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. 2012 Sep;11(9):1988-98.
doi: 10.1158/1535-7163.MCT-12-0167. Epub 2012 Jun 21.

YM155 reverses cisplatin resistance in head and neck cancer by decreasing cytoplasmic survivin levels

Bhavna Kumar  1 , Arti Yadav, James C Lang, Michael J Cipolla, Alessandra C Schmitt, Nicole Arradaza, Theodoros N Teknos, Pawan Kumar
Affiliations

YM155 reverses cisplatin resistance in head and neck cancer by decreasing cytoplasmic survivin levels

Bhavna Kumar et al. Mol Cancer Ther. 2012 Sep.

Abstract

Cisplatin is one of the commonly used chemotherapeutic drugs for the treatment of head and neck squamous cell carcinoma (HNSCC). However, acquisition of cisplatin resistance is common in patients with HNSCC, and it often leads to local and distant failure. In this study, we showed that survivin expression is significantly upregulated in HNSCC primary tumors and cell lines. In addition, survivin levels were significantly higher in human papilloma virus-negative patients that normally respond poorly to cisplatin treatment. Survivin expression was further increased in cisplatin-resistant cells (CAL27-CisR) as compared with its parent cells (CAL27). Therefore, we hypothesized that targeting of survivin in HNSCC could reverse the resistant phenotype in tumor cells, thereby enhancing the therapeutic efficacy of cisplatin. We used both in vitro and in vivo models to test the efficacy of YM155, a small molecule survivin inhibitor, either as a single agent or in combination with cisplatin. YM155 significantly decreased survivin levels and cell proliferation in a dose-dependent manner. In addition, YM155 pretreatment significantly reversed cisplatin resistance in cancer cells. Interestingly, YM155 treatment altered the dynamic localization of survivin in cells by inducing a rapid reduction in cytoplasmic survivin, which plays a critical role in its antiapoptotic function. In a severe combined immunodeficient mouse xenograft model, YM155 significantly enhanced the antitumor and antiangiogenic effects of cisplatin, with no added systemic toxicity. Taken together, our results suggest a potentially novel strategy to use YM155 to overcome the resistance in tumor cells, thereby enhancing the effectiveness of the chemotherapy in HNSCC.

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Conflict of interest statement

