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Axin gene methylation status correlates with radiosensitivity of lung cancer cells.

Yang LH, Han Y, Li G, Xu HT, Jiang GY, Miao Y, Zhang XP, Zhao HY, Xu ZF, Stoecker M, Wang E, Xu K, Wang EH - BMC Cancer (2013)

Bottom Line: The mechanisms, however, were not clear.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.Hypermethylated Axin gene was detected in 2 of 4 cell lines, and it correlated inversely with Axin expression.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology, First Affiliated Hospital and College of Basic Medical Sciences, China Medical University, Shenyang, Liaoning, China.

ABSTRACT

Background: 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. The mechanisms, however, were not clear.

Methods: Four 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.

Results: Hypermethylated 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.

Conclusions: X-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.

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X-ray induced over-expression of the Axin gene in lung cancer cell lines with hypermethylated Axin gene. A and B show the methylation status of the promoter and first intron of the Axin gene and levels of Axin transcripts in 4 lung cancer cell lines. Note low Axin mRNA expression (A) in H157 and H446 cells, which exhibit a hypermethylated Axin gene (B). Real-time PCR shows no up-regulation of Axin mRNA in LTE cells and H460 cells (C and F) but a marked increase in the Axin transcripts in H157 (D) and H446 (E) cell lines after X-ray treatment. Note time and dose dependent increase in Axin transcripts after the treatment in both H157 (D) and H446 (E) cell lines. Pro-M, amplification for the methylated Axin gene promoter; Pro-U, amplification for the unmethylated Axin gene promoter; Int1-M, amplification for the methylated first intron of the Axin gene; Int1-U, amplification for the unmethylated first intron of the Axin gene. * P<0.05, and ** P<0.01, in comparison with the group without X-ray irradiation.
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Figure 1: X-ray induced over-expression of the Axin gene in lung cancer cell lines with hypermethylated Axin gene. A and B show the methylation status of the promoter and first intron of the Axin gene and levels of Axin transcripts in 4 lung cancer cell lines. Note low Axin mRNA expression (A) in H157 and H446 cells, which exhibit a hypermethylated Axin gene (B). Real-time PCR shows no up-regulation of Axin mRNA in LTE cells and H460 cells (C and F) but a marked increase in the Axin transcripts in H157 (D) and H446 (E) cell lines after X-ray treatment. Note time and dose dependent increase in Axin transcripts after the treatment in both H157 (D) and H446 (E) cell lines. Pro-M, amplification for the methylated Axin gene promoter; Pro-U, amplification for the unmethylated Axin gene promoter; Int1-M, amplification for the methylated first intron of the Axin gene; Int1-U, amplification for the unmethylated first intron of the Axin gene. * P<0.05, and ** P<0.01, in comparison with the group without X-ray irradiation.

Mentions: Nested 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<0.01). This result suggests that hypermethylated Axin gene correlated inversely with Axin expression. Then all cell lines were treated with X-ray irradiation. Axin mRNA was apparently up-regulated in H157 and H446 cells that have hypermethylated Axin gene but not in LTE and H460 cells that have unmethylated Axin gene (Figure 1C-F). Interestingly, X-ray irradiation in H157 (Figure 1D) and H446 cells (Figure 1E) seems to demonstrate time dependent and dose dependent increases of Axin transcripts, with a more significant increase noted at the 72 hour point (P<0.01) and with 2 Gy. This time and dose dependent fashion of up-regulation of the Axin gene was not observed in LTE and H460 cells. Axin mRNA was not increased after X-ray irradiation in LTE (Figure 1C) or H460 cells (Figure 1F). 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.


Axin gene methylation status correlates with radiosensitivity of lung cancer cells.

Yang LH, Han Y, Li G, Xu HT, Jiang GY, Miao Y, Zhang XP, Zhao HY, Xu ZF, Stoecker M, Wang E, Xu K, Wang EH - BMC Cancer (2013)

X-ray induced over-expression of the Axin gene in lung cancer cell lines with hypermethylated Axin gene. A and B show the methylation status of the promoter and first intron of the Axin gene and levels of Axin transcripts in 4 lung cancer cell lines. Note low Axin mRNA expression (A) in H157 and H446 cells, which exhibit a hypermethylated Axin gene (B). Real-time PCR shows no up-regulation of Axin mRNA in LTE cells and H460 cells (C and F) but a marked increase in the Axin transcripts in H157 (D) and H446 (E) cell lines after X-ray treatment. Note time and dose dependent increase in Axin transcripts after the treatment in both H157 (D) and H446 (E) cell lines. Pro-M, amplification for the methylated Axin gene promoter; Pro-U, amplification for the unmethylated Axin gene promoter; Int1-M, amplification for the methylated first intron of the Axin gene; Int1-U, amplification for the unmethylated first intron of the Axin gene. * P<0.05, and ** P<0.01, in comparison with the group without X-ray irradiation.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 1: X-ray induced over-expression of the Axin gene in lung cancer cell lines with hypermethylated Axin gene. A and B show the methylation status of the promoter and first intron of the Axin gene and levels of Axin transcripts in 4 lung cancer cell lines. Note low Axin mRNA expression (A) in H157 and H446 cells, which exhibit a hypermethylated Axin gene (B). Real-time PCR shows no up-regulation of Axin mRNA in LTE cells and H460 cells (C and F) but a marked increase in the Axin transcripts in H157 (D) and H446 (E) cell lines after X-ray treatment. Note time and dose dependent increase in Axin transcripts after the treatment in both H157 (D) and H446 (E) cell lines. Pro-M, amplification for the methylated Axin gene promoter; Pro-U, amplification for the unmethylated Axin gene promoter; Int1-M, amplification for the methylated first intron of the Axin gene; Int1-U, amplification for the unmethylated first intron of the Axin gene. * P<0.05, and ** P<0.01, in comparison with the group without X-ray irradiation.
Mentions: Nested 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<0.01). This result suggests that hypermethylated Axin gene correlated inversely with Axin expression. Then all cell lines were treated with X-ray irradiation. Axin mRNA was apparently up-regulated in H157 and H446 cells that have hypermethylated Axin gene but not in LTE and H460 cells that have unmethylated Axin gene (Figure 1C-F). Interestingly, X-ray irradiation in H157 (Figure 1D) and H446 cells (Figure 1E) seems to demonstrate time dependent and dose dependent increases of Axin transcripts, with a more significant increase noted at the 72 hour point (P<0.01) and with 2 Gy. This time and dose dependent fashion of up-regulation of the Axin gene was not observed in LTE and H460 cells. Axin mRNA was not increased after X-ray irradiation in LTE (Figure 1C) or H460 cells (Figure 1F). 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.

Bottom Line: The mechanisms, however, were not clear.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.Hypermethylated Axin gene was detected in 2 of 4 cell lines, and it correlated inversely with Axin expression.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology, First Affiliated Hospital and College of Basic Medical Sciences, China Medical University, Shenyang, Liaoning, China.

ABSTRACT

Background: 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. The mechanisms, however, were not clear.

Methods: Four 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.

Results: Hypermethylated 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.

Conclusions: X-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.

Show MeSH
Related in: MedlinePlus