Cell proliferation, metabolism and death after masitinib treatment
Treatment of C2 cells with 100 nM masitinib induced a complete growth arrest with stable cell counts during 72 hours of treatment. In contrast, cell numbers in untreated C2 cells constantly increased during the experiment with an almost 3-fold rise of cell counts after 72 hours.
A WST-1 assay was performed to assess mitochondrial function before exposure, and after 24, 48 and 72 hours of masitinib exposure (Figure
). There was an approximately 60% reduction in WST-1 conversion of masitinib treated cells at all indicated time points when compared to the initial activity of the untreated cells. A starting point analysis was chosen to reduce the influence of variable cell concentrations of treated and untreated cell cultures and the different metabolic activity during the different growth curve phases of untreated cells.
Figure 1 Effect of masitinib on WST-1 conversion. Masitinib treatment (100 nM) reduced the metabolic activity of C2 cells by 54% after 24 hours of treatment. The metabolic activity further decreased up to 40% at 72 hours. Data shown represent the means+/−SD (more ...)
LDH-leakage assay identified a significant increase of 11.4% after 72 hours but no changes after 24 and 48 hours, indicating only a reduced level of cell death and cytotoxicity at the masitinib concentration used (Figure
Figure 2 Effect of masitinib on LDH-release. Masitinib induced a slight increase in LDH release of C2 cells following 72 h treatment indicating only a mild cytotoxic effect of the masitinib concentration (100 nM) used. Data shown represent the means+/−SD (more ...)
Changes in the transcriptome after masitinib treatment
Treatment of C2 cells with masitinib (100nM) induced a massive change in their global gene expression pattern. A total of 2,116, 3,087 and 3,502 genes had significant changes in their expression levels of > 1.5-fold after 12, 24 and 72 h, respectively (Figure
, Additional file
). Approximately 59% of these genes had decreased expression levels while the rest had increased expression. Approximately one third of these genes code for nuclear proteins while 18–26% of the gene products are expressed in the cytoplasm and in cell organelles (Figure
Figure 3 Microarray analysis of transcriptional changes after masitinib treatment. Masitinib treated cells showed substantial changes in the global gene expression pattern with up to 3,500 genes or approximately 16% of the canine genome having a significant change (more ...)
Cellular location of gene products differentially expressed after masitinib treatment.
Most nuclear factors were involved in mitosis and DNA replication (Figure
), which were mostly down-regulated after masitinib treatment. In addition, genes associated with stress response, glycolysis and the citrate cycle were significantly down-regulated (Figure
Figure 5 Gene functions enriched in the subset of down-regulated genes. Genes associated with functions in cell division, anti-apoptosis, stress response, carbohydrate metabolism were significantly enriched in the subset of down-regulated genes after masitinib (more ...)
An up-regulation of mRNA expression levels was mostly observed for genes associated with Golgi apparatus, endoplasmic reticulum and lysosomes and genes associated with apoptosis and proteolysis (Figure
). Of note, a set of pro-apoptotic genes were significantly enriched in both, up-regulated and down-regulated, groups of genes.
Gene functions enriched in the subset of up-regulated genes. Genes associated with functions in the Golgi-apparatus, the endoplasmic reticulum and apoptosis were significantly enriched in the subset of up-regulated genes after masitinib treatment.
Pathway analysis identified a significant down-regulation of gene expression levels associated with p53, steroid receptor and GTPase-associated signal transduction pathways. In contrast, there was a time dependent increase in the number of up-regulated genes associated with signal transduction pathways during masitinib treatment. After 12 hours of masitinib treatment there was a significant up-regulation of genes associated with three signal transduction pathways, i.e. T-cell receptor, insulin receptor and steroid hormone receptor. At 24 hours genes associated with five additional pathways were up-regulated, i.e. thyroid receptor, vitamin D receptor, Ras cascade, IL10 receptor and IGE receptor. Finally, at 72 hours up-regulation of genes associated with 15 signal transduction pathways were recorded, the aforementioned pathways and the signalling cascades of the B-cell receptor, MAP kinase, EGF receptor, focal adhesion, CXCR4 receptor, EPO signalling and PTEN signalling (Figure
Figure 7 Potentially KIT dependant pathways. Several pro-proliferative pathways have up-regulated mRNA expression levels after masitinib treatment and may thus reflect efforts of treated cells to activate alternative proliferation stimuli and circumvent growth (more ...)
