Mass spectrometry-based proteomic analysis to characterize cisplatin induced early signaling events in head and neck squamous cell carcinoma

Figures & data

Figure 1. (a) Response of HNSCC cell lines, A253, CAL27, Det562 and FaDu to cisplatin: HNSCC cells were treated with varying concentration of cisplatin for 72 h and IC₅₀ was determined by MTT assay. The IC₅₀ of A253, FaDu, Det562 and CAL27 was found to be 1.5 µM, 2 µM, 1.5 µM and 2 µM, respectively (b) Workflow employed to identify proteins altered in response to cisplatin. HNSCC cells were treated with cisplatin at their IC₅₀ for 24 h. Untreated cells served as control. Equal amount of protein from both the conditions was subjected to trypsin digestion followed by mass spectrometry analysis.

Table 1. Partial list of proteins that are differentially regulated in cisplatin treated HNSCC cell lines compared to untreated cells.

Gene Symbol	Description	Cisplatin treated (24 h)/untreated (Fold change)	Associated biological process
CREBBP	CREB-binding protein	16.9	Regulation of nucleobase, nucleoside, nucleotide and nucleic acid metabolism
PLK1	serine/threonine-protein kinase PLK1	7.5	Cell communication; Signal transduction
POLR2I	DNA-directed RNA polymerase II subunit RPB9	3.5	Regulation of nucleobase, nucleoside, nucleotide and nucleic acid metabolism
EIF4EBP1	eukaryotic translation initiation factor 4E-binding protein 1	2.2	Chromatin remodeling
AKT1	RAC-alpha serine/threonine-protein kinase	2.2	Cell communication ; Signal transduction
HMGA2	high mobility group AT-hook 2	0.3	Regulation of nucleobase, nucleoside, nucleotide and nucleic acid metabolism
TSSC4	tumor suppressing subtransferable candidate 4	0.4	spliceosomal snRNP assembly
KIF4A	kinesin family member 4A	0.4	Regulation of nucleobase, nucleoside, nucleotide and nucleic acid metabolism
SMARCA4	SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 4	0.3	Regulation of nucleobase, nucleoside, nucleotide and nucleic acid metabolism
THBS1	thrombospondin 1	0.2	Cell growth and/or maintenance

Figure 2. Protein-protein interaction (PPI) network of proteins implicated in DNA damage response and repair. Proteins associated with DNA damage and repair were shortlisted from list of proteins altered upon cisplatin treatment. An PPI network was created based on literature evidence using the tool, Pathvisio. The number on each protein indicates the fold change observed upon cisplatin treatment compared to untreated cells.

Table 2. Partial list of proteins involved in DNA damage response and repair and are differentially expressed upon cisplatin treatment.

Gene Symbol	Description	Cisplatin treated (24 h)/untreated (Fold change)	Associated Biological Process
MGMT	methylated-DNA-protein-cysteine methyltransferase	100	DNA Methylation
ATOX1	copper transport protein ATOX1	4	Copper ion transport
AKR1B10	aldo-keto reductase family 1 member B10	2.3	Cellular detoxification of aldehyde
NDUFA13	NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 13	3.2	Apoptotic signaling pathway
RAD50	DNA repair protein RAD50	2.3	DNA repair
CDK7	cyclin-dependent kinase 7	2.1	Cell cycle
HSPB1	heat shock protein family B (small) member 1	0.4	Signal transduction
SMARCA4	transcription activator BRG1	0.3	Chromatin remodeling
HNRNPC	heterogeneous nuclear ribonucleoprotein C	0.3	Chromatin remodeling
LGALS1	galectin 1	0.3	Apoptotic process
RELB	transcription factor RelB	0.3	Signal transduction
PTGS2	prostaglandin-endoperoxide synthase 2	0.4	Enzyme binding

Figure 3. Pathway enrichment analysis of proteins altered in response to cisplatin treatment was carried out using ClueGo plugin in Cytoscape. This analysis revealed enrichment of proteins implicated translation, mRNA splicing and DNA damage bypass pathways among others to be enriched upon cisplatin treatment. Proteins highlighted in red indicates upregulation and downregulated proteins are in green.

Figure 4. (a) Venn diagram showing the list of proteins that were common between this study and proteins associated with platinum resistance. (b-c) Development of cisplatin resistant cell lines. CAL27 and Det562 cells were treated with cisplatin at their IC₅₀ for 3 months. The IC₅₀ of both the cells were found to increase with chronic cisplatin treatment indicating acquisition of resistant phenotype. (d) CAL27 and Det562 cisplatin resistant cells exhibited higher cellular proliferation rate compared to untreated or parental cell lines; ((e-f)) Expression of TUBB3 in untreated and cisplatin treated CAL27 and Det562. TUBB3 was found to be overexpressed in cells exposed to cisplatin compared to untreated cells. Cisplatin resistant cells (R) also showed a higher expression of TUBB3 compared to cells treated with cisplatin for 24 h. The expression of TUBB3 was found to be statistically significant in Det562 cells. β- actin was used as loading control.