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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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The effect of 5-Aza and TSA treatment on lung cancer cell lines with hypermethylated Axin gene or unmethylated Axin gene. The Axin mRNA expression is significantly up-regulated in H157 cells after 5-Aza treatment or TSA treatment. Further increases in Axin transcripts are detected after combined use of 5-Aza and TSA in the H157 cells (A and B) but not in LTE cells (C and D). ** P<0.01, in comparison with control group (cells with no treatment).
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Figure 6: The effect of 5-Aza and TSA treatment on lung cancer cell lines with hypermethylated Axin gene or unmethylated Axin gene. The Axin mRNA expression is significantly up-regulated in H157 cells after 5-Aza treatment or TSA treatment. Further increases in Axin transcripts are detected after combined use of 5-Aza and TSA in the H157 cells (A and B) but not in LTE cells (C and D). ** P<0.01, in comparison with control group (cells with no treatment).

Mentions: Demethylation agent 5-Aza-2-Deoxycytidine (5-Aza, Sigma, 10 μM, 72 h) and deacetylase inhibitor TSA were used, and transcripts of the Axin gene were measured. Significant demethylation and increased Axin transcripts could be detected in H157 cells after 5-Aza treatment (P<0.01) (Figure 6A and B). When Trichostatin A (TSA, Sigma, 300 nmol/L, 24 h), an inhibitor of histone deacetylase, was used, the Axin mRNA expression was also up-regulated significantly (P<0.01) with no altered level of Axin gene methylation (Figure 6A and B). An additional increase in Axin transcripts was noted with combined use of 5-Aza and TSA in H157 (Figure 6A and B), suggesting a synergistic effect of demethylation and acetylation. In contrast, neither 5-Aza treatment nor TSA treatment could significantly up-regulate Axin expression in LTE cells and neither showed effects on methylation status of the Axin gene (Figure 6C and D).


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)

The effect of 5-Aza and TSA treatment on lung cancer cell lines with hypermethylated Axin gene or unmethylated Axin gene. The Axin mRNA expression is significantly up-regulated in H157 cells after 5-Aza treatment or TSA treatment. Further increases in Axin transcripts are detected after combined use of 5-Aza and TSA in the H157 cells (A and B) but not in LTE cells (C and D). ** P<0.01, in comparison with control group (cells with no treatment).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3750238&req=5

Figure 6: The effect of 5-Aza and TSA treatment on lung cancer cell lines with hypermethylated Axin gene or unmethylated Axin gene. The Axin mRNA expression is significantly up-regulated in H157 cells after 5-Aza treatment or TSA treatment. Further increases in Axin transcripts are detected after combined use of 5-Aza and TSA in the H157 cells (A and B) but not in LTE cells (C and D). ** P<0.01, in comparison with control group (cells with no treatment).
Mentions: Demethylation agent 5-Aza-2-Deoxycytidine (5-Aza, Sigma, 10 μM, 72 h) and deacetylase inhibitor TSA were used, and transcripts of the Axin gene were measured. Significant demethylation and increased Axin transcripts could be detected in H157 cells after 5-Aza treatment (P<0.01) (Figure 6A and B). When Trichostatin A (TSA, Sigma, 300 nmol/L, 24 h), an inhibitor of histone deacetylase, was used, the Axin mRNA expression was also up-regulated significantly (P<0.01) with no altered level of Axin gene methylation (Figure 6A and B). An additional increase in Axin transcripts was noted with combined use of 5-Aza and TSA in H157 (Figure 6A and B), suggesting a synergistic effect of demethylation and acetylation. In contrast, neither 5-Aza treatment nor TSA treatment could significantly up-regulate Axin expression in LTE cells and neither showed effects on methylation status of the Axin gene (Figure 6C and D).

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