Cancer stem cells in radiation resistance of esophageal cancer: role and molecular mechanism

Abstract

Objective To investigate the role of cancer stem cells in radiation resistance of esophageal cancer and its molecular mechanism, and to provide a theoretical basis for radiotherapy for esophageal cancer. Methods Esophageal cancer cell line TE1 was treated with 8 Gy of radiation. Esophageal cancer cell line with resistance to radiation, TE1-res, was established and screened. Cell counting was used to evaluate cell proliferation. Flow cytometry was used to determine the expression of CD44(high) CD24(-)CD133(+) and apoptosis in cells. The colony formation assay was used to determine the colony-forming rate and cell survival curve. Bisulfite sequencing PCR was used to determine the methylation status of cancer suppressor genes. Comparison of the data was made by group t test or analysis of variance. Results Compared with TE1 cells, TE1-res cells had significantly enhanced proliferation, a significantly higher proportion of CD44(high) CD24(-)CD133(+) cells, and significantly enhanced resistance to apoptosis (mean value 20.84×105vs. 4.46×105/day, P=0.008; (38.0±2.9)% vs. (10.1±1.3)%, P=0.001; mean value 33.23% vs. 10.50%, P=0.003). After treatment with 8 Gy of radiation, TE1-res cells had significantly higher colony-forming rate and D0 value than TE1 cells ((14.3±2.6)% vs. (0.9±0.3)%, P=0.011; 3.28 vs. 2.19 Gy, P=0.125). Moreover, the promoter methylation in cancer suppressor genes including SPINT2, CDKN1B, DKK1, TP53, and PPP2R1B was significantly enhanced in TE1-res cells than in TE1 cells ((89.7±4.9)% vs. (5.0±0.5)%, P=0.001; (92.3±4.7)% vs. (10.4±0.7)%, P=0.001; (90.7±3.7)% vs. (7.9±0.4)%, P=0.001; (83.4±5.7)% vs. (17.2±1.2)%, P=0.002; (90.2±6.7)% vs. (4.4±1.2)%, P=0.002). Conclusions Cancer stem cells play an important role in radiation resistance of esophageal cancer. The resistance to radiation is closely associated with promoter hypermethylation in cancer suppressor genes including SPINT2, CDKN1B, DKK1, TP53, and PPP2R1B. Key words: Esophageal neoplasms cell line; Irradioresistance; Cancer suppressor gene; Methylation