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FH535 increases the radiosensitivity and reverses epithelial-to-mesenchymal transition of radioresistant esophageal cancer cell line KYSE-150R.

Su H, Jin X, Zhang X, Zhao L, Lin B, Li L, Fei Z, Shen L, Fang Y, Pan H, Xie C - J Transl Med (2015)

Bottom Line: EMT phenotype was presented in the KYSE-150R cells with decreased E-cadherin and increased snail and twist expressions.The up-regulated expressions of Wnt/β-catenin signaling pathway proteins (Wnt1, FZD1-4, GSK3β, CTNNB1 and Cyclin D1), the increased phosphorylation of GSK3β, and the decreased phosphorylation of β-catenin were observed in KYSE-150R cells compared with KYSE-150 cells, implicating the activation of the Wnt pathway in KYSE-150R cells.The expression of nuclear β-catenin and nuclear translocation of β-catenin from the cytoplasm was decreased after FH535 treatment.

View Article: PubMed Central - PubMed

Affiliation: Radiotherapy and Chemotherapy Deparment, the 1st Affiliated Hospital of Wenzhou Medical University, No.2 Fuxue Lane, 325000, Wenzhou, China. 12602639@qq.com.

ABSTRACT

Background: Acquired radioresistance has significantly compromised the efficacy of radiotherapy for esophageal cancer. The purpose of this study is to investigate the roles of epithelial-mesenchymal transition (EMT) and the Wnt/β-catenin signaling pathway in the acquirement of radioresistance during the radiation treatment of esophageal cancer.

Methods: We previously established a radioresistant cell line (KYSE-150R) from the KYSE-150 cell line (a human cell line model for esophageal squamous cell carcinoma) with a gradient cumulative irradiation dose. In this study, the expression of EMT phenotypes and the Wnt/β-catenin signaling pathway proteins were examined by real-time PCR, western blot and immunofluorescence in the KYSE-150R cells. The KYSE-150R cells were then treated with a β-Catenin/Tcf inhibitor FH535. The expressions of nuclear and cytoplasmic β-catenin and EMT markers in KYSE-150R cells were assessed at both mRNA and protein level after FH535 treatment. The radiosensitization effect of FH535 on KYSE-150R was evaluated by CCK8 analysis and a colony forming assay. DNA repair capacities was detected by the neutral comet assays.

Results: KYSE-150R cell line displayed obvious radiation resistance and had a stable genetic ability. EMT phenotype was presented in the KYSE-150R cells with decreased E-cadherin and increased snail and twist expressions. The up-regulated expressions of Wnt/β-catenin signaling pathway proteins (Wnt1, FZD1-4, GSK3β, CTNNB1 and Cyclin D1), the increased phosphorylation of GSK3β, and the decreased phosphorylation of β-catenin were observed in KYSE-150R cells compared with KYSE-150 cells, implicating the activation of the Wnt pathway in KYSE-150R cells. The expression of nuclear β-catenin and nuclear translocation of β-catenin from the cytoplasm was decreased after FH535 treatment. FH535 also reversed EMT phenotypes by increasing E-cadherin expression. The cell proliferation rates of KYSE-150R were dose-dependent and the radiation survival fraction was significantly decreased upon FH535 treatment. Neutral comet assays indicated that FH535 impairs DNA double stranded break repair in KYSE-150R cells.

Conclusions: Acquisition of radioresistance and EMT in esophageal cancer cells is associated with the activation of the Wnt/β-catenin pathway. EMT phenotypes can be reduced and the radiosensitivity of esophageal cancer cells can be enhanced by inhibiting the Wnt/β-catenin pathway with FH535 treatment.

