The remarkable progress in understanding the molecular aspects of cancer has led to the development of novel therapies, which refer to new therapeutic agents targeting specific pathways in cancer cells (16, 28). Tyrosine kinases are the key mediators of the normal and cancer cell signalling network and they have been shown to be involved in the regulation of cellular proliferation, differentiation and angiogenesis. In addition, they also play an important role in the development, progression and metastasis of several types of cancer (2). Based on the role of receptor tyrosine kinases (RTKs) in the regulation of various cellular processes, especially dysregulation of RTK signalling in cancer, RTKs are considered to be a relevant target for anticancer drug research and development (15). Several oncogenic tyrosine kinases such as vascular endothelial growth factor (VEGF), epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR), c-KIT and c-Met have been found to be dysregulated in canine mammary tumours (11, 12). Although dysregulation of RTKs in dogs is not well known, similar mechanisms of RTK dysregulation were found in canine tumours when compared with their human counterparts. Clinically, canine mammary tumours (CMTs) and human breast tumours share many similar biological behaviours and the evidence of canine genome similarity to the human genome has emphasised the dog as an attractive alternative model for testing cancer therapeutic agents (9, 20). Based on the above-mentioned findings, it has been suggested that CMTs may be potential targets for tyrosine kinase inhibitor (TKI) therapy.
Mammary tumours are the most common neoplasm in female dogs and represent a serious problem in veterinary medicine (9). Development of mammary tumours depends on many factors such as alterations in the expression of hormone receptors, dysregulation of cellular differentiation or insufficient apoptosis (3). Despite recent advances multimodally combining surgery, supportive, radio-, and systemic chemotherapy in mammary cancer, the overall prognosis is still poor, the disease-free time short and survival rate low (9). Therefore, novel and innovative approaches are needed for the treatment of canine mammary cancer.
In the two decades since TKIs were discovered they have taken up a role in cancer treatment, and their therapeutic importance is increasing. These inhibitors are small molecules targeting a specific and/or broad spectrum of RTKs that block crucial intracellular signalling pathways in tumour cells, leading to deregulation of key cell functions (15). They show a much higher selectivity towards tumour cells than normal cells when compared with traditional chemotherapy. TKIs provide a wide therapeutic window with low toxicity to healthy tissues and improve the patient’s quality of life (2). Many
Masitinib mesylate, formerly known as AB 1010, is the first potent and selective TKI. It was approved by the European Medicines Agency in veterinary medicine for treatment of canine mast cell tumours about 15 years ago and targets mainly c-Kit receptor (c-KitR) and, to a lesser extent, other tyrosine kinase receptors such as lymphocyte-specific kinase (Lck), Lck/Yes-related protein (LYn), platelet-derived growth factor receptor-alpha/beta (PDGFRα/β), and fibroblast growth factor receptor 3 (FGFR3) (16). It is involved in controlling the proliferation, differentiation and degranulation of mast cells and therefore indirectly their released mediators,
To determine the effects of masitinib on cell viability of CMT cells, the MTT assay was used. The results showed that masitinib reduced the cell count at the end of the 24, 48 and 72 h treatments (Fig. 1). Significant decreases in cell viability were found at 4 μM concentrations of masitinib in both cell lines compared to the control group at 24 h, and at 48 and 72 h significant declines in cell viability were found at concentrations ≥8μM (P
Effects of masitinib treatment on cell viability of CMT-U309 and CMT-U27 cells. Data are expressed as mean percentage of cell viabilities ± standard error (SE) from three individual experiments. * -P < 0.05; ** -P < 0.01; *** -P < 0.001 compared to control
IC20, IC50 and IC80 values of masitinib in CMT-U27 and CMT-U309 cells as measured by the MTT assay
Cell line | Treatment | Time | IC20 (μM) | IC50 (μM) | IC80 (μM) |
---|---|---|---|---|---|
24 h | 2.00 | 9.129 | 41.668 | ||
CMT-U27 | Masitinib | 48 h | 3.099 | 7.607 | 18.670 |
72 h | 2.868 | 7.498 | 19.60 | ||
24 h | 2.497 | 15.032 | 90.485 | ||
CMT-U309 | Masitinib | 48 h | 3.097 | 8.871 | 25.405 |
72 h | 2.471 | 8.545 | 29.544 |
To determine whether apoptosis played a role in the masitinib-mediated weakening of CMT cell proliferation, the appearance of DNA fragmentation after 72 h incubation was quantitatively analysed. Masitinib at around IC50 (8 μM) in both cell lines significantly increased the fragmentation of histone-associated DNA in CMT cells and this was accompanied by a significant increase in OD values from 0.63 ± 0.07 to 2.69 ± 0.09 in CMT-U27 cells and from 0.97 ± 0.09 to 2.68 ± 0.16 in CMT-U309 cells (Fig. 2).
