Impact of combined treatment with nimesulide and cisplatin on oral carcinoma cells

Figures & data

Figure 1 SCC9 and SCC25 after exposure to nimesulide and cisplatin: induction of apoptosis (SCC9: A, SCC25: B) and necrosis (SCC9: C, SCC25: D) is detected by Annexin-V and 7-AAD using flow cytometry, respectively. Adenine nucleotide contents of SCC9 and SCC25 cells were measured after 48-h drug exposure. ATP and energy charge of SCC9 (E, F) and SCC25 (G, H) cells are illustrated. *P<0.05 versus control group indicates statistical significance. Data are mean ± SD of values from at least three experiments for each treatment. Line 1: control; line 2: cisplatin; lane 3: nimesulide, and line 4: nimesulide–cisplatin.

Abbreviations: SCC, squamous cell carcinoma; 7-AAD, 7-amino-actinomycin.

Figure 2 Immunohistochemical detection of apoptotic cells using M30 CytoDeath antibody in SCC9 and SCC25 cells. After 48 h, control SCC25 cells show only slight staining (A), whereas an enrichment of cytoskeletal staining is observed, together with a slight decrease in cell number, after treatment with 0.5 µmol/L cisplatin (B). Treatment with 100 µmol/L nimesulide alone (C) or in combination with 0.5 µmol/L cisplatin (D) reduced the SCC25 cell number and increased the number of apoptotic cells (clear enrichment of cytoskeletal staining). D contains a collage of apoptotic cells after incubation with nimesulide/cisplatin.

Figure 3 Proteomics data supports histone regulation by combination therapy. MS/MS spectra are shown for two iTRAQ-labeled peptides each from histones H2A and H2B, respectively. All peptides were identified with high (99%) confidence with ProteinPilot software. Tables illustrate b- and y-ions matched with experimental data. iTRAQ reporter ions are highlighted in the raw spectra panes. Reporter peaks at m/z =114.1, reflecting combination therapy are considerably more prominent than reporter peaks reflecting single-drug therapy (m/z =115.1 nimesulide, m/z =116.1 cisplatin) or control samples (m/z =117.1). Regulatory ratios were calculated with ProteinPilot software from raw reporter areas, properly accounting for isotope correction and normalization. As nuclei were removed by a centrifugation step during the experimental workflow, ratios observed in the shotgun proteomics experiment should reflect the relative abundance of proteins in the cytosol. We interpret the increased relative ratio of H2A and H2B in the combination therapy sample as further support for increased apoptosis in combination-treated cells, possibly leading to increased release of these histones from nuclei into the cytosolic environment.

Abbreviations: iTRAQ, isobaric tag for the relative and absolute quantization; MS, mass spectrometry; H2A, histone H2A; H2B, histone H2B.

Table 1 List of regulated proteins after exposure to nimesulide and cisplatin

Protein accession number	Protein name	Difference induced by treatment (compared to controls)
		Nimesulide	Cisplatin	Nimesulide + cisplatin
Q99878	Histone H2A type 1-J	0.9	1.2	3.0⇧*
Q99877	Histone H2B type 1-N	0.9	1.3	2.5⇧*
P62805	Histone H4	0.8	1.5	2.7⇧
P16403	Histone H1.2	0.8	0.8	1.0**
Q08043	Alpha-actinin-3	3.3⇧	1.8	1.3
P20618	Proteasome subunit beta type 1	0.5⇩	0.6	0.8
Q13707	ACTA2 protein	0.4⇩	0.8	1.0
P07910	Heterogeneous nuclear ribonucleoproteins C1/C2	0.2⇩	0.6	1.2
Q16643	Drebin	1.3	2.2⇧	1.2
P68363	Tubulin alpha-1B chain	1.9	2.2⇧	1.7
P14866	Heterogeneous nuclear ribonucleoprotein L	0.7	0.5⇩	0.8
Q8N4H5	Mitochondrial import receptor subunit TOM5 homolog	0.6	0.4⇩	0.7

Notes: For inclusion in the table, at least one of the three treatment ratios was required to exceed the threshold of 2-fold up- or down-regulation (respective ratios are highlighted ⇧ and ⇩ respectively).

* Statistical analysis of the data was performed as described in the methods section to account for multiple hypothesis testing: proteins with a local false discovery rate lfdr <0.2 are highlighted bold. These proteins are most likely to be truly regulated. Among all potentially regulated proteins, the lfdr was found under the cutoff 0.2 for two histones in the combination-treatment sample. We consider the fact that the entire group of histones H2 and H4 (but not histone H12) were found to be increased as further evidence for an effect of combination treatment on the cytosolic concentration of these histones.

** For comparison, histone H12 that was detected but found unregulated, was also included in the table.

Abbreviations: H2A, H2B, H4, H1.2, histons; TOM5, translocase complex of the outer mitochondrial membrane; ACTA2, alpha 2 actin.

Figure 4 Proteome analysis shows a slight regulation of prohibitin, particularly after combined treatment with nimesulide and cisplatin. MS/MS spectra are shown for two iTRAQ-labeled peptides from prohibitin with high (99%) confidence, using ProteinPilot software. Reporter peaks at m/z =114.1 reflecting combination therapy are considerably more prominent than reporter peaks reflecting single-drug therapy (m/z =115.1 nimesulide, m/z =116.1 cisplatin) or control samples (m/z =117.1).

Abbreviations: iTRAQ, isobaric tag for the relative and absolute quantization; MS, mass spectrometry.