Carboxyl terminal activating region 3 of latent membrane protein 1 encoded by the Epstein­Barr virus regulates cell proliferation and protein expression in NP69 cells.

In the present study, the mechanism by which carboxyl terminal activating region 3 (CTAR3) of latent membrane protein 1 (LMP1), encoded by the Epstein­Barr virus, regulated cell proliferation and protein expression was investigated in the nasopharyngeal epithelial cell line NP69. The deletion mutant LMP1 (LMP1Δ232­351; amino acid residues including 232­351 codons in CTAR3 deleted) was generated by polymerase chain reaction. An NP69­LMP1Δ232­351 cell line was established by retroviral infection. Finally, cell proliferation and protein expression of NP69 cells expressing LMP1Δ232­351 were examined using a cell growth curve and western blot analysis. The results demonstrated: i) The proliferation of NP69­LMP1Δ232­351 cells was significantly decreased compared with cells expressing wild type LMP1 (LMP1WT; n=3; P<0.05); ii) 17 proteins exhibited differential protein expression (>2­fold change) in NP69­LMP1Δ232­351 cells compared with NP69­LMP1WT cells; and iii) LMP1WT was involved in activating the Janus kinase 3 (JAK3) promoter and regulating the expression of JAK3 protein, while LMP1Δ232­351 was almost defective in ability to activate the JAK promoter. These results suggested that LMP1­CTAR3 may be an important functional domain for regulating cell proliferation and protein expression in nasopharyngeal epithelial cells.

MiR-101-3p inhibits EMT to attenuate proliferation and metastasis in glioblastoma by targeting TRIM44.

BACKGROUND: Glioblastoma (GBM) is the most common malignant tumor originating in the brain parenchyma. The invasive and infiltrative properties of glioblastoma result in poor clinical prognosis to conventional therapies. Emerging reports on microRNAs as important regulators during the process of EMT provide new insights into treating glioblastoma through new targets. However, underlying molecular mechanism of the regulation of miR-101-3p in glioblastoma remains unclear. METHODS: Level of miR-101-3p was determined in GBM cell lines by qRT-PCR. MTT, colony formation and transwell assays were utilized to evaluate functions of overexpression of miR-101-3p/knock down of TRIM44 on proliferation, migration and invasion in GBM cells. Direct interaction between miR-101-3p and TRIM44 was validated using dual luciferase reporter system and impacts of overexpression of miR-101-3p/knock down of TRIM44 on regulation of EMT markers were assessed by Western blotting. RESULTS: MiR-101-3p was validated to be repressed expressed in glioblastoma cancer cell lines. Both overexpression of miR-101-3p and knock down of TRIM44 attenuated proliferation, migration and invasion of glioblastoma cell lines in vitro. TRIM44 was shown to promote EMT in GBM progress and reverse inhibitory function of miR-101-3p. MiR-101-3p was found to suppress the expression of TRIM44 via directly targeting its 3'UTR. CONCLUSIONS: Our findings suggested miR-101-3p regulated proliferation and migration of glioblastoma cells through attenuating TRIM44 induced EMT via direct targeting 3'UTR of TRIM44, which provided preliminary study of potential therapeutic target in future GBM treatment.

Analysis of Differential Expressions of Long Non-coding RNAs in Nasopharyngeal Carcinoma Using Next-generation Deep Sequencing.

Background: Little knowledge about long non-coding RNAs(lncRNAs) in nasopharyngeal carcinoma (NPC) has been acquired. Methods: Next-generation sequencing was applied in 7 cases of NPC tissues and 7 cases of normal tissues in nasopharynx. PLEX, CNCI and CPAT soft-wares were used to predict novel lncRNAs. Real-time Quantitative PCR (qPCR) further validated the data in 20 cases of NPC tissues and 14 cases of normal tissues. Then the cis-regulators and trans-regulators and potential biological functions together with pathways were predicted by Bioinformatics. Results: Totally, 4248 novel lncRNAs were found to be expressed in our samples. And 2192 lncRNAs and 23342 mRNAs were considered to be differentially expressed in NPC. Among the results, 306 lncRNAs and 4599 mRNAs were significantly up-regulated, whereas 204 lncRNAs and 2059 mRNAs were significantly down-regulated, respectively. Moreover, 62 lncRNAs trans-regulated genes were involved in Epstein-Barr virus (EBV) infection pathway in our study. Jun proto-oncogene (JUN), which was related to a cis-regulator lncRNA RP4-794H19.1, was enriched in cancers and involved in Tumor Necrosis Factor (TNF) signaling pathway, might play a key role in NPC. Conclusion: These findings broadened the lncRNAs landscape of NPC tissues and shed light on the roles of these lncRNAs, which might be conducive to the comprehensive management of NPC.