Effects of masitinib on DNA fragmentation of CMT-U27 and CMT-U309 cells. Data are expressed as mean OD values ± standard error from three individual experiments. * – P < 0.05; ** – P < 0.01; *** – P < 0.001 compared to control
To measure and characterise cell death, a quantitative apoptotic cell death assay was performed via double-stained flow cytometry. Consistently with the MTT assay, flow cytometric analyses showed a dose-dependent increase in the percentage of apoptotic cells (the sum of early and late apoptotic cells) in masitinib-treated cells. Masitinib-treated CMT-U27 cells had a higher apoptosis rate of 66.70% compared to the 16.10% rate in CMT-U309 cells and the percentage of early apoptotic cells significantly increased at 8 μM and at 4 and 8 μM in masitinib-treated CMT-U27 and CMT-U309 cells, respectively (Fig. 3).
Flow cytometric analysis after incubation with masitinib (0–8 μM) for 72 h as representative profiles of annexin-V-FITC/PI staining of CMT-U27 cells and CMT-U309 cells. The lower left quadrant of the histogram shows viable cells (unstained by either fluorochrome) and the lower right one represents early apoptotic (annexin-V-positive) cells, indicating the translocation of phosphatidylserine to the external cell surface. The upper right quadrant represents late apoptotic (annexin-V- and PI-positive) cells, and the upper left one shows necrotic (PI-positive) cells. The numbers represent the mean percentage of cells (%) ± standard error
To examine additional mechanisms by which masitinib inhibits cell growth, the cell-cycle distribution in CMT cells was evaluated after drug treatment. At 8 μM concentration, which was approximately IC50 of masitinib in both cell lines, masitinib counteracted cell-cycle progression in CMT cells and increased the proportion of G0/G1 cells to 78% and 86% (
Effects of masitinib treatment on the percentage of the total cell population in each phase of the cell cycle of CMT-U27 and CMT-U309 cells
To assess tumour cell proliferation in correlation with the cell cycle, cells with Ki-67-positive nuclear immunostaining were counted. Immunocytochemical staining revealed that Ki-67 protein expression was suppressed with increasing concentrations of masitinib (Fig. 5A–F). A lower Ki-67 index was calculated after treatment with masitinib in comparison with the control (Fig. 6).
Immunocytochemical staining of Ki-67 in CMT-U27 and CMT-U309 cell lines. Bar = 20 μm. A – negative control, CMT-U27; B – negative control, CMT-U309; C – control high immunopositivity of Ki-67 in CMT-U27; D – control, high immunopositivity of Ki-67 in CMT-U309; E – IC50 masitinib, low immunopositivity of Ki-67 in CMT-U27; F – IC50 masitinib, low immunopositivity of Ki-67 in CMT-U309
Effects of masitinib treatment on the proliferation index of CMT-U27 and CMT-U309 cells measured from Ki-67-stained slides
To determine the effect of masitinib on angiogenesis, the concentration of VEGF was measured in control and masitinib-treated cells. The control cells expressed high levels of VEGF over the 72 h period in both cell lines. The VEGF levels in control cells for CMT-U27 and CMT-U309 were 536.5 pg/mL and 287.8 pg/mL, respectively. However, the measured cell culture supernatant VEGF levels fell with rising concentrations of masitinib (Table 2) and statistical significance was observed at much lower IC50 masitinib concentrations in both cell lines.
VEGF concentrations in the supernatant of control and masitinib treated CMT cells after 72 h
Masitinib concentration | CMT-U27 VEGF concentration (pg/mL) | CMT-U309 VEGF concentration (pg/mL) |
---|---|---|
Control | 536.5 ± 19.01 | 287.8 ± 14.25 |
0.25 μM | 504.64 ± 21.92 | 247.38 ± 14.98 |
0.5 μM | 439.48 ± 13.66* | 221.79 ± 10.32** |
1 μM | 440.28 ± 23.13* | 167.59 ± 8.05*** |
2 μM | 405.72 ± 21.05** | 154.90 ± 13.63*** |
4 μM | 399.87 ± 6.94*** | 99.59 ± 16.38*** |
8 μM | 102.74 ± 10.62*** | 67.97 ± 3.86*** |
* – P < 0.05; ** – P < 0.01; *** – P < 0.001 compared to control
Masitinib mesylate is a potent, orally bioavailable, and selective inhibitor of c-Kit receptor. It has demonstrated good clinical efficacy in unresectable mast cell tumours in dogs and is under clinical assessment as a therapeutic agent in several human cancers which involve c-Kit proto-oncogene mutations similar to those in canine neoplasms (16, 21). The promising clinical efficiency of masitinib in c-Kit-positive mast cell tumours has encouraged preclinical studies and clinical evaluations of masitinib in many other canine malignancies (6, 8, 18). The c-Kit proto-oncogene is reported to be frequently expressed in canine malignant mammary tumours (13). We have investigated the therapeutic potential of masitinib in canine mammary cancer prompted by these reports. In the present study, we analysed the effects of masitinib on cell proliferation, apoptosis, cell cycle and VEGF secretion in CMT cell lines. Masitinib induced a dose-dependent inhibition of cell growth in both CMT cell lines, and these results suggest that CMT cells are sensitive to masitinib and that it is an effective agent for suppressing the growth of CMT cells
To explore the mechanism of masitinib-induced growth inhibition in CMT cells, we examined the effects of masitinib on apoptosis in CMT cells. Apoptotic activity was shown to increase rapidly close to IC50 in both cell lines, and the flow cytometric analyses showed an increase in the percentage of apoptotic cells as the concentration increased, also suggesting that apoptosis is one of the mechanisms by which cell death occurs after exposure to masitinib. Previous data suggest that the masitinib induces growth inhibition
Although the pathophysiology of TKIs causing apoptosis has not been clearly defined, other mechanisms in the apoptotic pathway such as DNA fragmentation may contribute to CMT cell death alongside masitinib. To elucidate the apoptotic pathway induced by masitinib in CMT cells, DNA fragmentation was analysed by the cytoplasmic histone-associated cell death method using ELISA. The results of the present study have proven that DNA fragmentation could be responsible for masitinib-induced apoptosis. It is possible that TKIs may downregulate the anti-apoptotic factors or upregulate the apoptotic factors or may directly process downstream signalling proteins in the apoptotic pathway.
