Marked anti-tumour activity of the combination of YM155, a novel survivin suppressant, and platinum-based drugs

Abstract

Background: Survivin, a member of the inhibitor of apoptosis protein family, is an attractive target for cancer therapy. We have now investigated the effects of the combination of YM155, a novel small-molecule inhibitor of survivin expression, and platinum compounds (cisplatin and carboplatin) on human non-small cell lung cancer (NSCLC) cell lines.

Methods: The anti-cancer efficacy of YM155 in combination with platinum compounds was evaluated on the basis of cell death and progression of tumour xenografts. Platinum compound-induced DNA damage was evaluated by immunofluorescence analysis of histone gamma-H2AX.

Results: Immunofluorescence analysis of histone gamma-H2AX showed that YM155 delayed the repair of double-strand breaks induced in nuclear DNA by platinum compounds. The combination of YM155 and platinum compounds also induced synergistic increases both in the number of apoptotic cells and in the activity of caspase-3. Finally, combination therapy with YM155 and platinum compounds delayed the growth of NSCLC tumour xenografts in nude mice to an extent greater than that apparent with either treatment modality alone.

Conclusion: These results suggest that YM155 sensitises tumour cells to platinum compounds both in vitro and in vivo, and that this effect is likely attributable to the inhibition of DNA repair and consequent enhancement of apoptosis.

Figure 1

Effect of YM155 on survivin expression in human non-small cell lung cancer (NSCLC) cells. (A) H460, Calu6, H358, or PC14 cells were incubated in the absence (control, 0.1% dimethyl sulfoxide (DMSO)) or presence of the indicated concentrations of YM155 for 48 h. Cell lysates were then prepared and subjected to immunoblot analysis with antibodies to survivin or to β-actin (loading control). (B) H460, Calu6, H358, or PC14 cells were incubated in the absence or presence of 50 nM YM155 for 48 h, after which cell lysates were subjected to immunoblot analysis with antibodies to survivin, c-IAP1, XIAP, or β-actin.

Figure 2

Effects of YM155 on DNA-damaging agent-induced apoptosis and caspase-3 activity in H460, Calu6, H358, or PC14 cells. Cells were incubated with 50 nM YM155 or vehicle (0.1% dimethyl sulfoxide (DMSO)) for 48 h and then for the indicated times (upper panels) or for 24 h (lower panels) in the additional absence or presence of 10 μM CDDP (A) or CBDCA (B). The percentage of apoptotic cells was then determined by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining (upper panels), and cell lysates were assayed for caspase-3 activity (lower panels). All data are means±s.e. from three independent experiments; those for caspase-3 activity are expressed relative to the corresponding value for the control condition. ^*P<0.05, ^**P<0.001, ^***P<0.0001 vs the corresponding value for CDDP or CBDCA or for YM155 alone.

Figure 3

Effect of YM155 on the formation of γ-H2AX foci induced by DNA-damaging agents in non-small cell lung cancer (NSCLC) cells. (A) H460 cells were incubated with vehicle (0.1% dimethyl sulfoxide (DMSO)) or 50 nM YM155 for 48 h and then for the indicated times in the additional absence or presence of 10 μM CDDP or CBDCA . The cells were then fixed and subjected to immunofluorescence staining for γ-H2AX (green fluorescence). (B) H460 or Calu6 cells were incubated with vehicle or 50 nM YM155 for 48 h and then for the indicated times in the additional absence or presence of 10 μM CDDP or CBDCA . They were then fixed and subjected to immunofluorescence staining for γ-H2AX, and the percentage of cells containing γ-H2AX foci was determined. Data are means±s.e. from three independent experiments. ^*P<0.05, ^**P<0.001 vs the corresponding value for CDDP or CBDCA alone.

Figure 4

Effects of YM155 on the anti-tumour action of CDDP or CBDCA in vivo. Calu6 cells were injected into the right hind limb of nude mice and allowed to form tumours, after which the mice were assigned to one of four treatment groups (control, CDDP (A) or CBDCA (B) alone, YM155 alone, or the combination of YM155 and either CDDP (A) or CBDCA (B)) as described in Materials and Methods. Tumour volume was measured at the indicated times after the onset of treatment (left panels); values for mice in the control group are not shown for later time points to highlight differences among the other three groups. Body weight was also measured in each treatment group at the indicated times and is expressed relative to the corresponding value for time 0 (right panels). All data are means±s.e. from eight mice per group. ^*P<0.0001 vs the corresponding value for treatment with CDDP (A) or CBDCA (B) alone or with YM155 alone.