Ginnalin A from Kujin tea (Acer tataricum subsp. ginnala) exhibits a colorectal cancer chemoprevention effect via activation of the Nrf2/HO-1 signaling pathway.

Ginnalin A (also known as acertannin) is one of the most important phenolic compounds of several beverage Acer plants. In this study, it is reported for the first time that ginnalin A is an activator of the Nrf2 signaling pathway in human colon cancer cells. Ginnalin A, isolated from the leaves of Acer tataricum subsp. ginnala, exhibited promising preventive activity against colon cancer cells (HCT116, SW480 and SW620) with IC50 values of 24.8 µM, 22.0 µM and 39.7 µM, respectively. In addition, it significantly reduced the colony formation of these cells. Flow cytometry analysis indicated that ginnalin A suppressed cancer proliferation via the induction of cell cycle arrest at the S-phase. Real time PCR analysis demonstrated that ginnalin A can upregulate the mRNA expression levels of Nrf2-related antioxidant genes Nrf2, HO-1 and NQO1. Western blotting analysis revealed that ginnalin A promoted the Nrf2 nuclear translocation and upregulated the proteins Nrf2, HO-1 and NQO1. Moreover, the upregulation of p62 and the inhibition of Keap1 were also found by Western blotting analysis. Therefore, the activation of the Nrf2 signaling pathway was probably induced through the upregulation of p62 and the inhibition of Keap1.

Flotilin-1 promotes the tumorigenicity and progression of malignant phenotype in human lung adenocarcinoma.

Lung adenocarcinoma (LUAD) accounts for the most common histological subtype of lung cancer which remains the leading cause of cancer death worldwide. The discovery of more sensitive and specific novel target biomarkers for predicting the development and progression of LUAD is imperative. Flotillin-1 (Flot-1) has been reported to have important roles in the progression of several tumor types but not been reported in the progression of LUAD. Here, we demonstrated that the expression of flotillin-1 was upregulated in 5 LUAD cells. Moreover, multiple approaches were used to explore the tumorigenicity of flotillin-1 in LUAD cell lines. The expression levels of flotillin-1 were analyzed by immunoblotting after overexpression and siRNA-based knockdown. Cell proliferation, scratch wound healing, transwell migration and matrigel invasion and xenograft tumor growth assays were used to determine the role of flotillin-1 in LUAD progression. Downregulation of flotillin-1 reversed, whereas upregulation of flotillin-1 enhanced, the malignant phenotype of LUAD cells in vitro. Consistently, cells with flotillin-1 knockdown formed smaller tumors in nude mice than cells transfected with the empty vector. Furthermore, the control group demonstrated significantly more tumorigenic effects compared to the flotillin-1-silenced group in the xenograft model of LUAD. In all, there draws a conclusion that flotillin-1 is a tumorigenic protein that plays an important role in promoting the proliferation and tumorigenicity of LUAD, suggesting that flotillin-1 may represent a novel the therapeutic target to LUAD.

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.

Identification of integrin ß1 as a prognostic biomarker for human lung adenocarcinoma using 2D-LC-MS/MS combined with iTRAQ technology.

To discover novel lung adenocarcinoma (AdC) biomarkers, isobaric tags for relative and absolute quantitation (iTRAQ)-tagging combined with 2D-LC-MS/MS analysis was used to identify differentially expressed plasma membrane proteins in lung AdC and paired paraneoplastic normal lung tissues (PNLTs) adjacent to tumors. In this study, significant caveolin-1 downregulation and integrin ß1 upregulation was observed in primary lung AdC vs. PNLT. As there has been no report on the association of integrin ß1 with lung AdC, immunohistochemical staining was performed to detect the expression of integrin ß1 in an independent set of archival tissue specimens including 42 cases of PLNT, 46 cases of without lymph node metastasis primary AdC (non-LNM AdC) and 62 cases of LNM AdC; the correlation of their expression levels with clinicopathological characteristics and clinical outcomes were evaluated. Based on the data, upregulation of integrin ß1 was significantly correlated with advanced clinical stage and lymph node metastasis. Integrin ß1 overexpression was significantly associated with advanced clinical stage (P<0.05), lymph node metastasis (P<0.05), increased relapse rate (P<0.05) and decreased overall survival (P<0.05) in AdCs. Cox regression analysis indicated that integrin ß1 overexpression is an independent prognostic factor. The data suggest that integrin ß1 is a potential biomarker for LNM and prognosis of AdC and integrin ß1 upregulation may play an important role in the pathogenesis of AdC.

Quantitative proteomic study of human lung squamous carcinoma and normal bronchial epithelial acquired by laser capture microdissection.

