Identification of chemoresistant factors by protein expression analysis with iTRAQ for head and neck carcinoma

Abstract

Background: Cisplatin and other anticancer drugs are important in the treatment of head and neck squamous cell carcinoma; however, some tumours develop drug resistance. If chemoresistance could be determined before treatment, unnecessary drug administration would be avoided. Here, we investigated chemoresistance factors by comprehensive analyses at the protein level.

Methods: Four human carcinoma cell lines were used: cisplatin-sensitive UM-SCC-23, UM-SCC-23-CDDPR with acquired cisplatin resistance, naturally cisplatin-resistant UM-SCC-81B, and UM-SCC-23/WR with acquired 5-fluorouracil resistance. Extracted proteins were labelled with iTRAQ and analysed by tandem mass spectrometry to identify resistance. Protein expression was confirmed by western blotting and functional analysis was carried out using siRNA.

Results: Thirteen multiple-drug resistance proteins were identified, as well as seven proteins with specific resistance to cisplatin, including α-enolase. Differential expression of these proteins in cisplatin-resistant and -sensitive cell lines was confirmed by western blotting. Functional analysis for α-enolase by siRNA showed that cisplatin sensitivity significantly was increased in UM-SCC-81B and slightly in UM-SCC-23-CDDPR but not in UM-SCC-23/WR cells.

Conclusions: We identified proteins thought to mediate anticancer drug resistance using recent proteome technology and identified α-enolase as a true cisplatin chemoresistance factor. Such proteins could be used as biomarkers for anticancer agent resistance and as targets of cancer therapy.

Figure 1

Flowchart of iTRAQ proteomics approach. Samples were treated according to the iTRAQ protocol, including the blocking of cysteines. They were then labelled with iTRAQ tags and analysed by the nano-LC and Triple TOF5600 system. Protein expression was confirmed by western blotting.

Figure 2

Characterisation of cell lines. Assessment of cell survival following treatment with different concentrations of CDDP according to the WST-1 assay. Black diamonds represent UM-SCC-23 cells, light grey squares represent UM-SCC-23-CDDPR cells, and dark gray triangles represent UM-SCC-81B cells.

Figure 3

MS/MS spectra of select peptides with their reporter ions for the four proteins XRCC1, calreticulin, GST, and α-enolase. Representative peptide sequencing and quantification using iTRAQ with indicated amino acid sequences, annotated b-ion and y-ion series, and an expanded view of the reporter ion region showing representative relative abundances of signature iTRAQ ions at m/z 114, 115, 116, and 117.

Figure 4

Validation of iTRAQ results by western blotting for XRCC1, calreticulin, GST, and α-enolase. The revelation was checked for these proteins by western blotting, with the same result obtained as for ProteinPilot. The density of each band was measured and normalized against that of β-actin. Beta-actin was used as an internal loading control.

Figure 5

Expression of NOTCH1 by western blot analysis in each cell line. The expression of NOTCH1 was seen in all the cell lines but slightly decreased in UM-SCC-81B.

Figure 6

Alpha-enolase knockdown effect using siRNA was analyzed by western blot analysis in UM-SCC-23-CDDPR and UM-SCC-81B cell lines. High α-enolase expression was detected in both cell lines. The effect of knockdown of α-enolase was determined in UM-SCC-23-CDDPR and UM-SCC-81B cells. The density of each band was measured and normalised against that of β-actin. Beta-actin was used as an internal loading control. As a result of rectifying by β-actin, this experiment is conducted correctly (Image Quant TL version 7.0 GE Healthcare, England).

Figure 7

Changes to anticancer drug sensitivity after α-enolase siRNA in each cell line. Bars represent the standard deviation. (A) UM-SCC-23/WR+(5-FU) (solid line), UM-SCC-23/WR+(5-FU+α-enolase siRNA) (dashed line). The effect of combination treatment using α-enolase siRNA and 5-FU on the proliferation of UM-SCC-23/WR was examined by WST-1 assay. (B) UM-SCC-23CDDPR+(CDDP) (solid line), UM-SCC-23CDDPR+(CDDP+α-enolase siRNA) (dashed line). The effect of combination treatment using α-enolase siRNA UM-SCC-23CDDPR was examined by WST-1 assay. (C) UM-SCC-81B+(CDDP) (solid line), UM-SCC-81B+(CDDP +α-enolase siRNA (dashed line). The effect of combination treatment using α-enolase siRNA UM-SCC-81B was examined by WST-1 assay. (D) Comparison for IC50. ^†, Student's t-test was used for comparison of log-transformed IC50 between with and without α-enolase.