Purpose: Survivin, a member of the inhibitor of apoptosis protein family, is an attractive target for cancer therapy. We have now investigated the effect of YM155, a small-molecule inhibitor of survivin expression, on the sensitivity of human non–small cell lung cancer (NSCLC) cell lines to γ-radiation.
Experimental Design: The radiosensitizing effect of YM155 was evaluated on the basis of cell death, clonogenic survival, and progression of tumor xenografts. Radiation-induced DNA damage was evaluated on the basis of histone H2AX phosphorylation and foci formation.
Results: YM155 induced down-regulation of survivin expression in NSCLC cells in a concentration- and time-dependent manner. A clonogenic survival assay revealed that YM155 increased the sensitivity of NSCLC cells to γ-radiation in vitro. The combination of YM155 and γ-radiation induced synergistic increases both in the number of apoptotic cells and in the activity of caspase-3. Immunofluorescence analysis of histone γ-H2AX also showed that YM155 delayed the repair of radiation-induced double-strand breaks in nuclear DNA. Finally, combination therapy with YM155 and γ-radiation delayed the growth of NSCLC tumor xenografts in nude mice to a greater extent than did either treatment modality alone.
Conclusions: These results suggest that YM155 sensitizes NSCLC cells to radiation both in vitro and in vivo, and that this effect of YM155 is likely attributable, at least in part, to the inhibition of DNA repair and enhancement of apoptosis that result from the down-regulation of survivin expression. Combined treatment with YM155 and radiation warrants investigation in clinical trials as a potential anticancer strategy.
Survivin is a potentially important molecular target for cancer therapy. Reflecting the many mechanisms that seem to regulate survivin expression, diverse approaches have been evaluated for targeting survivin in experimental models. YM155 is a novel small, imidazolium-based compound that specifically inhibits survivin expression in various types of cancer cell lines in vitro. In addition, YM155 has been shown to distribute preferentially to tumor tissues rather than to plasma as well as to exert pronounced antitumor activity in tumor xenograft models in vivo. The use of YM155 as a single agent in phase I clinical trials did not reveal significant toxicity. Although phase II studies of YM155 use as a single agent for certain types of cancer are currently under way, the effects of YM155 in combination with radiation have not been reported. We now show that inhibition of survivin expression by YM155 sensitizes tumor cells to radiation in vitro and in vivo. Therefore, our preclinical results provide a rationale for future clinical investigation of the therapeutic efficacy of YM155 in combination with radiotherapy.
Survivin is a 16.5-kDa member of the inhibitor of apoptosis protein (IAP) family. It blocks the mitochondrial pathway of apoptosis by inhibiting caspases (1, 2) and regulates cell division through interaction with the proteins INCENP and Aurora B (3). It is abundant in many types of cancer cells but not in the corresponding normal cells (4–6). High levels of survivin expression in cancer cells are associated with poor patient prognosis and survival as well as with resistance to therapy and an increased rate of cancer recurrence (7–9). Survivin has therefore become a therapeutic target and potentially important prognostic marker for many tumor types, including non–small cell lung cancer (NSCLC; refs. 7, 10). Molecular antagonists of survivin including antisense oligonucleotides, and dominant negative mutants have been shown to induce apoptosis in cancer cells in vitro and in vivo as well as to enhance chemotherapy-induced cell death (11–13). Although antisense oligonucleotides and ribozymes can be engineered to be highly specific for survivin, they may be difficult to deliver in the clinical setting.
YM155, a small imidazolium-based compound, was identified by high-throughput screening of chemical libraries for inhibitors of the activity of the survivin gene promoter in a reporter assay (14). This compound specifically inhibits the expression of survivin at both the mRNA and protein levels and exhibits pronounced anticancer activity in preclinical models (14). An advantage of YM155 compared with previously investigated suppressors of survivin expression (15–20) is that it is active in the subnanomolar range. Pharmacokinetic analysis also revealed that YM155 was highly distributed to tumor tissue in tumor xenograft models in vivo (14). YM155 is thus an attractive candidate drug for cancer therapy, and clinical trials of YM155 in single-agent therapy are currently under way for some types of cancer.