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EMT suppress in KYSE-150R cell line with FH535. (A) The effect of FH535 on EMT target genes expression determined by RT-PCR. All samples were normalized to the signal generated from housekeeping gene (β-actin). (B) Western blot analysis of EMT protein levels treated with FH535 for 24 h, GAPDH was used as normalization control. (C) Immunofluorescence analysis showed protein levels of E-cadherin and Vimentin. Nuclei were counterstained with DAPI (blue). The images were zoomed in 400×.
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Fig5: EMT suppress in KYSE-150R cell line with FH535. (A) The effect of FH535 on EMT target genes expression determined by RT-PCR. All samples were normalized to the signal generated from housekeeping gene (β-actin). (B) Western blot analysis of EMT protein levels treated with FH535 for 24 h, GAPDH was used as normalization control. (C) Immunofluorescence analysis showed protein levels of E-cadherin and Vimentin. Nuclei were counterstained with DAPI (blue). The images were zoomed in 400×.

Mentions: Next, we sought to test whether FH535 could affect the EMT markers in the treated KYSE-150R cell line. A statistically significant increase of E-cadherin mRNA and decreases of Vimentin (VIM)、Snail and GSK3β mRNAs after FH535 treatment were observed in the KYSE-150R cells (Figure 5A). Consistently, Western blot and immunofluorescence analysis results showed significantly increase of protein levels of E-cadherin in the KYSE-150R cells (Figure 5B). The increased expression of E-cadherin in radioresistant KYSE-150R cells treated with FH535 was 0.081 ± 0.0028, and the average optical density of group without FH535 treatment was 0.056 ± 0.0025 (p = 0.01). However, no significant difference in the expression of Vimentin was observed between FH535 treated groups and controls in both western blot and immunofluorescence analysis. The expression of Vimentin in radioresistant KYSE-150R cells treated with FH535 was 0.042 ± 0.0016, and the average optical density of group without FH535 treatment was 0.040 ± 0.0005 (p = 0.222). Interestingly, the Wnt pathway target gene Cyclin D1 was down-regulated specifically in the FH535 treatment group in both mRNA and protein level, indicating the inhibition of the Wnt/β-catenin pathway.Figure 5


FH535 increases the radiosensitivity and reverses epithelial-to-mesenchymal transition of radioresistant esophageal cancer cell line KYSE-150R.

Su H, Jin X, Zhang X, Zhao L, Lin B, Li L, Fei Z, Shen L, Fang Y, Pan H, Xie C - J Transl Med (2015)

EMT suppress in KYSE-150R cell line with FH535. (A) The effect of FH535 on EMT target genes expression determined by RT-PCR. All samples were normalized to the signal generated from housekeeping gene (β-actin). (B) Western blot analysis of EMT protein levels treated with FH535 for 24 h, GAPDH was used as normalization control. (C) Immunofluorescence analysis showed protein levels of E-cadherin and Vimentin. Nuclei were counterstained with DAPI (blue). The images were zoomed in 400×.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4384308&req=5

Fig5: EMT suppress in KYSE-150R cell line with FH535. (A) The effect of FH535 on EMT target genes expression determined by RT-PCR. All samples were normalized to the signal generated from housekeeping gene (β-actin). (B) Western blot analysis of EMT protein levels treated with FH535 for 24 h, GAPDH was used as normalization control. (C) Immunofluorescence analysis showed protein levels of E-cadherin and Vimentin. Nuclei were counterstained with DAPI (blue). The images were zoomed in 400×.
Mentions: Next, we sought to test whether FH535 could affect the EMT markers in the treated KYSE-150R cell line. A statistically significant increase of E-cadherin mRNA and decreases of Vimentin (VIM)、Snail and GSK3β mRNAs after FH535 treatment were observed in the KYSE-150R cells (Figure 5A). Consistently, Western blot and immunofluorescence analysis results showed significantly increase of protein levels of E-cadherin in the KYSE-150R cells (Figure 5B). The increased expression of E-cadherin in radioresistant KYSE-150R cells treated with FH535 was 0.081 ± 0.0028, and the average optical density of group without FH535 treatment was 0.056 ± 0.0025 (p = 0.01). However, no significant difference in the expression of Vimentin was observed between FH535 treated groups and controls in both western blot and immunofluorescence analysis. The expression of Vimentin in radioresistant KYSE-150R cells treated with FH535 was 0.042 ± 0.0016, and the average optical density of group without FH535 treatment was 0.040 ± 0.0005 (p = 0.222). Interestingly, the Wnt pathway target gene Cyclin D1 was down-regulated specifically in the FH535 treatment group in both mRNA and protein level, indicating the inhibition of the Wnt/β-catenin pathway.Figure 5

