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. 2017 Feb 21;8(8):13600-13619.
doi: 10.18632/oncotarget.14618.

Telomerase antagonist imetelstat increases radiation sensitivity in esophageal squamous cell carcinoma

Xuping Wu  1 , Jing Zhang  2 , Sijun Yang  3 , Zhihui Kuang  2 , Guolei Tan  1 , Gang Yang  1 , Qichun Wei  4 , Zhigang Guo  2
Affiliations

Telomerase antagonist imetelstat increases radiation sensitivity in esophageal squamous cell carcinoma

Xuping Wu et al. Oncotarget. .

Abstract

The morbidity and mortality of esophageal cancer is one of the highest around the world and the principal therapeutic method is radiation. Thus, searching for sensitizers with lower toxicity and higher efficiency to improve the efficacy of radiation therapy is critical essential. Our research group has previously reported that imetelstat, the thio-phosphoramidate oligonucleotide inhibitor of telomerase, can decrease cell proliferation and colony formation ability as well as increase DNA breaks induced by radiation in esophageal cancer cells. Further study in this project showed that imetelstat significantly sensitized esophageal cancer cells to radiation in vitro. Later study showed that imetelstat leads to increased cell apoptosis. We also measured the expression level of several DNA repair and apoptosis signaling proteins. pS345 CHK1, γ-H2AX, p53 and caspase3 expression were up-regulated in imetelstat treated cells, identifying these factors as molecular markers. Mouse in vivo model using imetelstat at clinically achievable concentrations and fractionated irradiation scheme yielded results demonstrating radiosensitization effect. Finally, TUNEL assay, caspase 3 and Ki67 staining in tumor tissue proved that imetelstat sensitized esophageal cancer to radiation in vivo through promoting cell apoptosis and inhibiting cell proliferation. Our study supported imetelstat increase radiation sensitivity of esophageal squamous cell carcinoma through inducing cell apoptosis and the specific inhibitor of telomerase might serve as a potential novel therapeutic tool for esophageal squamous cell carcinoma therapy.

Keywords: apoptosis; esophageal squamous cell carcinoma; radiosensitization; telomerase.

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Conflict of interest statement

CONFLICTS OF INTEREST

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1. Imetelstat radiosensitizes Kyse410 and Kyse520 cells by using clonogenic cell survival following imetelstat and/or X-ray irradiation (2, 4, 6 and 8 Gy)
(A) shows survival fraction as a function of imetelstat, radiation dose and their combination. (B) shows survival plots as a function of radiation dose with or without imetelstat. Survival data were fitted to S = exp (-αD-βD2) where D is the dose in Gy and α and β are fitting parameters, n ≥ 6, Error bars represent standard deviation. All imetelstat treatment had P-values less than 0.05 except imetelstat + 6 Gy treatment for Kyse410.
Figure 2
Figure 2. Imetelstat increases cell apoptosis of Kyse410 and Kyse520 cell
Kyse410 and Kyse520 cells were incubated and treated with 5 μM sense or imetelstat for 72 h and at the same time exposed to 10 μM TMZ. Results of early and late apoptosis were added together to calculate the total amount of apoptosis. Values represent the mean±SD and are representative of three independent experiments. *p < 0.05, statistical significance was established by t test.
Figure 3
Figure 3. DNA repair and apoptosis signaling protein were upregulated by the treatment of imetelstat in Kyse410 and Kyse520 cells
(A) Kyse410 and Kyse520 cells were treated with 5 μM imetelstat or 5 μM sense for 72 h and meanwhile exposed to 10 μM TMZ. The expressions of phosphorylation of CHK1 at S345 were detected, taking the expression of TBB5 as inner control. (B and C) Kyse410 and Kyse520 cells were treated with 5 μM imetelstat or 5 μM sense for 72 h together with TMZ (0, 5, 10, 20 μM). Expression levels of γ-H2AX, p53 and caspase 3 were detected by immunoblotting. The intensity of each protein band was quantified using the BandScan software (Glyko) and normalized against tublin. *represents statistical significant (p < 0.05).
Figure 3
Figure 3. DNA repair and apoptosis signaling protein were upregulated by the treatment of imetelstat in Kyse410 and Kyse520 cells
(A) Kyse410 and Kyse520 cells were treated with 5 μM imetelstat or 5 μM sense for 72 h and meanwhile exposed to 10 μM TMZ. The expressions of phosphorylation of CHK1 at S345 were detected, taking the expression of TBB5 as inner control. (B and C) Kyse410 and Kyse520 cells were treated with 5 μM imetelstat or 5 μM sense for 72 h together with TMZ (0, 5, 10, 20 μM). Expression levels of γ-H2AX, p53 and caspase 3 were detected by immunoblotting. The intensity of each protein band was quantified using the BandScan software (Glyko) and normalized against tublin. *represents statistical significant (p < 0.05).
Figure 4
Figure 4. Imetelstat sensitizes esophageal cancer cell Kyse520 to radiation in vivo
Kyse520 cells were pretreated with imetelstat or sense for 7 weeks before injected into nude mice to form tumors. When the size of tumor reached approximately 50 mm3, mice were randomly divided into 6 different groups, including untreated group, radiation group, sense group, imetelstat group, sense + radiation group and imetelstat + radiation group. Tumors in nude mice were subjected to 2 Gy of irradiation for 5 consecutive days to mimic a fractionated weekly scheme. The sizes of tumor were measured 3 times per week to obtain cell growth curve. At days 13, 31 and 45 postinjection, imetelstat were injected intraperitoneally to ensure inhibition of telomerase activity. Data are presented as mean tumor volume ± SE. Imetelstat with irradiation significantly decreased tumor growth compared with mice receiving irradiation alone or irradiation with sense (p < 0.05).
Figure 5
Figure 5. Imetelstat increases cell apoptosis and decreases cell proliferation in vivo
Pathological changes of esophageal squamous carcinoma were evaluated by H&E staining. Apoptotic cells were labeled by TUNEL assay and further evaluated by immunofluorescence staining of caspase 3. Imetelstat promoted radiation-induced cell apoptosis compared with radiation-treated tumor. The cell proliferation was evaluated by Ki67 staining in tumor tissue. Imetelstat combined with radiation showed less Ki67 expression compared to other treatment groups.
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