Identification of the proteins related to p53-mediated radioresponse in nasopharyngeal carcinoma by proteomic analysis.
ABSTRACT 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.
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ABSTRACT: Curcumin, one of the main bioactive components extracted from a traditional Chinese medicinal herb, exhibits potent anticancer activity against many types of cancer cells including nasopharyngeal carcinoma (NPC). However, the detailed molecular mechanism underlying this is not clearly understood. In this study, we showed that curcumin significantly inhibited the growth of NPC cells in a dose- and time-dependent manner as determined by MTT assays, while increasing apoptosis was also observed as measured by flow cytometry for the FITC-Annexin V and propidium iodide (PI) label and Hoechst 33258 staining. To further explore the potential mechanism, we showed that curcumin increased the phosphorylation of ERK1/2 but not p38 MAPK in a time-dependent manner, and induced protein expression of the tumor suppressors FOXO3a and p53 in a dose‑dependent manner, which were not observed in the presence of PD98059, an inhibitor of ERK1/2. Furthermore, silencing of FOXO3a and p53 genes by siRNAs overcame the inhibitory effect of curcumin on cell proliferation. Silencing or blockade of p53 using siRNA or chemical inhibitor abrogated the effect of curcumin on expression of FOXO3a protein; silencing or overexpression of FOXO3a had no further effect on curcumin-induced p53 protein expression. Furthermore, blockade of ERK1/2 and exogenous expression of FOXO3a restored the effect of curcumin on growth of cells. Together, our studies show that curcumin inhibits growth and induces apoptosis of NPC cells through ERK1/2-mediated increase in the protein expression and interaction of p53 and FOXO3a. p53 is upstream of FOXO3a, which form a regulatory loop that mediates the effect of curcumin. This study unveils a new mechanism by which curcumin inhibits the proliferation and induces apoptosis of human NPC cells.International Journal of Oncology 05/2014; · 2.66 Impact Factor
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ABSTRACT: The purpose of this study was to identify miRNAs and genes involved in nasopharyngeal carcinoma (NPC) radioresistance, and explore the underlying mechanisms in the development of radioresistance. We used microarrays to compare the differences of both miRNA and mRNA expression profiles in the radioresistant NPC CNE2-IR and radiosensitive NPC CNE2 cells, applied qRT-PCR to confirm the reliability of microarray data, adopted databases prediction and anticorrelated analysis of miRNA and mRNA expression to identify the miRNA target genes, and employed bioinformatics tools to examine the functions and pathways in which miRNA target genes are involved, and construct a miRNA-target gene regulatory network. We further investigated the roles of miRNA-23a and its target gene IL-8 in the NPC radioresistance. THE MAIN FINDINGS WERE FOURFOLD: (1) fifteen differential miRNAs and 372 differential mRNAs were identified, and the reliability of microarray data was validated for randomly selected eight miRNAs and nine genes; (2) 174 miRNA target were identified, and most of their functions and regulating pathways were related to tumor therapeutic resistance; (3) a posttranscriptional regulatory network including 375 miRNA-target gene pairs was constructed, in which the ten genes were coregulated by the six miRNAs; (4) IL-8 was a direct target of miRNA-23a, the expression levels of IL-8 were elevated in the radioresistant NPC tissues and showed inverse correlation with miRNA-23a expression, and genetic upregulation of miRNA-23a and antibody neutralization of secretory IL-8 could reduce NPC cells radioresistance. We identified fifteen differential miRNAs and 372 differential mRNAs in the radioresistant NPC cells, constructed a posttranscriptional regulatory network including 375 miRNA-target gene pairs, discovered the ten target genes coregulated by the six miRNAs, and validated that downregulated miRNA-23a was involved in NPC radioresistance through directly targeting IL-8. Our data form a basis for further investigating the mechanisms of NPC radioresistance.PLoS ONE 01/2014; 9(1):e87767. · 3.53 Impact Factor
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ABSTRACT: Nasopharyngeal carcinoma (NPC) is one of the most common malignant tumors in southern China and southern Asia, and poses one of the most serious public health problems in these areas. Early diagnosis, predicting metastasis, recurrence, prognosis and therapeutic response of NPC remain a challenge. Discovery of diagnostic and predictive biomarkers is an ideal way to achieve these objectives. Proteomics has great potential in identifying cancer biomarkers. Comparative proteomics has identified a large number of potential biomarkers associated with NPC, although the clinical performance of such biomarkers needs to be further validated. In this article, we review the latest discovery and progress of biomarkers for early diagnosis, predicting metastasis, recurrence, prognosis and therapeutic response of NPC, inform the readers of the current status of proteomics-based NPC biomarker findings and suggest avenues for future work.Expert Review of Proteomics 03/2014; · 3.90 Impact Factor