Bottom Line: EMT phenotype was presented in the KYSE-150R cells with decreased E-cadherin and increased snail and twist expressions.The up-regulated expressions of Wnt/β-catenin signaling pathway proteins (Wnt1, FZD1-4, GSK3β, CTNNB1 and Cyclin D1), the increased phosphorylation of GSK3β, and the decreased phosphorylation of β-catenin were observed in KYSE-150R cells compared with KYSE-150 cells, implicating the activation of the Wnt pathway in KYSE-150R cells.The expression of nuclear β-catenin and nuclear translocation of β-catenin from the cytoplasm was decreased after FH535 treatment.

View Article: PubMed Central - PubMed

Affiliation: Radiotherapy and Chemotherapy Deparment, the 1st Affiliated Hospital of Wenzhou Medical University, No.2 Fuxue Lane, 325000, Wenzhou, China. 12602639@qq.com.

ABSTRACT

Background: Acquired radioresistance has significantly compromised the efficacy of radiotherapy for esophageal cancer. The purpose of this study is to investigate the roles of epithelial-mesenchymal transition (EMT) and the Wnt/β-catenin signaling pathway in the acquirement of radioresistance during the radiation treatment of esophageal cancer.

Methods: We previously established a radioresistant cell line (KYSE-150R) from the KYSE-150 cell line (a human cell line model for esophageal squamous cell carcinoma) with a gradient cumulative irradiation dose. In this study, the expression of EMT phenotypes and the Wnt/β-catenin signaling pathway proteins were examined by real-time PCR, western blot and immunofluorescence in the KYSE-150R cells. The KYSE-150R cells were then treated with a β-Catenin/Tcf inhibitor FH535. The expressions of nuclear and cytoplasmic β-catenin and EMT markers in KYSE-150R cells were assessed at both mRNA and protein level after FH535 treatment. The radiosensitization effect of FH535 on KYSE-150R was evaluated by CCK8 analysis and a colony forming assay. DNA repair capacities was detected by the neutral comet assays.

Results: KYSE-150R cell line displayed obvious radiation resistance and had a stable genetic ability. EMT phenotype was presented in the KYSE-150R cells with decreased E-cadherin and increased snail and twist expressions. The up-regulated expressions of Wnt/β-catenin signaling pathway proteins (Wnt1, FZD1-4, GSK3β, CTNNB1 and Cyclin D1), the increased phosphorylation of GSK3β, and the decreased phosphorylation of β-catenin were observed in KYSE-150R cells compared with KYSE-150 cells, implicating the activation of the Wnt pathway in KYSE-150R cells. The expression of nuclear β-catenin and nuclear translocation of β-catenin from the cytoplasm was decreased after FH535 treatment. FH535 also reversed EMT phenotypes by increasing E-cadherin expression. The cell proliferation rates of KYSE-150R were dose-dependent and the radiation survival fraction was significantly decreased upon FH535 treatment. Neutral comet assays indicated that FH535 impairs DNA double stranded break repair in KYSE-150R cells.

Conclusions: Acquisition of radioresistance and EMT in esophageal cancer cells is associated with the activation of the Wnt/β-catenin pathway. EMT phenotypes can be reduced and the radiosensitivity of esophageal cancer cells can be enhanced by inhibiting the Wnt/β-catenin pathway with FH535 treatment.

Show MeSH
Related in: MedlinePlus