Glioblastoma cells that overexpress survivin were found to be less responsive to radiation than survivin-negative cells in a preclinical model (21). Clinically, high levels of survivin expression have been associated with an increased risk of local treatment failure after radiochemotherapy in patients with rectal cancer (9). These observations suggest that survivin plays a role in resistance to radiotherapy. Indeed, suppression of survivin expression with the use of antisense oligonucleotides or ribozymes has been shown to increase the radiosensitivity of cancer cells in vitro (20, 22–26). We have now examined the effects of the combination of YM155 and radiation on NSCLC cell lines in vitro and in vivo.
Materials and Methods
Cell culture and reagents. The human NSCLC cell lines NCI-H460 (H460) and Calu6 were obtained from the American Type Culture Collection. The cells were cultured under an atmosphere of 5% CO2 at 37°C in RPMI 1640 (Sigma) supplemented with 10% fetal bovine serum. YM155 (Astellas Pharma, Inc.) was dissolved in DMSO.
Immunoblot analysis. Cells were washed twice with ice-cold PBS and then lysed in a solution containing 20 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl, 1 mmol/L EDTA, 1% Triton X-100, 2.5 mmol/L sodium PPi, 1 mmol/L phenylmethylsulfonyl fluoride, and leupeptin (1 μg/mL). The protein concentration of lysates was determined with the Bradford reagent (Bio-Rad), and equal amounts of protein were subjected to SDS-PAGE of a 15% gel. The separated proteins were transferred to a nitrocellulose membrane, which was then exposed to 5% nonfat dried milk in PBS for 1 h at room temperature before incubation overnight at 4°C with rabbit polyclonal antibodies to human survivin (1:1000 dilution; R&D Systems), to human c-IAP1 (1:1,000 dilution; MBL International), to human XIAP (1:1,000 dilution; Cell Signaling), to human STAT3 (1:1,000 dilution; Cell Signaling), or to β-actin (1:500 dilution; Sigma), or with mouse monoclonal antibodies to human p53 (1:1,000 dilution; Santa Cruz Biotechnology). The membrane was then washed with PBS containing 0.05% Tween 20 before incubation for 1 h at room temperature with horseradish peroxidase–conjugated goat antibodies to rabbit (Sigma) or mouse (Santa Cruz Biotechnology) IgG. Immune complexes were finally detected with chemiluminescence reagents (Perkin-Elmer Life Science).
Clonogenic survival assay. Exponentially growing cells in 25-cm2 flasks were harvested by exposure to trypsin and counted. They were diluted serially to appropriate densities and plated in triplicate in 25-cm2 flasks containing 10 mL of complete medium in the presence of 50 nmol/L YM155 or vehicle (final DMSO concentration of 0.1%; we confirmed that this DMSO concentration did not affect the proliferation of NSCLC cell lines). After incubation for 48 h, the cells were exposed at room temperature to various doses of γ-radiation with a 60Co irradiator at a rate of ∼0.82 Gy/min. The cells were then washed with PBS, cultured in drug-free medium for 10 to 14 d, fixed with methanol:acetic acid (10:1, v/v), and stained with crystal violet. Colonies containing >50 cells were counted. The surviving fraction was calculated as: (mean number of colonies)/(number of inoculated cells × plating efficiency). Plating efficiency was defined as the mean number of colonies divided by the number of inoculated cells for nonirradiated control cells. The surviving fraction for combined treatment was corrected by that for YM155 treatment alone. Cell survival was corrected according to the equation S = 1 − (1 − f)1/N, where S is the single-cell survival rate, f is the measured surviving fraction, and N is multiplicity, which was defined as the average number of cells per microcolony at the time of radiation and which ranged from 2.4 to 6.7 for the cell lines studied under the described conditions. The dose enhancement factor was then calculated as the dose (Gy) of radiation that yielded a surviving fraction of 0.1 for vehicle-treated cells divided by that for YM155-treated cells (after correction for drug toxicity).