Conflict of interest: The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1. Survivin expression is upregulated in HNSCC cell lines and patient’s primary tumor samples
A: Survivin expression in primary tumor samples (n=10) and in adjacent normal mucosa (n=10) of head and neck cancer patients. B: Survivin stain intensity was analyzed in p16 negative (n=56) and p16 positive primary tumor samples (n=169). C: Survivin stain intensity was analyzed in tumors from patients with ≤10 pack (pk)-years smoking history (n=58) and compared with survivin stain intensity in tumors from patients with >10 pack-years smoking history (n=157). Blue box, interquartile range (25th to 75th percentile); black diamond, mean; black horizontal bar, median; top and bottom whiskers, maximum and minimum values, respectively. D–E: Survivin expression was analyzed in normal keratinocytes and head and neck cancer cell lines by RT-PCR (D) and Western blotting (E). For survivin Western blot, equal protein loading was verified by stripping the blots and reprobing with GAPDH antibody.
Figure 2
Figure 2. Survivin expression is upregulated in cisplatin resistant cells and YM155 inhibits survivin expression in a dose dependent manner
A: CAL27-CisR and its parental cell line CAL27 were treated with different concentrations of cisplatin (CDDP) and cell proliferation was assessed by MTT assay. B–C: Survivin expression in CAL27-CisR and its parental cell line CAL27 was examined by RT-PCR (B) and Western blotting (C). *, represents a significant difference (p<0.05). D–F: Survivin expression was knocked down in CAL27-CisR cells by siRNA. D: Survivin knockdown was verified by Western blotting. E–F: CAL27-CisR cells with survivin knockdown or treated with scrambled RNA (SC) were treated with cisplatin (CDDP) and examined for cell proliferation (E; MTT assay) or colony formation (F). G: Chemical structure of YM155. H–I: UM-SCC-74A and CAL27-CisR cells were treated with different doses of YM155 and survivin expression was examined by Western Blotting. Equal protein loading was verified by stripping the blots and reprobing with GAPDH antibody.
Figure 3
Figure 3. YM155 enhances the anti-proliferative effects of cisplatin treatment
A: CAL27, CAL27-CisR cells were treated with YM155 or cisplatin (CDDP) alone or in combination and cell proliferation was assessed by MTT assay. *, represents a significant difference (p<0.05) as compared to no treatment group and **, represents a significant difference (p<0.05) as compared to single treatment groups. B: CAL27-CisR cells were treated with cisplatin (CDDP) or YM155 alone or in combination. After 72 hours, cells were stained with annexin V and analyzed by flow cytometry. C–D: CAL27-CisR cells were treated with YM155 or cisplatin (CDDP) alone or in combination for 72 hours. Four thousand viable cells from each group were cultured for additional 10 days 6 cm plates. Each assay was photographed and the number of colonies analyzed. E–F: UM-SCC-74A and CAL27-CisR cells were treated with YM155 or cisplatin (CDDP) alone or in combination and survivin expression was examined by Western Blotting. Equal protein loading was verified by stripping the blots and reprobing with GAPDH antibody. G: CAL27-CisR cells were cultured in 4-well labtech chambers and treated with YM155 (10 nM) for different time points. At the end of YM155 treatment, cells were fixed and stained with survivin (red), β–catenin (green, membrane staining), and DAPI (blue, nucleus). H: CAL27-CisR cells were treated with YM155 (10 nM) for different time points and cytosolic and nuclear protein fractions were isolated and probed for survivin expression by Western Blot. Purity of cytosolic and nuclear fractions was verified by the presence of GAPDH and lamin A/C, respectively.
Figure 4
Figure 4. YM155 inhibits tumor growth in a dose-dependent manner
A–B: Tumor bearing animals (n=5) were treated with YM155 at different doses (1 mg/kg, 3 mg/kg or 10 mg/kg) as described in methods. A: Representative photomicrographs of tumors from untreated, YM155 1 mg/kg, YM155 3 mg/kg and YM155 10 mg/kg groups. B: Tumor growth curves for UM-SCC-74A tumors treated with different doses of YM155. C: Survivin levels in UM-SCC-74A tumors at the end of the in vivo experiments. *, represent a significant difference (p<0.05).
Figure 5
Figure 5. YM155 and cisplatin combination significantly inhibits tumor growth
Animals bearing CAL27, CAL27-CisR or UM-SCC-74A were treated with YM155 (3 mg/kg) or cisplatin (CDDP, 5 mg/kg) alone or in combination. A: Representative photomicrographs of tumors from untreated, cisplatin (CDDP), YM155, or CDDP and YM155 treated groups of CAL27 or CAL27-CisR. B: B: Tumor growth curves for CAL27 and CAL27-CisR tumors treated with cisplatin (CDDP), YM155, or CDDP and YM155. C: Representative photomicrographs of tumors from untreated, cisplatin (CDDP), YM155, or CDDP and YM155 treated groups of UM-SCC-74A. D: Tumor growth curves for UM-SCC-74A tumors treated with cisplatin (CDDP), YM155, or CDDP and YM155. *, represents a significant difference (p<0.05) as compared to no treatment group and **, represents a significant difference (p<0.05) as compared to single treatment groups.
Figure 6
Figure 6. YM155 and cisplatin combination treatment significantly inhibits tumor angiogenesis and VEGF mediated endothelial cell tube formation
A: Representative photomicrographs of tumor blood vessel staining for untreated, cisplatin (CDDP) or YM155 alone or combination groups for UM-SCC-74A tumors. B–C: Microvessel density in the tumor samples was calculated by counting 5 random fields (200x) and expressed as vessel density ± SE. *, represents a significant difference (p<0.05) as compared to no treatment group and **, represents a significant difference (p<0.05) as compared to single treatment groups. D: Representative photomicrographs of in vitro tube formation assay for untreated, VEGF, VEGF + cisplatin (V + CDDP), VEGF + YM155 (V + YM155), and VEFG + CDDP + YM155. E: Quantitative data for tube formation expressed as angiogenic score ± SE from three independent experiments. E: Endothelial cells were treated with VEGF and cisplatin (5 μM) or YM155 (10 nM) alone or in combination for 48 hour. Whole cell lysates from each group were Western blotted and probed for survivin. Equal sample loading was verified by stripping the blots and reprobing with anti-GAPDH antibody.
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