YM155 enhances the cytotoxic activity of etoposide against canine osteosarcoma cells

Abstract

Canine osteosarcoma (OSA) is an aggressive and highly malignant primary bone tumor. Its poor survival outcome remains problematic despite recent advances in anti-cancer therapy, therefore highlighting the need for alternative treatment options or drug repositioning. The aim of this study was to determine if YM155, a small-molecule survivin inhibitor, potentiates the chemotherapeutic efficacy of etoposide against canine OSA in vitro and in vivo. In cell culture, YM155 enhanced the cytotoxic effect of etoposide against canine OSA cell lines; however, the molecular mechanism behind this effect was heterogeneous, as only one cell line had an elevated apoptotic level. In addition, this effect was not associated with survivin suppression in two of the cell lines. These results suggest that the molecular target of YM155 is not restricted to survivin alone. When tested on a murine xenograft model, the average tumor volume of the combination treatment group (YM155, 5 mg/kg, intraperitoneally, 5 consecutive days/week; and etoposide, 20 mg/kg, intraperitoneally, every 5 days) was 66% smaller than the control group, although this difference was not statistically significant (P=0.17). Further studies to improve the treatment protocol are necessary to confirm the findings of this study.

Keywords: YM155; canine osteosarcoma; etoposide; survivin; synergism.

Fig. 1.

Expression of survivin and actin visualized by western blot analysis. YM155 suppressed the endogenous survivin protein expression in a dose-dependent manner 24 hr after treatment. Actin was used as a loading control.

Fig. 2.

Effect of etoposide and YM155 combination treatment on the viability of canine osteosarcoma cell lines, determined using the Cell Counting Kit-8 assay. Data are expressed as means ± SDs. The figures are representative of three independent experiments. Drug concentrations for etoposide and YM155 are in µM and nM, respectively. Bonferroni test; ***†††P<0.001; *compared with control; †compared with etoposide alone; E, etoposide; Y, YM155.

Fig. 3.

Inhibitory effect of the treatments on colony formation in HMPOS, POS, and HOS cell lines. Data are expressed as means ± SDs. Data are representative of three independent experiments. Drug concentrations for etoposide and YM155 are in µM and nM, respectively. Bonferroni test; ***†††P<0.001; *compared with control; †compared with etoposide alone; E, etoposide; Y, YM155.

Fig. 4.

Average percentages of sub-G₁ cells after treatment for 72 hr. Data are expressed as means ± SDs. Drug concentrations for etoposide and YM155 are in µM and nM, respectively. Bonferroni test; **P<0.01; ***†††P<0.001; *compared with control; †compared with etoposide alone; E, etoposide; Y, YM155.

Fig. 5.

Apoptosis assay by FACS in HMPOS, POS, and HOS cells treated with etoposide, YM155, or etoposide and YM155 combined. (A) Dot plots from annexin V/PI assay are representative of three independent experiments performed on HMPOS, POS and HOS cell lines. (B) Average percentages of canine OSA cells that underwent early apoptosis at 48 hr. Data are expressed as means ± SDs. Drug concentrations for etoposide and YM155 are in µM and nM, respectively. Bonferroni test; *†P<0.01; **P<0.01; ***P<0.001; *compared with control; †compared with etoposide alone; E, etoposide; Y, YM155.

Fig. 6.

Western blot analyses of canine osteosarcoma cells exposed to treatments for 24 hr (HMPOS and POS cell lines) or 72 hr (HOS cell line). Treatment with YM155 at IC₅₀ either alone or in combination with etoposide did not down-regulate survivin expression of HMPOS and POS cells. Combination treatment enhanced the expression of cleaved poly (ADP-ribose) polymerase (PARP) in HOS cell line only.

Fig. 7.

Effect of treatments on xenograft tumor progression and body weight. (A) Average tumor mass of the combination treatment group was smaller than the control group but did not reach statistical significance. (B) The body weight of the combination treatment group was reduced on day 13 and 19 after initiation of treatment. Data are expressed as means ± SDs. Dunnett’s test; *P<0.05 compared with control.

Fig. 8.

Representative photomicrographs showing immunoreactivity for (A) Ki-67, (B) survivin, and (C) TUNEL analysis in the xenograft tumors. (D) Effects of the treatments on cell proliferation activity. Both etoposide single agent and combination treatment regimens reduced the proliferation index values of the xenograft tumors. (E) Effects of the treatments on the expression of survivin. Combination treatment suppressed survivin protein expression. (F) Effects of treatments on apoptosis. A slight increase in apoptosis was observed in the tumors from etoposide-treated mice. Data are expressed as means ± SDs. Student’s t test for unpaired data; *P<0.05 compared with control, **P<0.01 compared with control; bar=100 µm.