Abstract
BackgroundWe previously reported that Axin1 (Axin) is down-regulated in many cases of lung cancer, and X-ray irradiation increased Axin expression and inhibited lung cancer cells. The mechanisms, however, were not clear.MethodsFour lung cancer cell lines were used to detect the methylation status of Axin with or without X-ray treatment. Real-time PCR was used to quantify the expression of Axin, and western blot analysis was applied to measure protein levels of Axin, β-catenin, Cyclin D1, MMP-7, DNMTS, MeCP2 and acetylated histones. Flow cytometric analysis, colony formation assay, transwell assay and xenograft growth experiment were used to study the biological behavior of the cells with hypermethylated or unmethylated Axin gene after X-ray treatment.ResultsHypermethylated Axin gene was detected in 2 of 4 cell lines, and it correlated inversely with Axin expression. X-ray treatment significantly up-regulated Axin expression in H446 and H157 cells, which possess intrinsic hypermethylation of the Axin gene (P<0.01), but did not show up-regulation in LTE and H460 cells, which have unmethylated Axin gene. 2Gy X-ray significantly reduced colony formation (from 71% to 10.5%) in H157 cells, while the reduction was lower in LTE cells (from 71% to 20%). After X-ray irradiation, xenograft growth was significantly decreased in H157 cells (from 1.15 g to 0.28 g) in comparison with LTE cells (from 1.06 g to 0.65 g). Significantly decreased cell invasiveness and increased apoptosis were also observed in H157 cells treated with X-ray irradiation (P<0.01). Down-regulation of DNMTs and MeCP2 and up-regulation of acetylated histones could be detected in lung cancer cells.ConclusionsX-ray-induced inhibition of lung cancer cells may be mediated by enhanced expression of Axin via genomic DNA demethylation and histone acetylation. Lung cancer cells with a different methylation status of the Axin gene showed different radiosensitivity, suggesting that the methylation status of the Axin gene may be one important factor to predict radiosensitivity of the tumor.
Highlights
We previously reported that Axin1 (Axin) is down-regulated in many cases of lung cancer, and X-ray irradiation increased Axin expression and inhibited lung cancer cells
Effect of X-ray irradiation on axin mrna expression and methylation in lung cancer cells with hypermethylated or unmethylated Axin gene Nested methylation specific PCR (MSP) showed that the promoter and first intron regions of the Axin gene are hypermethylated in H157 and H446 cells but unmethylated in LTE and H460 cells, and correspondingly, Real-time RT-PCR demonstrated that H157 and H446 cells had a mean level of Axin mRNA significantly lower than LTE and H460 cells (Figure 1A and B) (P
These results suggest that X-ray irradiation could possibly up-regulate Axin expression in the cells with hypermethylated Axin gene but not in the cells with unmethylated Axin gene
Summary
We previously reported that Axin (Axin) is down-regulated in many cases of lung cancer, and X-ray irradiation increased Axin expression and inhibited lung cancer cells. X-ray irradiation has been shown to induce demethylation of the whole genome by inhibiting DNA methyltransferases (DNMTs) and methyl-binding protein 2 (MeCP2) [14,15,16,17,18,19,20,21]. These previous studies raise the possibility that X-ray irradiation triggers apoptosis of lung cancer cells via demethylationand acetylation-mediated up-regulation of the Axin gene by inhibiting DNMTs and MeCP2 [22]. We studied the effects of X-ray irradiation on expression of Axin, DNMTs, and MeCP2, its effect on the methylation status of the Axin gene, and the associated changes in cell proliferation, invasiveness, apoptosis and tumor progression
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