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Nuclear factor-kappa B inhibition can enhance apoptosis of differentiated thyroid cancer cells induced by 131I.

Meng Z, Lou S, Tan J, Xu K, Jia Q, Zheng W - PLoS ONE (2012)

Bottom Line: Western blot also demonstrated significant up-regulation of XIAP, cIAP1, and Bcl-xL after (131)I therapy.And inhibition of NF-κB could significantly down-regulate these factors.NF-κB inhibition by Bay 11-7082 or by p65 siRNA transfection was effective in suppressing NF-κB regulated anti-apoptotic changes and in combined regimen apoptosis was achieved synergistically.

View Article: PubMed Central - PubMed

Affiliation: Department of Nuclear Medicine, Tianjin Medical University General Hospital, Tianjin, People's Republic of China. james_mencius@hotmail.com

ABSTRACT

Objective: To evaluate changes of nuclear factor-kappa B (NF-κB) during radioiodine 131 ((131)I) therapy and whether NF-κB inhibition could enhance (131)I-induced apoptosis in differentiated thyroid cancer (DTC) cells in a synergistic manner.

Methods: Three human DTC cell lines were used. NF-κB inhibition was achieved by using a NF-κB inhibitor (Bay 11-7082) or by p65 siRNA transfection. Methyl-thiazolyl-tetrazolium assay was performed for cell viability assessment. DNA-binding assay, luciferase reporter assay, and Western blot were adopted to determine function and expression changes of NF-κB. Then NF-κB regulated anti-apoptotic factors XIAP, cIAP1, and Bcl-xL were measured. Apoptosis was analyzed by Western blot for caspase 3 and PARP, and by flow cytometry as well. An iodide uptake assay was performed to determine whether NF-κB inhibition could influence radioactive iodide uptake.

Results: The methyl-thiazolyl-tetrazolium assay showed significant decrease of viable cells by combination therapy than by mono-therapies. The DNA-binding assay and luciferase reporter assay showed enhanced NF-κB function and reporter gene activities due to (131)I, yet significant suppression was achieved by NF-κB inhibition. Western blot proved (131)I could increase nuclear NF-κB concentration, while NF-κB inhibition reduced NF-κB concentration. Western blot also demonstrated significant up-regulation of XIAP, cIAP1, and Bcl-xL after (131)I therapy. And inhibition of NF-κB could significantly down-regulate these factors. Finally, synergism induced by combined therapy was displayed by significant enhancements of cleaved caspase 3 and PARP from Western blot, and of Annexin V positively staining from flow cytometry. The iodine uptake assay did not show significant changes when NF-κB was inhibited.

Conclusion: We demonstrated that (131)I could induce NF-κB activation, which would attenuate (131)I efficacy in DTC cells. NF-κB inhibition by Bay 11-7082 or by p65 siRNA transfection was effective in suppressing NF-κB regulated anti-apoptotic changes and in combined regimen apoptosis was achieved synergistically.

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Effect of different treatments on apoptotic cascade.KTC-1 cells were left untreated as controls (A), or treated with 20 MBq/ml 131I (B), 5 µmol/L Bay 11-7082 (C) or combination (D) for 24 hours. Then whole cell lysates were examined by Western blot for caspase 3 and PARP. In the second set of experiments, KTC-1 cells were treated with no oligonucleotide control (E), scrambled oligonucleotides (F) or p65 siRNA transfection (G) first. Then these three groups of cells were exposed to 131I (20 MBq/ml) for 24 hours (H–J). Caspase 3 and PARP protein levels were examined by Western blot. β-actin was used as a loading control. Similar results were obtained in three independent experiments.
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pone-0033597-g006: Effect of different treatments on apoptotic cascade.KTC-1 cells were left untreated as controls (A), or treated with 20 MBq/ml 131I (B), 5 µmol/L Bay 11-7082 (C) or combination (D) for 24 hours. Then whole cell lysates were examined by Western blot for caspase 3 and PARP. In the second set of experiments, KTC-1 cells were treated with no oligonucleotide control (E), scrambled oligonucleotides (F) or p65 siRNA transfection (G) first. Then these three groups of cells were exposed to 131I (20 MBq/ml) for 24 hours (H–J). Caspase 3 and PARP protein levels were examined by Western blot. β-actin was used as a loading control. Similar results were obtained in three independent experiments.

Mentions: First, Western blot was applied to detect changes of caspase 3 (a key apoptosis executioner) and PARP (a main target of caspase 3). We showed that although any intervention could induce cleavages of caspase 3 (subunits p19 and p17) and PARP (p89), their levels increased significantly further by combined treatments (Figure 6). In Table 1, ANOVA and LSD showed significant enhancements in combined therapies than in mono-therapies (P<0.05). Then, to further confirm the effects on apoptosis, double staining flow cytometry was performed. Results showed that while any therapy could induce apoptosis, combined treatments increased apoptosis synergistically due to significant enhancements of Annexin V positively stained cells (Figure 7 and Table 1).


