FMS-Related Tyrosine Kinase 3 Ligand Promotes Radioresistance in Esophageal Squamous Cell Carcinoma

Abstract

Aim: The FMS-related tyrosine kinase 3 ligand (FL) has an important role in regulating FMS-related tyrosine kinase 3 (Flt-3) activity. Serum FL levels are markedly increased among patients with hematopoietic disease. However, its role in radiation treatment remains unclear. In this study, we investigated the effects of FL on radiotherapy for esophageal squamous cell carcinoma (ESCC). Methods: KYSE150 and KYSE450 cells were stimulated with FL (200 ng/ml). mRNA expression was analyzed using qRT-PCR. Cell viability was checked using CCK-8 assay kits. Proliferation was determined using the EdU assay. Radiosensitivity was detected through a colony-forming assay. Flow cytometry was used to evaluate cell apoptosis. The number of γH2AX foci was verified using an immunofluorescence assay. The change in relative proteins was determined by western blot analysis. The growth of transplanted tumors was demonstrated in nude mice. Results: Our results showed that FL increased the radiation resistance of ESCC cells by promoting clone formation, increasing EdU incorporation, enhancing DNA damage repair, and inhibiting apoptosis. Moreover, the Flt-3 receptor expression significantly increased in ESCC cells after radiation, which may have been an important factor in their radioresistance. Conclusion: Our results suggest that FL increases the radioresistance of esophageal cancer cells and that FL-Flt-3 could be a potential target for enhancing radiosensitivity in ESCC.

Keywords: DNA damage; apoptosis; esophageal squamous cell carcinoma; fms-related tyrosine kinase 3 ligand; radiotherapy.

FIGURE 1

FL promotes viability and tumor growth of ESCCs in vitro and in vivo. (A) The cell viability of ESCC cells in different concentration (0, 5, 10, 20, 40, 80, 160, 320, and 640 ng/ml) of FL (n = 3). (B) Cell viability of different time-point after 200 ng/ml FL treatment (n = 3). (C) The tumor volume assessed every 2 days (n = 5). (D) The tumor weight after sacrificed (n = 5). (E) Images of nude mice (n = 5). Mean ± SEM, *p < 0.05, **p < 0 0.01.

FIGURE 2

FL promotes radioresistance of ESCC cells in vitro. (A) The clonogenic survival assays in PBS groups or 200 ng/ml FL administration for 24 h groups after 24 h of 8Gy radiation. (B) The survival curves calculated and fitted to a multi-target single-hit model from (A). (C) the EdU incorporation assays in ESCC cells (Red fluorescence: EdU-positive cells; Blue fluorescence: total cells). (D) Mean rate of EdU positive cells in histogram. Mean ± SEM, N = 3, *p < 0.05, **p < 0.01.

FIGURE 3

FL reduces IR-induced DNA damage level and increases DNA repair. (A,B) Immunofluorescence detection of γH2AX foci in PBS groups or 200 ng/ml FL administration for 24 h groups after 8 Gy radiation different time. Mean ± SEM, N = 3, *p < 0.05, **p < 0 0.01 (C,D) PARP1 protein expression in PBS groups or 200 ng/ml FL administration for 24 h groups after 24 h of 8 Gy radiation. GAPDH is an internal reference.

FIGURE 4

FL decreases IR-induced apoptosis. (A,B) Apoptosis ratio in PBS groups or 200 ng/ml FL administration for 24 h groups after 24 h of 8 Gy radiation. Mean ± SEM, N = 3, *p < 0.05.

FIGURE 5

FL induces radioresistance in ESCC cells through Flt-3/AKT/Bad signal pathway. (A) mRNA levels of Flt-3 receptor with or without IR in ESCC cells. (B,C) Expression and the quantitative analysis results of Flt-3 and P-Flt-3 in ESCC cells after different doses (0, 2, 4, 6, 8, and 10 Gy) radiation. (D,E) Expression and the quantitative analysis results of Flt-3 and P-Flt-3 in ESCC cells administrated with 200 ng/ml FL for different time after 24 h of 8 Gy radiation. (F,G) The levels and the quantitative analysis results of P-Flt-3/P-PI3K/P-AKT signal proteins and apoptosis-related proteins Bad and P-Bad in PBS groups or 200 ng/ml FL administration for 30 min groups after 24 h of 8 Gy radiation. GAPDH is an internal reference. (H) IHC images of nude mice and IHC analysis of P-Flt-3 and Ki 67 in tumor sections of KYSE150 bearing nude mice. Data show mean ± SEM *p < 0.05.

FIGURE 6

Relevant mechanism of FL mediated the radioresistance of ESCCs during the radiotherapy. Radiation significantly increase Flt-3 receptor expression in ESCCs. FL phosphorylate the increased Flt-3 receptor, which further activate the PI3K/Akt pathway leading to BAD phosphorylation.