The cell cycle distribution profiles of masitinib-treated CMT cells were examined over 72 h to investigate the mechanism underlying masitinib-mediated antiproliferative effects. The histograms of cell cycle analysis allow us to evaluate any change in the distribution of cell cycle phases by masitinib. Tyrosine kinase inhibitors appear to be more potent in arresting cancer cells in the G0/G1 phase, as reported previously for many other cancer cell lines (29, 30). Similarly to study findings previously reported, there was cell cycle arrest in G0/G1 with an associated decrease in the number of cells in the S phase, with the highest percentage of G0/G1 cells resulting when masitinib was used at nearly its IC50. The results of our study show that masitinib induces apoptosis and blocks cell-cycle progression in CMT cells. Both mechanisms may contribute to drug-induced growth inhibition in these cells. Additionally, it is plausible that alterations in cell cycle regulatory proteins such as downregulation of cyclin D1 may lead to the arrest of masitinib-treated CMT cells in the G0/G1 phase. Further investigation is needed to determine the expression of the relevant cell cycle regulatory proteins in masitinib‐ treated CMT cells.
In relation to the cell cycle results, the Ki-67 marker allowing the estimation of the proportion of proliferative cells was detected (24). It has been reported that Ki-67 is the most frequently used prognostic biomarker in studies concerning canine mammary carcinomas and that its expression is the highest in tumours with poor clinical and histopathological characteristics (10, 19). It appears in the active phases of the cell cycle (G1, S, G2, and M), but it is not expressed in the quiescent G0 phase (24). In the present study, consistently with our cell cycle analyses, less Ki-67 was also revealed to be in masitinib-treated cells due to the induction of cell arrest in G0/G1 phase. An explanation could be that the Ki-67 protein is absent in non-dividing masitinib-treated cells; the fact that cells which are not actively dividing cannot express Ki-67 has already been described in many cells (24). Battistello
Angiogenesis inhibition is an attractive anticancer strategy in cancer chemoprevention and therapy as it targets the vessels that provide oxygen and nutrients to actively proliferating cells. Vascular endothelial growth factor is a basic prerequisite for sustainable growth and proliferation of tumours and supports angiogenesis and proliferation and inhibition of apoptosis mediated by autocrine or paracrine loops between it and its receptors (17). Previous studies have demonstrated that dogs with mammary cancer have higher plasma VEGF concentrations than healthy dogs (22). In canine mammary tumours, overexpression of VEGF has been correlated with metastasis, and its role as a marker of malignancy and as a prognostic indicator has already been documented in canine mammary tumours (11, 22). It has been demonstrated in experimental studies that the inhibition of VEGF production by administration of TKIs was associated with decreased cell proliferation (4, 27). In the present study, VEGF was secreted heavily by CMT cells, predominantly by CMT-U27 cells, and treatment with masitinib contributed to a dose-dependent inhibition of VEGF production by CMT-U27 and CMT-U309 cells. The difference between the VEGF levels of the two cell lines could be related to the doubling times of the CMT-U27 and CMT-U309 cells. The population-doubling time of the CMT-U27 cells is 48 h; however, this value for CMT-U309 is 103.8 h. Since the treatment period was 72 h, the doubling time had been exceeded for CMT-U27 cells; however, the CMT-U309 cells had not yet completed this proliferation period. Additionally, the decrease in the levels of VEGF in the masitinib-treated cell lines may be related to a reduction in the density of the CMT cells. In contrast to our findings, Fahey
In conclusion, masitinib inhibited the proliferation of CMT cells through induction of apoptosis and cell cycle arrest. Decreased immunocytochemical staining of masitinib-treated cells with Ki-67 confirmed the antiproliferative effect of masitinib, in addition the reduced VEGF levels prove the ability of masitinib to modulate angiogenesis. Although the data obtained from this