Activity of tyrosine kinase inhibitor Dasatinib in neuroblastoma cells in vitro and in orthotopic mouse model

Abstract

Stage 4 neuroblastoma (NB) is a devastating childhood cancer whose poor outcome has remained essentially unchanged in the last 20 years. Receptor tyrosine kinases have important roles in the control of proliferation, differentiation and apoptosis of NB cells. Thus, we tested the activity of second-generation tyrosine kinase inhibitor Dasatinib in human NB cell lines in vitro and in an orthotopic mouse model. Dasatinib inhibited cell viability with an IC(50) in the submicromolar range in 7 of 10 tested cell lines. In sensitive cells, Dasatinib reduced anchorage-independent growth and, in some instances, induced senescence and apoptosis. In HTLA-230 cells, Dasatinib treatment caused down-regulation of c-Kit and c-Src phosphorylation in conjunction with strong inhibition of Erk1/2 and Akt activity. To test the efficacy of Dasatinib in vivo, HTLA-230 and SY5Y cells were orthotopically injected in the adrenal gland of nude mice and drug treatments carried out until day 40. In mice injected with HTLA-230 cells, tumour growth was significantly inhibited at the dose of 30 mg/(kg day) when treatment was started 7 days after injection. In animals injected with SY5Y cells that were exquisitely sensitive in vitro (IC(50)= 92 nM), the antitumour effect of Dasatinib was observed at the dose of 60 mg/(kg day) but only when treatment was started 1 day after injection. However, the anti-tumour effect of Dasatinib in vivo was partial in both orthotopic models, emphasizing the importance of testing candidate new drugs in animal environments closely mimicking the human tumour.

Figure 1.

A: Cell viability assays were carried out by exposing the indicated NB cell lines to increasing concentrations of Dasatinib (10, 100 and 1000 nM) for 48 hours. IC₅₀ values (in grey triangles on the top of each column) were calculated by regression analysis in the cell lines where at least one drug concentration caused a 50 % decrease in cell viability. Triplicate assays were performed for each drug concentration. Values ± SE are given. B: top panels show beta-galactosidase staining of untreated (un) and Dasatinib-treated SH-EP cells. Percentages of beta-galactosidase positive cells counted in randomly selected microscopic fields (at least 1000 elements) are reported in the bottom graph. C: invasion assays were carried out in vitro in Matrigel® - coated chambers (see Materials and Methods) using the indicated NB cell lines treated with increasing concentration of Dasatinib (10, 100 and 1000 nM) for 20 hours. Percent invasion was arbitrarily set to 100 in untreated cells. Experiments were carried out in triplicate. Values ± SE are given.

Figure 2.

A and C: western blot analyses for phospho-Akt (p-Akt), Akt, phospho-Erk1/2 (p-Erk1/2) and Erk1/2 detection in Dasatinib-treated HTLA-230 (500 nM) and SY5Y (550 nM) cells. In HTLA-230 cells experiments were carried out either by adding Dasatinib to complete medium (lanes on the left) or by starvation in the absence of serum (20 hours), followed by treatment with Dasatinib (2 hours) and serum stimulation (1 hour).SY5Y cells in complete medium were treated with Dasatinib (550 nM) for 2 or 4 hours. B and D: Densitometric analyses of phospho-Akt and phospho-Erk1/2 in untreated and Dasatinib-treated HTLA-230 and SY5Y cells from blots in panels A and C. Densitometric values were normalized for the levels of total Akt and Erk1/2. Expression levels in untreated cells were arbitrarily set to 100. D: detection of phospho-c-Src (p-c-Src), c-Src, phospho-c-Kit (p-c-Kit) and c-Kit in untreated or Dasatinib-treated (2 hours) HTLA-230 and SY5Y cells. HSP-70 expression was utilized to normalize the amount of protein extracts loaded in each lane in A, C and in E.

Figure 3.

A: HTLA-230 (A) and SY5Y (B) cells were orthotopically injected in nude mice (see text). Forty days after injection, animals were sacrificed and mean tumour volume (bars ± SE) was calculated in untreated and in Dasatinib-treated groups. p was determined by Student’s t test. Dasatinib treatment was given at the dose of 30mg/Kg/day starting 7 days after orthotopic injection of HTLA-230 cells (A) or at 60mg/Kg/day starting either 1 or 7 days after injection of SY5Y cells (B). C and D: Body weight was periodically measured until the last day of treatment (day 40). The graph in D shows the comparison of body weight of untreated and Dasatinib-treated animals (60mg/Kg/day from day 1 to day 40). In all groups, body weight differences between untreated and Dasatinib-treated animals throughout the experiments were not significant.

Figure 4.

Histological sections of untreated (A and E) and Dasatinib-treated (B and F) tumours from animals orthotopically injected with HTLA-230 (A and B) and SY5Y (E and F) cells; t: tumour; k: kidney. Immunohistochemical staining for Ki-67 expression in untreated (C and G) and Dasatinib-treated (D and H) tumours from animals injected with HTLA-230 (C and D) or SY5Y cells (G and H). Inset in D shows the negative control in which staining with primary anti-Ki-67 antibody is replaced by staining with normal serum.

Figure 5.

A: western blot analyses to detect phospho-Akt (p-Akt), Akt, phospho-Erk1/2 (p-Erk1/2) and Erk1/2, BAX and BAD in cell extracts from tumours of untreated and Dasatinib-treated animals transplanted with HTLA-230 (blots on the left) or with SY5Y cells (blots on the right). Protein amount loaded in each lane was normalized by HSP-70 levels. B: Densitometric analysis of phospho-Akt and phospho-Erk1/2 in untreated and Dasatinib-treated animals injected with HTLA-230 cells. Each densitometric value represents the mean of 2 untreated and 2 Dasatinib-treated samples from blot in panel A. Values were normalized for the levels of total Akt and Erk1/2. Expression levels in untreated samples were arbitrarily set to 100.