Detection of apoptotic cells. Cells were fixed with 4% paraformaldehyde for 1 h at room temperature, after which a minimum of 1,000 cells per sample was evaluated for apoptosis with the use of the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) technique (In situ Cell Death Detection Kit; Boehringer Mannheim).
Assay of caspase-3 activity. The activity of caspase-3 in cell lysates was measured with the use of a CCP32/Caspase-3 Fluometric Protease Assay Kit (MBL). Fluorescence attributable to cleavage of the DEVD-AFC substrate was measured at excitation and emission wavelengths of 390 and 460 nm, respectively.
Immunofluorescence staining of γ-H2AX. Cells were grown to 50% confluence in two-well Lab-Tec Chamber Slides (Nunc) and then cultured for 48 h in the presence of 50 nmol/L YM155 or vehicle before exposure to 3 Gy of γ-radiation. At various times thereafter, they were fixed with 4% paraformaldehyde for 10 min at room temperature, permeabilized with 0.1% Triton X-100 for 10 min at 4°C, and exposed to 5% nonfat dried milk for 10 min at room temperature. The slides were washed with PBS and then incubated at room temperature first for 2 h with mouse monoclonal antibodies to histone γ-H2AX (Upstate Biotechnology) at a dilution of 1:300 and then for 1 h with Alexa 488–labeled goat antibodies to mouse IgG (Molecular Probes) at a dilution of 1:700. The slides were mounted in fluorescence mounting medium (Dako Cytomation), and fluorescence signals were visualized with a confocal laser-scanning microscope (Axiovert 200M; Carl Zeiss) equipped with the LSM5 PASCAL system (Carl Zeiss). Three random fields each containing =50 cells were examined at a magnification of × 100. Nuclei containing ≥10 immunoreactive foci were counted as positive for γ-H2AX, as previously described (27), and percentage of positive cells was calculated.
Evaluation of tumor growth in vivo. All animal studies were done in accordance with the Recommendations for Handling of Laboratory Animals for Biomedical Research compiled by the Committee on Safety and Ethical Handling Regulations for Laboratory Animal Experiments, Kyoto University. The ethical procedures followed met the requirements of the United Kingdom Coordinating Committee on Cancer Research guidelines (28). Tumor cells (2 × 106) were injected s.c. into the right hind leg of 6-week-old female athymic nude mice (BALB/c nu/nu). Tumor volume was determined from caliper measurement of tumor length (L) and width (W) according to the formula LW2/2. Treatment was initiated when the tumors in each group of animals achieved an average volume of ∼200 to 250 mm3. Treatment groups (each containing eight mice) consisted of vehicle control (physiologic saline), YM155 alone, vehicle plus radiation, and YM155 plus radiation. Vehicle or YM155 at a dose of 5 mg/kg of body mass was administered over 7 consecutive days (days 1-7) with the use of an implanted micro-osmotic pump (Alzet model 1003D; Durect). Mice in the radiation groups received 10 Gy of γ-radiation from a cobalt irradiator either as a single fraction on day 3 of drug treatment or fractionated over 5 consecutive days (days 3 to 7); the radiation was targeted to the tumor, with the remainder of the body shielded with lead. Growth delay (GD) was calculated as the time required to achieve a 5-fold increase in volume for treated tumors minus that for control tumors. The enhancement factor was then determined as: (GDcombination − GDYM155)/GDradiation.
Statistical analysis. Data are presented as means ± SD or SE and were compared with the unpaired Student's t test. A P value of <0.05 was considered statistically significant.
