Cheng AL, Huang WG, Chen ZC, Zhang PF, Li MY, Li F, Li JL, Li C, Yi H, Peng F, Duan CJ, Xiao ZQIdentifying cathepsin D as a biomarker for differentiation and prognosis of nasopharyngeal carcinoma by laser capture microdissection and proteomic analysis. J Proteome Res 7: 2415-2426
In this study, we applied laser capture microdissection and a proteomic approach to identify novel nasopharyngeal carcinoma (NPC) biomarkers. Proteins from pooled microdissected NPC and normal nasopharyngeal epithelial tissues (NNET) were separated by two-dimensional gel electrophoresis, and differential proteins were identified by mass spectrometry. Expression of the differential protein cathepsin D in the above two tissues as well as four NPC cell lines was determined by Western blotting. Next, siRNA was used to inhibit the expression of cathepsin D in highly metastatic NPC cell line 5-8F to examine whether it associates with NPC metastasis. Immunohistochemistry was also performed to detect the expression of cathepsin D in 72 cases of primary NPC, 28 cases of NNET, and 20 cases of cervical lymph node metastases, and the correlation of its expression level with clinicopathologic features and clinical outcomes were evaluated. Thirty-six differential proteins between the NPC and NNET were identified. The expression level of cathepsin D in the two types of tissues was confirmed by Western blotting and related to differentiation degree and metastatic potential of the NPC cell lines. Down-regulated cathepsin D expression by siRNA significantly decreased in vitro invasive ability of 5-8F cells. Significant cathepsin D down-regulation was observed in NPC versus NNET, whereas significant cathepsin D up-regulation was observed in lymph node metastasis versus primary NPC. In addition, cathepsin D down-regulation was significantly correlated with poor histological differentiation, whereas cathepsin D up-regulation was significantly correlated with advanced clinical stage, recurrence, and lymph node and distant metastasis. Furthermore, survival curves showed that patients with cathepsin D up-regulation had a poor prognosis. Multivariate analysis confirmed that cathepsin D expression was an independent prognostic indicator. The data suggest that cathepsin D is a potential biomarker for the differentiation and prognosis of NPC, and its dysregulation might play an important role in the pathogenesis of NPC.
"It is upregulated in some malignant tumor metastases , gastric cancer with lymphatic and/or blood vessel invasion , breast cancer , , colorectal cancer , , liver cancer  and pancreatic cancer . Furthermore, Cheng et al  observed significant CTSD upregulation in lymph node metastasis versus primary NPC, which was significantly correlated with advanced clinical stage, recurrence, and lymph node and distant metastasis. "
[Show abstract][Hide abstract] ABSTRACT: Nasopharyngeal carcinoma (NPC) has a high metastatic character in the clinic, but its mechanism is not clear. As a carcinogen with organ specificity for the nasopharyngeal epithelium, N,N'-Dinitrosopiperazine (DNP) is involved in NPC metastasis. Herein, our data revealed that anterior gradient 2 (AGR2) was overexpressed in human NPC tissues, particularly in cervical lymph node metastatic NPC (LMNPC). High AGR2 expression was associated with NPC metastasis. Importantly, DNP induced AGR2 expression, and increased cell motility and invasion in the NPC cell line 6-10B. However, DNP-mediated cell motility and invasion was dramatically decreased when transfected with siRNA-AGR2. Further, AGR2 directly regulated cathepsin (CTS) B and D by binding them in vitro. These results indicate that DNP induces AGR2 expression, regulates CTSB and CTSD, increases cell motility and invasion, and promotes NPC tumor metastasis. Therefore, DNP-mediated AGR2 expression may be an important factor in prolific NPC metastasis.
