of 34 differential proteins were identified. Among them, 14-3-3sigma and Maspin were downregulated and GRP78 and Mn-SOD were upregulated in the radioresistant CNE2-IR compared with control CNE2, which was conformed by Western blot. Immunohistochemistry was performed to detect the expression of the four validated proteins in the 39 radioresistant and 51 radiosensitive NPC tissues and their value for predicting NPC response to radiotherapy were evaluated by receiver operating characteristic analysis. The results showed that the downregulation of 14-3-3sigma and Maspin and the upregulation of GRP78 and Mn-SOD were significantly correlated with NPC radioresistance and the combination of the four proteins achieved a sensitivity of 90% and a specificity of 88% in discriminating radiosensitive from radiaoresistant NPC. Furthermore, the resistance to ionizing radiation can be partially reversed by the overexpression of 14-3-3sigma in the CNE2-IR. The data suggest that 14-3-3sigma, Maspin, GRP78, and Mn-SOD are potential biomarkers for predicting NPC response to radiotherapy and their dysregulation may be involved in the radioresistance of NPC.

Identification of tyrosine phosphoproteins in signaling pathway triggered TGF-a by using functional proteomics technology.

Although several groups had conducted proteomics of nasopharyngeal carcinoma (NPC), little study was involved in phosphoproteomics, oncogenic signaling, and cancer research about NPC. Analysis of phosphotyrosine proteins from transforming growth factor alpha (TGF-a) triggered phosphotyrosine proteome permitted the identification of novel downstream substrates of the epidermal growth factor receptor (EGFR). Using functional proteomics technology based on 2-DE, 2-D western blotting, and mass spectrometry, we identified and quantified the tyrosine phosphorylation levels of 16 proteins between control and TGF-a-treated CNE2 human NPC cells. Among these proteins, tyrosine phosphorylated levels of ten proteins were increased, and those of six proteins were decreased in TGF-a-treated CNE2 cells compared with control. In addition, among these identified proteins, ANXA3, KRT8, and KRT18 were validated to be novel tyrosine-phosphorylation targets of EGFR signaling by IP-western blotting and part of a complex EGFR phosphotyrosine signaling network. These novel findings will provide new insights into the complex EGFR phosphorylation signaling and may have implications in molecular cancer therapy of NPC.

Identification of Rack1, EF-Tu and Rhodanese as aging-related proteins in human colonic epithelium by proteomic analysis.

The aging process of human colonic epithelium involves a slow decline in physiological vigor and an increasing susceptibility to age-related diseases, especially, colon cancer, but the mechanisms still remain to be elucidated. To reveal the molecular bases of colonic epithelial aging, a proteomic approach was used to screen for differential proteins in the human normal colonic epithelial tissues from young and old people. As a result, 17 differential proteins were identified by two-dimensional electrophoresis and mass spectrometry, and the partial differential proteins were confirmed by immunohistochemistry. Rack1, EF-Tu and Rhodanese, three validated differential proteins, were further investigated for their role in the in vitro cell senescence. Western blot showed that the expression of all the three proteins was downregulated in the senescent NIH/3T3 cells induced by D-galactose as compared to the control cells. Furthermore, knockdown of Rack1 by siRNA could promote NIH/3T3 cell senescence. Taken together, our results suggest that Rack1, EF-Tu and Rhodanese are aging-related proteins in human colonic epithelium, and injury of mitochondrial function and decline of antioxidant capability are important reasons for the aging of human colonic epithelium.