Inhibition of survivin expression in NSCLC cells by YM155. We first examined the effect of YM155 on survivin expression in human NSCLC cell lines by immunoblot analysis. Treatment of H460 or Calu6 cells with YM155 at 1 to 100 nmol/L for 48 hours inhibited survivin expression in a concentration-dependent manner (Fig. 1A). In contrast, YM155 had no effect on the abundance of other members of the IAP family including XIAP and c-IAP1 (Fig. 1A), suggesting that YM155 specifically inhibits survivin expression in the NSCLC cell lines. The mechanism by which YM155 inhibits survivin expression remains to be elucidated. Previous observations have shown that p53 and signal transducer and activator of transcription 3 (STAT3) regulate survivin expression at the transcriptional level (29). We therefore examined the effect of YM155 on the abundance of p53 and STAT3 in NSCLC cell lines. YM155 showed no marked effect on the amounts of p53 and STAT3 in H460 or Calu6 cells (Fig. 1A), suggesting that the inhibition of survivin expression by YM155 is independent of these transcriptional regulators. Monitoring of the time course of survivin expression in cells exposed to 50 nmol/L YM155 for up to 72 hours revealed that the abundance of survivin in Calu6 cells had decreased by 2 hours and that survivin was virtually undetectable in H460 cells after 24 hours (Fig. 1B). In both cell lines, treatment with 50 nmol/L YM155 resulted in time-dependent inhibition of survivin expression.
YM155-induced sensitization of NSCLC cells to radiation. To examine the effect of YM155 on cell survival, we first did a clonogenic survival assay. Exposure to the drug at concentrations of 1 to 500 nmol/L for 48 hours revealed that YM155 inhibited the survival of H460 cells with a median inhibitory concentration (IC50) of 64 nmol/L and that of Calu6 cells with an IC50 of 43 nmol/L (Fig. 2A). On the basis of these data, we adopted treatment with 50 nmol/L YM155 for 48 hours as the standard protocol for radiation experiments. We next examined whether YM155 might affect the sensitivity of NSCLC cell lines to radiation. Treatment with 50 nmol/L YM155 for 48 hours shifted the survival curves for both H460 and Calu6 cells to the left (Fig. 2B), with a dose enhancement factor of 1.57 and 1.61, respectively, suggesting that YM155 increased the radiosensitivity of both cell lines.
Enhancement of radiation-induced apoptosis in NSCLC cells by YM155. We next examined the effect of YM155 on radiation-induced apoptosis in H460 or Calu6 cells with the use of the TUNEL assay. Combined treatment of either cell line with YM155 and γ-radiation resulted in an increase in the number of apoptotic cells at 24 and 48 hours that was greater than the sum of the increases induced by YM155 or radiation alone (Fig. 3A). To confirm the results of the TUNEL assay, we measured the activity of caspase-3 in cell lysates. Again, the combined treatment of H460 or Calu6 cells with YM155 and γ-radiation induced a synergistic increase in caspase-3 activity (Fig. 3B). These data thus suggested that YM155 promotes radiation-induced apoptosis in NSCLC cell lines.
Inhibition of DNA repair in irradiated NSCLC cells by YM155. Defects in DNA repair have been associated with enhanced sensitivity of cells to radiation (30, 31), and survivin is thought to play a direct or indirect role in DNA repair (21). We therefore next investigated the effect of YM155 on DNA repair by immunostaining of cells with antibodies to the phosphorylated form (γ-H2AX) of histone H2AX, foci of which form at DNA double-strand breaks (DSBs). The formation of γ-H2AX foci in H460 cells was apparent between 30 minutes and 6 hours after γ-irradiation (Fig. 4A). In the presence of YM155, however, these foci persisted for at least 24 hours after irradiation. Evaluation of the percentage of H460 or Calu6 cells with γ-H2AX foci at 24 hours after irradiation revealed that YM155 significantly inhibited the repair of DSBs (Fig. 4B). These results thus suggested that down-regulation of survivin expression by YM155 results in the inhibition of the repair of radiation-induced DSBs in NSCLC cells, possibly accounting for the observed radiosensitization by this drug.
