In vitro and in vivo inhibition of neuroblastoma tumor cell growth by AKT inhibitor perifosine

Abstract

Background: Activated AKT is a marker of decreased event-free or overall survival in neuroblastoma (NB) patients. The aim of this study was to investigate the effect of perifosine, a nontoxic AKT inhibitor, as a single agent on NB cell growth in vitro and in vivo.

Methods: Four human NB cell lines (AS, NGP, BE2, and KCNR) were treated with increasing concentrations of perifosine, and a quantitative analysis of cell death (apoptosis) was performed by using MTS and caspase-3/7 activity assays. Survival of mice carrying xenograft NB tumors that were treated with perifosine (n = 6-7 mice per group) was compared with that of untreated mice (n = 7 mice per group) using Kaplan-Meier analysis. Tumor volumes were calculated to determine the effect of perifosine on NB tumor growth. Phosphorylation of AKT and expression of cleaved caspase-3 were measured in proteins from the tumors. All statistical tests were two-sided.

Results: Perifosine, at 30 muM concentration, decreased AKT phosphorylation and increased apoptosis in all four NB cell lines in vitro. Perifosine-treated mice bearing xenograft NB tumors had longer survival than untreated mice (untreated vs treated, median survival: AS, 13 days, 95% confidence interval [CI] = 11 to 16 days vs not reached, P = .003; NGP, 22 days, 95% CI = 20 to 26 days vs not reached, P = .013; BE2, 24 days, 95% CI = 21 to 27 days vs not reached, P < .001; and KCNR, 18 days, 95% CI = 18 to 21 days vs not reached, P < .001). Perifosine treatment induced regression in AS tumors, growth inhibition in BE2 tumors, and slower growth in NGP and KCNR tumors. Inhibition of AKT phosphorylation and induction of caspase-dependent apoptosis were noted in tumors of perifosine-treated mice in all four in vivo NB tumor models.

Conclusions: Perifosine inhibited the activation of AKT and was an effective cytotoxic agent in NB cells in vitro and in vivo. Our study supports the future clinical evaluation of perifosine for the treatment of NB tumors.

Figure 1

Effect of perifosine on the survival of neuroblastoma (NB) cells. A) Perifosine induced cell death in NB cells. AS, NGP, BE2, and KCNR cells and the control NIH3T3 fibroblasts were treated with different concentrations of perifosine for 48 hours. MTS assay was used to detect cell survival. The percentage of surviving cells was calculated by normalizing the absorbance value of the treated cells by the absorbance value of the control cells within every cell line. Data are representative of two independent experiments (for data shown, the number of replicates [n] for NIH3T3 = 5–6; for AS, n = 6; for BE2, n = 3; for KCNR, n = 3; and for NGP, n = 3). Means and 95% confidence intervals are shown. B) Effect of perifosine on the cell cycle of NB cells. Four NB cell lines were either untreated or treated with different concentrations of perifosine (10, 20, and 30 µM) for 48 hours. The cells were then stained with propidium iodide and analyzed by flow cytometry. The results are shown in histograms, with the percentages of cells in sub-G1 phase, G1 phase, S phase, and G2/M phase indicated. C) Induction of caspase-3/7 activity by perifosine in NB cells. AS, NGP, BE2, and KCNR cells were treated with 20 µM perifosine for 16 hours. Caspase-Glo 3/7 Assay Kit was used to detect caspase-3/7 activity. Data represent means and 95% confidence intervals of two independent experiments. #P < .001 for perifosine-treated cells vs control cells (two-sided analysis of variance [ANOVA]). D) Rescue of perifosine-induced decreased survival by Z-VAD-FMK. AS, NGP, BE2, and KCNR cells were pretreated with 10 µM Z-VAD for 3 hours, followed by treatment with perifosine (20 µM for AS and 30 µM for NGP, BE2, and KCNR) for 48 hours, or treated with perifosine or Z-VAD alone for 48 hours. MTS assay was used to detect cell survival. Data represent means and 95% confidence intervals of two independent experiments. *P ≤ .005 for perifosine/Z-VAD-FMK–treated cells vs perifosine-treated cells (two-sided ANOVA).

