Raltitrexed increases radiation sensitivity of esophageal squamous carcinoma cells

Abstract

Background: Radiation therapy remains an important therapeutic modality, especially for those patients who are not candidates for radical resection. Many strategies have been developed to increase the radiosensitivity of esophageal cancer, with some success.

Methods: This study was conducted to determine whether raltitrexed can enhance radiosensitivity of esophageal squamous cell carcinoma (ESCC). ESCC cell lines 24 h were incubated with raltitrexed or DMSO with or without subsequent irradiation. Cell Counting Kit assay-8 assay and clonogenic survival assay were used to measure the cell proliferation and radiosensitization, respectively. Flow cytometry was utilized to examine cell apoptosis and cell cycle distribution in different groups. Immunofluorescence analysis was performed to detect deoxyribonucleic acid (DNA) double-strand breaks. In addition, the expression levels of proteins that are involved in radiation induced signal transduction including Bax, Cyclin B1, Cdc2/pCdc2, and Cdc25C/pCdc25C were examined by western blot analysis.

Results: The results indicated that raltitrexed enhanced radiosensitivity of ESCC cells with increased DNA double-strand breaks, the G2/M arrest, and the apoptosis of ESCC cells induced by radiation. The sensitization enhancement ratio of 1.23-2.10 was detected for ESCC cells with raltitrexed treatment in TE-13 cell line. In vitro, raltitrexed also increased the therapeutic effect of radiation in nude mice.

Conclusion: Raltitrexed increases the radiosensitivity of ESCC. This antimetabolite drug is promising for future clinical trials with concurrent radiation in esophageal cancer.

Keywords: Cell apoptosis; Cell cycle arrest; Esophageal cancer; Radiosensitization; Raltitrexed.

Fig. 1

Raltitrexed (Ral) inhibited cell viability of esophageal squamous cancer cell lines and potentiated the inhibitory effect on cell viability and colony formation by irradiation (IR). The effects of different doses of Ral on the cell viability 24, 48 or 72 h’ treatment were determined in TE-13 (a) and Kyse150 cell lines (b); the effects of IR in different doses with or without Ral (4 nM) pretreatment (24 h) on the proliferation of TE-13 (c) and Kyse150 cell lines (d) were studied after 48 h using Cell counting kit 8 assay; the radiosensitization effect of Ral (4 nM, 8 nM) was studied in TE-13 (e) and Kyse150 cell lines (f) using clonogenic survival assay. Error bar: standard deviation; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 2

Raltitrexed (Ral) promoted irradiation (IR) induced cell cycle distribution and protein expression of TE-13 and Kyse150 cell lines. The effect of different doses of IR on cell cycle distribution in TE-13 (a) and Kyse150 cell lines (b); the effects of IR (4 Gy) with or without Ral (4 nM) pretreatment (24 h) on cell cycle were studied in TE-13 (c, e) and Kyse150 (d, f) cell lines 24 h after IR; the protein expression related to G2/M arrest in TE-13 (g) and Kyse150 (h) cell lines. Error bar: standard deviation; *p < 0.05, ***p < 0.001, ^#p < 0.0001

Fig. 3

Raltitrexed (Ral) increased cell apoptosis in esophageal squamous cancer cells. The percentages of apoptosis cells were measured by flow cytometry after Ral (4 nM) treatment, irradiation (IR, 8 Gy, 24 h or 48 h) or combination of IR (8 Gy, 24 h or 48 h) + Ral (4 nM) in TE-13 (a, c) and Kyse150 (b, d) cell lines. Expression of apoptosis related proteins after different treatments was studied in TE-13 and Kyse150 (e). *p < 0.05, ***p < 0.001, ****p < 0.0001

Fig. 4

Raltitrexed (Ral) enhanced irradiation (IR) induced DNA damage in TE-13 and Kyse150 cell lines. γ-H2AX staining with Ral (4 nM) or without Ral (4 nM) pretreatment (24 h) in TE-13 (a, c) and Kyse150 (b, d) cell lines at each time point after IR exposure; TE-13 and Kyse150 cells were incubated with 0.1% DMSO or 4 nM Raltitrexed for 24 h and then exposed to 6 Gy of X ray for 2 h, 4 h or 24 h followed by immunofluorescence staining of γ-H2AX (green). Immunoreactive foci were ≥ 10 in nuclei that considered as positive for γ-H2AX. e Protein expression of P53, γ-H2AX, FPGS and TS in TE-13 and Kyse150 cells; f the effect of Ral (4 nM), IR (6 Gy) or combination of Ral (4 nM) and IR (6 Gy) in TE-13 and Kyse150 cells. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 5

Raltitrexed (Ral) sensitized radiation (IR) induced anticancer effect in TE-13 xenografts. a The sizes of xenograft tumors in four different groups, including the control group, Ral group (7.5 mg/kg/day, day 0–4 and day 7–11), IR group (8 Gy day 0), and combination group; b tumor volumes in four groups; c T/C(%), tumor volume of treatment Group (T) compared with the control group (C); d mice weight in the four groups during the treatment; e comparison of tumor weight of the four groups; f, g immunohistochemistry of xenograft tumor for Ki-67 and PCNA. Error bar: standard deviation; *p < 0.05, ***p < 0.001, ****p < 0.0001