Enhancement of radiation-induced tumor regression by YM155. To determine whether the YM155-induced radiosensitization of NSCLC cells observed in vitro might also be apparent in vivo, we injected H460 or Calu6 cells into nude mice to elicit the formation of solid tumors. After tumor formation, the mice were treated with YM155, γ-radiation, or both modalities. YM155 was infused continuously for 7 days with the use of an implanted osmotic pump system, and mice were subjected to local irradiation with a single dose of 10 Gy on day 3 of YM155 administration. Combined treatment with radiation and YM155 inhibited H460 or Calu6 tumor growth to a markedly greater extent than did either modality alone (Fig. 5). The tumor growth delays induced by treatment with radiation alone, YM155 alone, or both YM155 and radiation were 2.9, 5.6, and 14.8 days, respectively, for H460 cells, and 8.9, 41.0, and 76.0 days, respectively, for Calu6 cells. The enhancement factor for the effect of YM155 on the efficacy of radiation was 3.3 for H460 cells and 3.5 for Calu6 cells, revealing the effect to be greater than additive. No pronounced tissue damage or toxicity such as weight loss was observed in mice in any of the four treatment groups.
Finally, we evaluated whether the combination of YM155 and fractionated radiation treatment would result in the inhibition of tumor growth similar to that observed with YM155 plus single-fraction radiation. Mice bearing H460 tumors were thus again subjected to continuous YM155 infusion for 7 days, but local irradiation was done in 2-Gy fractions on days 3 to 7 of drug administration (for a total dose of 10 Gy). The tumor growth delays induced by treatment with radiation alone, YM155 alone, or both YM155 and radiation were 3.8, 5.3, and 16.6 days, respectively (Fig. 6). The enhancement factor for the effect of YM155 on the efficacy of radiation was 3.0. Again, there was no evidence of toxicity on the basis of body weight loss, and there were no animal deaths in any of the four groups. These data suggested that YM155 enhances the tumor response to both single-dose and fractionated radiotherapy in vivo.
Survivin is a potentially important molecular target for cancer therapy. Reflecting the many mechanisms that seem to regulate survivin expression, diverse approaches have been evaluated for targeting survivin in experimental models. Although certain drugs, such as inhibitors of histone deacetylases, mitogen-activated protein kinases, and cyclin-dependent kinases, have been shown to suppress survivin expression by targeting various signaling pathways, these drugs inhibit survivin expression nonspecifically (15–17, 19, 32). Gene therapy strategies based on small interfering RNA or other antisense oligonucleotides are specific for survivin, but the effective delivery of these molecules remains a challenge for the transition to the clinic (33). YM155 is a small-molecule agent that specifically inhibits survivin expression in various types of cancer cell lines in vitro (14). In addition, YM155 has been shown both to distribute preferentially to tumor tissues rather than to plasma as well as to exert pronounced antitumor activity in tumor xenograft models in vivo (14). The use of YM155 as a single agent in phase I clinical trials did not reveal significant toxicity (34). Although phase II studies of YM155 use as a single agent for certain types of cancer are currently under way, the effects of YM155 in combination with radiation have not been reported. We now show that YM155 increased the sensitivity of tumor cells to radiation in vitro and in vivo.
Clonogenic survival analysis, the most reliable approach for assessing the ability of genotoxic agents to induce cell death (35), revealed that YM155 markedly potentiated the decrease in NSCLC cell survival induced by γ-radiation. Given that induction of apoptosis is a key mechanism of cytotoxicity for most antitumor agents, including γ-radiation, defects in apoptotic signaling may underlie resistance to such agents (36). Radiation-sensitive tumors undergo radiation-induced apoptosis in vitro more readily than do radiation-resistant tumors (37–40). Treatment with caspase inhibitors has been shown to protect tumor cells against radiation-induced apoptosis and to increase their radioresistance (21, 41, 42), suggesting that radiation-induced apoptosis is caspase-dependent and that caspases contribute to radiosensitivity. The antiapoptotic activity of survivin is mostly attributable to inhibition of the activation of downstream effectors of apoptosis such as caspase-3 and caspase-7 (25). We have now shown that radiosensitization of NSCLC cells by YM155 was associated with increases both in the activity of caspase-3 and in the proportion of apoptotic cells. Our findings thus suggest that YM155 sensitized tumor cells to radiation at least in part by enhancing radiation-induced apoptosis.