Nuclear factor-kappa B inhibition can enhance apoptosis of differentiated thyroid cancer cells induced by 131I.

Meng Z, Lou S, Tan J, Xu K, Jia Q, Zheng W - PLoS ONE (2012)

Effect of different treatments on apoptotic cascade.KTC-1 cells were left untreated as controls (A), or treated with 20 MBq/ml 131I (B), 5 µmol/L Bay 11-7082 (C) or combination (D) for 24 hours. Then whole cell lysates were examined by Western blot for caspase 3 and PARP. In the second set of experiments, KTC-1 cells were treated with no oligonucleotide control (E), scrambled oligonucleotides (F) or p65 siRNA transfection (G) first. Then these three groups of cells were exposed to 131I (20 MBq/ml) for 24 hours (H–J). Caspase 3 and PARP protein levels were examined by Western blot. β-actin was used as a loading control. Similar results were obtained in three independent experiments.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3306418&req=5

pone-0033597-g006: Effect of different treatments on apoptotic cascade.KTC-1 cells were left untreated as controls (A), or treated with 20 MBq/ml 131I (B), 5 µmol/L Bay 11-7082 (C) or combination (D) for 24 hours. Then whole cell lysates were examined by Western blot for caspase 3 and PARP. In the second set of experiments, KTC-1 cells were treated with no oligonucleotide control (E), scrambled oligonucleotides (F) or p65 siRNA transfection (G) first. Then these three groups of cells were exposed to 131I (20 MBq/ml) for 24 hours (H–J). Caspase 3 and PARP protein levels were examined by Western blot. β-actin was used as a loading control. Similar results were obtained in three independent experiments.
Mentions: First, Western blot was applied to detect changes of caspase 3 (a key apoptosis executioner) and PARP (a main target of caspase 3). We showed that although any intervention could induce cleavages of caspase 3 (subunits p19 and p17) and PARP (p89), their levels increased significantly further by combined treatments (Figure 6). In Table 1, ANOVA and LSD showed significant enhancements in combined therapies than in mono-therapies (P<0.05). Then, to further confirm the effects on apoptosis, double staining flow cytometry was performed. Results showed that while any therapy could induce apoptosis, combined treatments increased apoptosis synergistically due to significant enhancements of Annexin V positively stained cells (Figure 7 and Table 1).

Bottom Line: Western blot also demonstrated significant up-regulation of XIAP, cIAP1, and Bcl-xL after (131)I therapy.And inhibition of NF-κB could significantly down-regulate these factors.NF-κB inhibition by Bay 11-7082 or by p65 siRNA transfection was effective in suppressing NF-κB regulated anti-apoptotic changes and in combined regimen apoptosis was achieved synergistically.

View Article: PubMed Central - PubMed

Affiliation: Department of Nuclear Medicine, Tianjin Medical University General Hospital, Tianjin, People's Republic of China. james_mencius@hotmail.com

ABSTRACT

Objective: To evaluate changes of nuclear factor-kappa B (NF-κB) during radioiodine 131 ((131)I) therapy and whether NF-κB inhibition could enhance (131)I-induced apoptosis in differentiated thyroid cancer (DTC) cells in a synergistic manner.

Methods: Three human DTC cell lines were used. NF-κB inhibition was achieved by using a NF-κB inhibitor (Bay 11-7082) or by p65 siRNA transfection. Methyl-thiazolyl-tetrazolium assay was performed for cell viability assessment. DNA-binding assay, luciferase reporter assay, and Western blot were adopted to determine function and expression changes of NF-κB. Then NF-κB regulated anti-apoptotic factors XIAP, cIAP1, and Bcl-xL were measured. Apoptosis was analyzed by Western blot for caspase 3 and PARP, and by flow cytometry as well. An iodide uptake assay was performed to determine whether NF-κB inhibition could influence radioactive iodide uptake.

Results: The methyl-thiazolyl-tetrazolium assay showed significant decrease of viable cells by combination therapy than by mono-therapies. The DNA-binding assay and luciferase reporter assay showed enhanced NF-κB function and reporter gene activities due to (131)I, yet significant suppression was achieved by NF-κB inhibition. Western blot proved (131)I could increase nuclear NF-κB concentration, while NF-κB inhibition reduced NF-κB concentration. Western blot also demonstrated significant up-regulation of XIAP, cIAP1, and Bcl-xL after (131)I therapy. And inhibition of NF-κB could significantly down-regulate these factors. Finally, synergism induced by combined therapy was displayed by significant enhancements of cleaved caspase 3 and PARP from Western blot, and of Annexin V positively staining from flow cytometry. The iodine uptake assay did not show significant changes when NF-κB was inhibited.

Conclusion: We demonstrated that (131)I could induce NF-κB activation, which would attenuate (131)I efficacy in DTC cells. NF-κB inhibition by Bay 11-7082 or by p65 siRNA transfection was effective in suppressing NF-κB regulated anti-apoptotic changes and in combined regimen apoptosis was achieved synergistically.

Show MeSH
Related in: MedlinePlus