Analysis of the differential secretome of nasopharyngeal carcinoma cell lines CNE-2R and CNE-2

Abstract

Radioresistance is the major cause of poor prognosis in nasopharyngeal carcinoma (NPC). To identify and characterize the secretome associated with NPC radioresistance, we compared the conditioned serum-free medium of radioresistant CNE-2R cells with that of the parental radiosensitive CNE-2 cells using isobaric tags for relative and absolute quantitation (iTRAQ) with liquid chromatography-electrospray tandem mass spectrometry (LC-ESI-MS/MS) quantitative proteomics. Before proceeding to quantitative proteomics, we investigated the survival curves of CNE-2R and CNE-2 cells by colony formation assay, and the CNE-2R survival curves were significantly higher than those for CNE-2. In total, 3,581 proteins were identified in the quantitative proteomics experiments, and 40 proteins exhibited significant differences between the CNE-2R and CNE-2 cells. Twenty-six of the 40 proteins were secreted by classical, non-classical, or exosomal secretion pathways. To verify the reliability of iTRAQ quantitative proteomics, we applied western blotting (WB) to study the secretory protein expression of fibrillin-2, CD166, sulfhydryl oxidase 1 and cofilin-2, which are involved in cell adhesion, migration and invasion. The WB results showed that fibrillin-2 (p=0.017) and sulfhydryl oxidase 1 (p=0.000) were highly expressed in the CNE-2 cells, while CD166 (p=0.012) and cofilin-2 (p=0.003) were highly expressed in the CNE-2R cells, which was in accordance with iTRAQ quantitative proteomics. Finally, a phenotypic subset of CD166-positive NPC cells was verified by immunocytochemistry. In summary, we defined a collection of secretory proteins that may be relevant to the radioresistance in NPC cells, and we determined that CD166, which is widely used as a positive marker of cancer stem cells, is expressed in NPC cells.

Reduced QSOX1 enhances radioresistance in nasopharyngeal carcinoma

“…To identify biological markers that can predict the response of NPC to radiotherapy, in a previous study, we analyzed the profiles of secretory proteins in NPC cell lines with variable radiosensitivities using the quantitative iTRAQ method [ 8 ]. The resultant data identified 26 differentially secreted proteins, including QSOX1, that displayed a dramatic difference in expression between the radiosensitive and radioresistant NPC cells.…”

“…Cells were cultured in RPMI-1640 medium (HyClone, Logan, UT, USA) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA) and 1% penicillin/streptomycin (Beyotime Bio, China) (100 μg /mL), at 37°C in a humidified incubator with 5% CO 2 . The preparation of conditioned media (CM) was performed as previously described [ 8 ]. For lentiviral infection, CNE-2 cells were cultured with QSOX1-shRNA-encoded lentivirus (QSOX1-shRNA group) or empty vector-encoded lentivirus (NC group) at a multiplicity of infection (MOI) of 40 for 48 h. To assess the transduction efficiency, the expression of GFP was evaluated using an inverted fluorescence microscope (Olympus, Tokyo, Japan).…”

“…In our previous study, we analyzed the total secretory protein profiles in NPC cell lines with variable radiosensitivities and compared the conditioned serum-free medium of radioresistant CNE-2R cells with that of the parental radiosensitive CNE-2 cells using isobaric tags for relative and absolute quantitation (iTRAQ) with liquid chromatography-electrospray tandem mass spectrometry (LC-ESI-MS/MS) quantitative proteomics and found that the expression of quiescin sulfhydryl oxidase 1 (QSOX1) was significantly different between these cell lines [ 8 ]. Therefore, QSOX1 may be associated with the radiosensitivity of NPC.…”

Serum CD166: A novel biomarker for predicting nasopharyngeal carcinoma response to radiotherapy

“…The proteomic technology is an effective method to search for effective serum biomarkers. In previous studies [ 7 ], some differentially secreted proteins were identified by proteomic techniques. CD166 is one of the 26 differentially secreted proteins, which is of interest.…”

“…Identification of differentially expressed proteins by proteomic methods is an effective and widely used technique [ 6 ]. In previous studies [ 7 ], the radioresistant CNE-2R and its parental CNE-2 cell line were cultured in vitro . Some differentially secreted proteins were identified by proteomic techniques, and CD166 is one of them.…”