We examined further the mechanism by which YM155 induces radiosensitization. Survivin is essential for the proper execution of mitosis and cell division, with disruption of survivin expression resulting in cell division defects that can lead to polyploidy and the formation of multinucleated cells (43, 44). Although treatment with 50 nmol/L YM155 for 48 hours inhibited survivin expression in NSCLC cells, it did not induce polyploidy (data not shown), suggesting that YM155-induced radiosensitization in the present study was not attributable to cell division defects caused by survivin depletion. Survivin was previously suggested to enhance tumor cell survival after radiation exposure through regulation of DSB repair (21). We therefore investigated the effect of YM155 on the repair of radiation-induced DSBs by immunofluorescence imaging of γ-H2AX foci. H2AX is a histone that is phosphorylated by ataxia telangiectasia mutated and DNA-dependent protein kinase in response to the generation of DSBs (45, 46). This reaction occurs rapidly, with half-maximal amounts of γ-H2AX generated within 1 minute and maximal amounts within 10 minutes (47), and a linear relation has been shown between the number of γ-H2AX foci and that of DSBs (48). The number of γ-H2AX foci is thus a sensitive and specific indicator of the existence of DSBs, with a decrease in this number reflecting DSB repair. We found that YM155 inhibited the repair of radiation-induced DSBs in NSCLC cells. If left unrepaired, DSBs can result in chromosome loss or cell death; agents that inhibit such repair thus increase the sensitivity of cells to ionizing radiation (49, 50). Our results therefore suggest that inhibition of DSB repair by YM155 contributes to the radiosensitization induced by this drug. Given that suppression of survivin expression impairs the repair of radiation-induced DNA damage (9, 21), our results further suggest that inhibition of DNA repair by YM155 is attributable to down-regulation of survivin expression.
The antitumor activity of YM155 has previously been shown to be time-dependent, with continuous infusion of the drug resulting in greater antitumor activity and less systemic toxicity compared with bolus injection in tumor xenograft models in vivo (14). Ongoing clinical trials of YM155 are thus being done with the drug administered on a continuous schedule. We also administered YM155 by continuous infusion in our in vivo experiments. The combination of YM155 with single-dose radiotherapy resulted in a marked increase in tumor growth delay compared with that apparent with either radiation or YM155 alone, indicating that YM155 enhanced the antitumor effect of ionizing radiation in vivo. Given that standard radiation therapy in the clinic is delivered according to a fractionated schedule, we also examined whether YM155 enhanced the tumor response to clinically relevant fractionated doses (2 Gy) of radiation. Indeed, YM155 was also effective in enhancing the tumor response to such fractionated radiation. The enhancement factor with fractionated radiation (3.0) was similar to that observed with single-dose radiation (3.3) for H460 tumor xenografts.
Resistance to cytotoxic drugs and radiation is a major limiting factor in the treatment of cancer patients. Cross-resistance has been noted between radiotherapy and chemotherapy and has been attributed to defects in apoptosis signaling or to an enhanced capacity for DNA repair (51, 52). Our findings provide evidence that YM155 may break radioresistance by promoting apoptosis and inhibiting DNA repair. Previous studies have shown that suppression of survivin expression increases the sensitivity of tumor cells to chemotherapy(18, 53). It will therefore be of interest to determine whether YM155 also sensitizes tumor cells to chemotherapy.
In conclusion, we have shown that YM155 sensitizes NSCLC cells to radiation both in vitro and in animal models in vivo. The radiosensitization induced by YM155 seems to be attributable to the promotion of caspase-mediated apoptosis and inhibition of the repair of radiation-induced DNA damage. Our preclinical results provide a rationale for future clinical investigation of the therapeutic efficacy of YM155 in combination with radiotherapy.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank S. Ono for technical assistance.