OBJECTIVE: To investigate the differential protein profile of human lung squamous carcinoma (HLSC) and normal bronchial epithelium (NBE) and provide preliminary results for further study to explore the carcinogenic mechanism of HLSC. METHODS: Laser capture microdissection (LCM) was used to purify the target cells from 10 pairs of HLSC tissues and their matched NHBE, respectively. A stable-isotope labeled strategy using iTRAQ, followed by 2D-LC/Q-STAR mass spectrometry, was performed to separate and identify the differential expression proteins. RESULTS: A total of 96 differential expression proteins in the LCM-purified HLSC and NBE were identified. Compared with NBE, 49 proteins were upregulated and 47 proteins were downregulated in HLSC. Furthermore, the expression levels of the differential proteins including HSPB1, CKB, SCCA1, S100A8, as well as S100A9 were confirmed by western blot and tissue microarray and were consistent with the results of quantitative proteomics. CONCLUSION: The different expression proteins in HLSC will provide scientific foundation for further study to explore the carcinogenic mechanism of HLSC.

STGC3 inhibits xenograft tumor growth of nasopharyngeal carcinoma cells by altering the expression of proteins associated with apoptosis.

STGC3 is a potential tumor suppressor that inhibits the growth of the nasopharyngeal carcinoma cell line CNE2; the expression of this protein is reduced in nasopharyngeal carcinoma compared with normal nasopharyngeal tissue. In this study, we investigated the tumor-suppressing activity of STGC3 in nude mice injected subcutaneously with Tet/pTRE-STGC3/CNE2 cells. STGC3 expression was induced by the intraperitoneal injection of doxycycline (Dox). The volume mean of Tet/pTRE-STGC3/CNE2+Dox xenografts was smaller than that of Tet/pTRE/CNE2+Dox xenografts. In addition, Tet/pTRE-STGC3/CNE2+Dox xenografts showed an increase in the percentage of apoptotic cells, a decrease in Bcl-2 protein expression and an increase in Bax protein expression. A proteomic approach was used to assess the protein expression profile associated with STGC3-mediated apoptosis. Western blotting confirmed the differential up-regulation of prohibitin seen in proteomic analysis. These results indicate that overexpression of STGC3 inhibits xenograft growth in nude mice by enhancing apoptotic cell death through altered expression of apoptosis-related proteins such as Bcl-2, Bax and prohibitin. These data contribute to our understanding of the function of STGC3 in human nasopharyngeal carcinoma and provide new clues for investigating other STGC3-associated tumors.

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.

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.

STGC3 inhibits xenograft tumor growth of nasopharyngeal carcinoma cells by altering the expression of proteins associated with apoptosis

STGC3 is a potential tumor suppressor that inhibits the growth of the nasopharyngeal carcinoma cell line CNE2; the expression of this protein is reduced in nasopharyngeal carcinoma compared with normal nasopharyngeal tissue. In this study, we investigated the tumor-suppressing activity of STGC3 in nude mice injected subcutaneously with Tet/pTRE-STGC3/CNE2 cells. STGC3 expression was induced by the intraperitoneal injection of doxycycline (Dox). The volume mean of Tet/pTRE-STGC3/CNE2+Dox xenografts was smaller than that of Tet/pTRE/CNE2+Dox xenografts. In addition, Tet/pTRE-STGC3/CNE2+Dox xenografts showed an increase in the percentage of apoptotic cells, a decrease in Bcl-2 protein expression and an increase in Bax protein expression. A proteomic approach was used to assess the protein expression profile associated with STGC3-mediated apoptosis. Western blotting confirmed the differential up-regulation of prohibitin seen in proteomic analysis. These results indicate that overexpression of STGC3 inhibits xenograft growth in nude mice by enhancing apoptotic cell death through altered expression of apoptosis-related proteins such as Bcl-2, Bax and prohibitin. These data contribute to our understanding of the function of STGC3 in human nasopharyngeal carcinoma and provide new clues for investigating other STGC3-associated tumors.

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.

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.

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.

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.

Identification of Galectin-1 as a novel biomarker in nasopharyngeal carcinoma by proteomic analysis.

Nasopharyngeal carcinoma (NPC) is one of the most common malignant tumors in southern China and Southeast Asia. It is characterized as a multistep process involved in multiple genetic and epigenetic events. The mechanism of carcinogenesis still needs to be further clarified. In this study, two-dimensional gel electrophoresis, matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS), RT-PCR, Western blot and immunohistochemical (IHC) analyses were used to detect Galectin-1 expression in NPC compared with normal nasopharyngeal epithelial tissues (NNET). We found that Galectin-1 was expressed at a significantly higher level in NPC compared with NNET. Our results indicated that high expression level of Galectin-1 might correlate with the development of NPC and Galectin-1 may serve as a potential diagnostic marker or therapeutic target for 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.