A correlation analysis of expression levels with the different timepoints identified 89 genes with a time dependent, continuous up-regulation in gene expression levels during masitinib treatment, including the cyclin-dependent kinase inhibitor 1A (p21, Cip1), parathyroid hormone (PTH) and platelet/endothelial cell adhesion molecule 1 (PECAM1) ( Additional file
). DAVID analysis identified a significant enrichment of the functional annotations apoptosis, ATM-signalling pathway, RAS protein signal transduction, aging, B-cell proliferation and unfolded protein response in this group of genes. A correlation analysis identified 55 genes that had a time dependent, continuous decrease in expression levels during masitinib treatment, including EIF2 and EIF5. Enriched functional annotations in this gene subset were butyrate and pyruvate metabolism, mitochondrial functions, cell migration, apoptosis and mitosis ( Additional file
Changes in the proteome after masitinib treatment
2D-DIGE and MALDI-TOF analyses of the proteome after 24 and 72 hours of masitinib treatment identified 24 proteins with significant differences in protein expression levels when compared to the proteome before masitinib treatment (Figure
A and 8B). Three proteins, TAR-DNA binding protein (TARDBP, 1.52-fold, p = 0.012), eukaryotic translation initiation factor 3 (EIF3, 1.30-fold, p = 0.014) and the actin related protein 2 (ACTR 2, 1.09-fold, p = 0.0054) were down-regulated after 24 hours of masitinib treatment (Table
). Only two proteins, annexin A1 (ANXA1, 1.66-fold, p = 0.0087) and the gelsolin-like capping protein (CAPG, 1.66-fold, p = 0.0039) were up-regulated after 24 hours of masitinib treatment.
Figure 8 Differentially expressed proteins in masitinib treated C2 cells after 24 (A) and 72 hours (B) when compared to untreated cells. D1–8: proteins down-regulated in treated cells; U1–U25 (red): proteins up-regulated in treated cells. Two-dimensional (more ...)
Down- or up-regulated proteins after 24 hours masitinib treatment
The effect of masitinib treatment on all five proteins was confirmed by comparing the proteome at 72 hours of treatment with the pre-treatment proteome. All five proteins were identified as significantly regulated at 24 hours and having an even increased expression level after 72 hours treatment (Figure
). Nineteen additional proteins had significant changes in expression levels after 72 hours treatment (Table
). Proteins with the highest down-regulation were the eukaryotic translation initiation factor 4a (EIF4A, 1.66-fold, p = 0.005), T-complex protein 1 alpha (TCP1A, 1.63-fold, p = 0.019) and the inorganic pyrophosphatase 1 (PPA1, 1.25-fold, p = 0.021). In addition to the two proteins with increased expression levels after 24 hours, 14 up-regulated proteins were identified after 72 hours of masitinib treatment. Of these, iroquois homeobox 6 (IRX6, 1.74-fold, p = 0.0018), selenium binding protein 1 (SELENBP1, 1.65-fold, p = 0.0011), ubiquitin carboxyl-terminal esterase L3 (UCHL3, 1.51-fold, p = 0.027) and annexin A6 (ANXA6, 1.50-fold, p = 0.031) had the highest up-regulation in protein expression levels.
Down- or up-regulated proteins after 72 hours masitinib treatment
Comparison with the set of genes identified in the transcriptome analysis identified 15 gene products to be present in the list of mRNA and proteins with significant changes in expression levels. mRNA expressions from 6 of the 8 down-regulated proteins after masitinib treatment were also down-regulated. Furthermore, mRNA from 9 of the 15 proteins up-regulated in C2 treated cells was also present in the transcriptome analysis. However, only five of the transcripts were up-regulated whereas four were down-regulated, in contrast to the situation at the protein level.