PLoS ONE 05/2014; 9(4):e92081. DOI:10.1371/journal.pone.0092081 · 3.23 Impact Factor
"It is up-regulated in metastasis of some malignant tumors, including primary laryngeal cancers correlated with neck lymph node involvement , gastric cancer with lymphatic and/or blood vessel invasion , and breast cancer. Furthermore, Cheng et al.  found that significant cathepsin D expression occurred in lymph node metastasis versus primary NPC and was significantly correlated with advanced clinical stage, recurrence, and lymph node and distant metastasis. AGR2 was reported to be linked with several human cancers and induced metastasis . "
[Show abstract][Hide abstract] ABSTRACT: Background
Nasopharyngeal carcinoma (NPC) has a high metastatic feature. N,N′-Dinitrosopiperazine (DNP) is involved in NPC metastasis, but its mechanism is not clear. The aim of this study is to reveal the pathogenesis of DNP-involved metastasis. 6-10B cells with low metastasis are from NPC cell line SUNE-1, were used to investigate the mechanism of DNP-mediated NPC metastasis.
6-10B cells were grown in DMEM containing 2H4-L-lysine and 13C 6 15 N4-L-arginine or conventional L-lysine and L-arginine, and identified the incorporation of amino acid by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Labeled 6-10B cells were treated with DNP at 0 -18 μM to establish the non-cytotoxic concentration (NCC) range. NCC was 0 -10 μM. Following treatment with DNP at this range, the motility and invasion of cells were detected in vitro, and DNP-mediated metastasis was confirmed in the nude mice. DNP increased 6-10B cell metastasis in vitro and vivo. DNP-induced protein expression was investigated using a quantitative proteomic. The SILAC-based approach quantified 2698 proteins, 371 of which showed significant change after DNP treatment (172 up-regulated and 199 down-regulated proteins). DNP induced the change in abundance of mitochondrial proteins, mediated the status of oxidative stress and the imbalance of redox state, increased cytoskeletal protein, cathepsin, anterior gradient-2, and clusterin expression. DNP also increased the expression of secretory AKR1B10, cathepsin B and clusterin 6-10B cells. Gene Ontology and Ingenuity Pathway analysis showed that DNP may regulate protein synthesis, cellular movement, lipid metabolism, molecular transport, cellular growth and proliferation signaling pathways.
DNP may regulate cytoskeletal protein, cathepsin, anterior gradient-2, and clusterin expression, increase NPC cells motility and invasion, is involved NPC metastasis.
"The detailed approach of two-dimensional gel electrophoresis (2-DE), image analysis and mass spectrometer (MS) analysis was described by Cheng et al (20). Briefly, 10 pairs of radioresistant and radiosensitive NPC tissues were dissolved in lysis buffer (7 mol/l urea, 2 mol/l thiourea, 100 mmol/l DTT, 4% CHAPS, 0.5 mmol/l EDTA, 40 mmol/l Tris, 2% NP40, 1% Triton X-100, 5 mmol/l PMSF, and 2% phamarlyte) at 4˚C for 1 h. "
[Show abstract][Hide abstract] ABSTRACT: Radioresistance continues to be a major problem in the treatment of nasopharyngeal carcinoma (NPC). This study aimed to identify novel proteins associated with NPC radio-resistance. We used a mass spectrometry driven-proteomic strategy to identify novel proteins associated with NPC radio-resistance, and differential proteins were subsequently processed by bio-informatic analysis. As a result, twelve proteins were identified with aberrant expression in radioresistant (RR) NPC tissues compare to radiosensitive (RS) NPC tissues. Among these proteins, ERp29, Mn-SOD, HSP27 and GST ω1 were found to be significantly up-regulated in RR NPC tissues, and ERp29 was selected for further validation. Immunohistochemistry analysis confirmed that ERp29 was overexpressed in RR NPC tissues compared with RS NPC tissues. To prove the role of ERp29 in the induction of NPC radioresistance, ERp29 was down-regulated in the ERp29 enriched NPC cells CNE-1 and 6-10B by specific shRNA. Radiosensitivity was measured using cell proliferation assay and clonogenic survival assay, and cell apoptosis was measured using flow cytometric analysis. We found that ERp29 knockdown attenuated CNE-1 and 6-10B cell radioresistance and enhanced cell apoptosis. These results suggest that ERp29 associates with radioresistance in NPC, and ERp29 could be a potential biomarker for predicting NPC response to radiotherapy.
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