Identificating cathepsin D as a biomarker for differentiation and prognosis of nasopharyngeal carcinoma by laser capture microdissection and proteomic analysis.
ABSTRACT 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.
- [show abstract] [hide abstract]
ABSTRACT: This study investigated the association between the results of nasopharyngeal (NPX) biopsies and clinical manifestations in patients with suspected nasopharyngeal carcinoma (NPC). Retrospective cohort study. Four hundred seventy-three patients with 512 NPX biopsies were enrolled. The statistical analyses were conducted to evaluate clinical significance and screening performance for suspected NPC. The negative rate of all NPX biopsies was 69.7% (345/495), and the majority of the noncancerous group revealed lymphoid hyperplasias (208/345, 60.3%). The three diagnostic capabilities of NPX mass, epistaxis (EPI), neck mass (NM), EPI-NM, EPI-NPX mass, NM-NPX mass, and EPI-NM-NPX mass were 0.595, 0.557, 0.735, 0.609, 0.566, 0.748, and 0.600, respectively. Although NPX mass, EPI, and NM were significant to identify suspected NPC, the diagnostic capabilities of combining EPI, NM, and NPX were still low. A large number of noncancerous biopsy results were obtained due to lymphoid hyperplasias often being mistaken as NPC.The Laryngoscope 07/2012; 122(9):1988-93. · 1.98 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Recently, the tumor microenvironment is increasingly recognized as playing an important role in cancer proliferation, invasion, and metastasis. To screen stroma-associated proteins involved in nasopharyngeal carcinoma (NPC) carcinogenesis, laser capture microdissection (LCM) and quantitative proteomic analysis were employed to assess different protein expression of the stroma between NPC and normal nasopharyngeal mucosa (NNM). In this study, periostin was identified to be significantly up-regulated in NPC stroma compared with NNM stroma and the result was further confirmed by Western blotting. Immunohistochemistry showed that over-expression of periostin was frequently observed in the stroma of NPC and matched lymph node metastases (LNM) compared with the stroma of NNM. Statistical analysis showed over-expression of periostin was significantly associated with advanced clinical stage (P < 0.001) and lymph node metastasis (P < 0.001) and decreased overall survival (P < 0.001) in NPC. Cox regression analysis indicated over-expression of periostin was an independent prognostic factor. Furthermore, ectopic expression of periostin was used to examine its effect on invasiveness of NPC cell in vitro and the result showed that periostin was able to promote invasiveness of NPC cell. In conclusion, periostin expression is correlated with tumor stage, lymph node metastasis, and patient survival. Periostin is a potential biomarker for the differentiation and prognosis of NPC, and it might play an important role in the progression of NPC.Clinical and Experimental Metastasis 06/2012; · 3.46 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: BACKGROUND: Nasopharyngeal carcinoma (NPC) has a high metastatic feature. N,N[prime]-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. RESULTS: 6-10B cells were grown in DMEM containing 2H4-L-lysine and 13C615N4-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 muM to establish the non-cytotoxic concentration (NCC)range. NCC was 0 -10 muM. 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. CONCLUSION: DNP may regulate cytoskeletal protein, cathepsin, anterior gradient-2, and clusterin expression, increase NPC cells motility and invasion, is involved NPC metastasis.BMC Biochemistry 11/2012; 13(1):25. · 1.78 Impact Factor