Figure 2

Inhibition of AKT phosphorylation by perifosine. Neuroblastoma cells (AS, NGP, BE2, and KCNR) were cultured in medium containing 10% fetal bovine serum for 24 hours and then treated with perifosine at different concentrations (2.5, 5, 10, 15, and 20 μM) for 16 hours. Total proteins (40 μg) were analyzed for phosphorylated (P)-AKT (S473), total (T)-AKT, P-ERK1/2 (T202/Y204), T-ERK 1/2, P-FKHRL1 (T32), T-FKHRL1, P-S6 (S235/236), and T-S6 by immunoblotting. Densitometric analysis was performed. The density ratios of the P-AKT/T-AKT in perifosine-treated samples in each cell line were normalized by the density ratio of the P-AKT/T-AKT of respective control sample. The numbers shown are the relative density unit.

Figure 3

Effect of perifosine on the survival of tumor-bearing mice. Subcutaneous xenografts were established by injecting 2 × 10⁶ of neuroblastoma cells (AS, NGP, BE2, and KCNR) into the right flank of 5- to 6-week-old female nude mice. Perifosine treatment was initiated when tumors reached around 100–200 mm³. Each group had seven mice, except the perifosine-treated AS tumor-bearing group that had six mice. Perifosine (24 mg/kg/d) and placebo were administered once daily 7 d/wk for up to 30–32 days by oral gavage. Survival curve was plotted by Kaplan–Meier analysis. After 10 days of treatment, there were seven control and six perifosine mice in AS groups, seven control and seven perifosine mice in NGP groups, seven control and seven perifosine mice in BE2 groups, and seven control and seven perifosine mice in KCNR groups; after 20 days of treatment, there were zero control and five perifosine mice in the AS groups, five control and seven perifosine mice in NGP groups, seven control and seven perifosine mice in BE2 groups, and two control and seven perifosine mice in KCNR groups; after 30 days of treatment, there were zero control and four perifosine mice in AS groups, zero control and five perifosine mice in NGP groups, zero control and seven perifosine mice in BE2 groups, and zero control and six perifosine mice in KCNR groups. P values were calculated using a two-sided log-rank test.

Figure 4

Effect of perifosine on subcutaneous tumor growth. Subcutaneous xenografts were established as described in Figure 3. Perifosine (24 mg/kg/d) and placebo were administered once daily 7 d/wk for up to 30–32 days. The tumor sizes were measured at least three times a week. A) The tumor size changes of individual mice were plotted according to the time of treatment. Each cell line had a plotted graph indicating the control and perifosine-treated mice. B) The comparison of mean tumor volumes between control and perifosine-treated groups in each cell line. Data represent means and 95% confidence intervals (where n = 4–7, where n < 4, no summary statistics are shown.

Figure 5

Effect of perifosine on orthotopic tumor growth. We injected 5 × 10⁵ AS-luciferase or AS cells into the fat pad around the left adrenal gland in severe combined immunodeficiency–Beige mice. One week after inoculation, perifosine or placebo treatment (24 mg/kg) was initiated and was given once daily 7 d/wk by oral gavage. A) In mice bearing AS-luciferase tumors, luciferase-based bioluminescent imaging was performed at 1 and 3 weeks of perifosine treatment. B) Photon counts of control mice (n = 6) and perifosine-treated mice (n = 6). Values for individual mice are shown (symbols), as are the mean values for each group (bars). C) After 5 weeks of treatment, mice bearing AS tumors were killed and the tumor weight in each mouse was measured. Tumor weights of control (median = 2.98 g; n = 10) and perifosine-treated (median = 0.18 g; n = 10) groups are shown in the box-and-whiskers plot. The boxes represent the interquartile range (25th and 75th percentiles), and the horizontal line in the box represent the median value. The whiskers show the range of largest and smallest values. P < .001 (two-sided Wilcoxon rank sum test).

Figure 6

Effect of perifosine on tumor histopathology and AKT phosphorylation. Subcutaneous xenografts were established as described in Figure 4. A) Hematoxylin–eosin staining of AS tumors at 6 days of perifosine treatment and at the end of perifosine treatment. For the control mouse, histopathology sections were prepared after 15 days of placebo treatment when the tumor reached 20 mm in length; for the perifosine-treated mouse, sections were stained at the end of the experiment, 32 days after perifosine treatment. The magnification of the pictures is ×20 (scale bar = 50 μm). The magnification of the insets is ×100 (scale bar = 10 μm). B and C) Mice in each group were killed at 6 days (AS cells) or 14 days (BE2, NGP, and KCNR cells) of perifosine treatment. Tumors were excised and proteins extracted. Total proteins (40 μg) were analyzed for phosphorylated (P)-AKT (S473), total (T)-AKT, P-ERK1/2 (T202/Y204), T-ERK1/2, P-FKHRL1 (T32), T-FKHRL1, P-S6 (S235/236), T-S6, and cleaved caspase-3 by immunoblotting.