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EGCG regulates the cross-talk between JWA and topoisomerase IIα in non-small-cell lung cancer (NSCLC) cells.

Li Y, Shen X, Wang X, Li A, Wang P, Jiang P, Zhou J, Feng Q - Sci Rep (2015)

Bottom Line: The results revealed that EGCG up-regulated JWA while decreased topoisomerase IIα expression in both human non-small cell lung cancer (NSCLC) cells and an NSCLC xenograft mice model.Topoisomerase IIα overexpression reduced JWA at the translational level.These results may serve a novel mechanism for cancer prevention.

View Article: PubMed Central - PubMed

Affiliation: Department of Nutrition and Food Hygiene, School of Public Health, Nanjing Medical University, Nanjing, Jiangsu, 211166, China.

ABSTRACT
(-)-epigallocatechin-3-gallate (EGCG) is a well-known cancer chemopreventive agent. The potential mechanisms include regulation of multiple molecules. Carcinogenesis in lung cancer is related to the imbalance of tumor suppressor and oncogene. JWA is a structurally novel microtubule-binding protein and is a potential tumor suppressor. DNA topoisomerase IIα is a nuclear enzyme that governs DNA topology and is usually highly expressed in many types of cancer. It serves as a target of anticancer drugs. In the current study, the regulation of JWA and topoisomerase IIα by EGCG, and thereafter the mutual interaction between them was investigated. The results revealed that EGCG up-regulated JWA while decreased topoisomerase IIα expression in both human non-small cell lung cancer (NSCLC) cells and an NSCLC xenograft mice model. There was a negative correlation between JWA and topoisomerase IIα in NSCLC as well as in human NSCLC tissue specimens. Topoisomerase IIα overexpression reduced JWA at the translational level. Meanwhile, JWA-induced topoisomerase IIα degradation was regulated both in the transcriptional and post-translational level. Interestingly, JWA and topoisomerase IIα regulated each other in the cells arrested in G2/M. Furthermore, JWA and topoisomerase IIα synergistically affected NCI-H460 cells invasion. These results may serve a novel mechanism for cancer prevention.

No MeSH data available.


Related in: MedlinePlus

EGCG regulated the expression of JWA and topoisomerase IIα (topo IIα) in vitro (NCI-H460 and A549 cells) and in vivo.(a) Cells transfected with Flag-JWA (4 μg) or without transfection were incubated with EGCG (20–40 μM) for 24 h. JWA protein expression was assessed by Western blot analysis. β-actin expression served as a loading control. (b) NCI-H460 cells were treated with EGCG (20–40 μM) for 24 h and total cellular RNA was extracted. mRNA level of JWA was detected by real-time PCR. GAPDH was used as an internal control. (c) Western blot analysis of the protein level of JWA in EGCG-treated A549 cells for 24 h. β-actin expression served as a loading control. (d) After A549 cells were incubated with EGCG (20–40 μM) for 24 h, total RNAs were prepared and real-time PCR was applied to measure the JWA mRNA level. GAPDH was used as an internal control. (e) NCI-H460 cells were transfected with or without Flag-topoisomerase IIα plasmid (4 μg) and then treated with EGCG (20–40 μM) for 24 h. Protein from cell was subjected to western blot analysis. β-actin expression was served as a loading control. (f) Total RNAs from NCI-H460 cells incubated with EGCG (20–40 μM) for 24 h were extracted and subjected to real-time PCR using primer for topoisomerase IIα. GAPDH was used as an internal control. (g) A549 cells were treated with EGCG (20–40 μM) for 24 h and then protein from cell lysate was subjected to western blot analysis. β-actin expression was served as a loading control. (h) A549 cells were incubated in the absence or presence of EGCG (20–40 μM) for 24 h. Then cells were lysed for the detection expression of topoisomerase IIα mRNA by real-time PCR. GAPDH was used as an internal control. The A549 xenograft nude mice model was established and treated with EGCG or normal saline. (i) Tumor size was checked twice per week. (j) Protein obtained from tumor tissues was subjected to western blot. β-tubulin expression was served as a loading control. Error bars represent the mean ± SD of triplicate experiments. Statistical differences to the controls were shown as *p < 0.05, **p < 0.01.
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f1: EGCG regulated the expression of JWA and topoisomerase IIα (topo IIα) in vitro (NCI-H460 and A549 cells) and in vivo.(a) Cells transfected with Flag-JWA (4 μg) or without transfection were incubated with EGCG (20–40 μM) for 24 h. JWA protein expression was assessed by Western blot analysis. β-actin expression served as a loading control. (b) NCI-H460 cells were treated with EGCG (20–40 μM) for 24 h and total cellular RNA was extracted. mRNA level of JWA was detected by real-time PCR. GAPDH was used as an internal control. (c) Western blot analysis of the protein level of JWA in EGCG-treated A549 cells for 24 h. β-actin expression served as a loading control. (d) After A549 cells were incubated with EGCG (20–40 μM) for 24 h, total RNAs were prepared and real-time PCR was applied to measure the JWA mRNA level. GAPDH was used as an internal control. (e) NCI-H460 cells were transfected with or without Flag-topoisomerase IIα plasmid (4 μg) and then treated with EGCG (20–40 μM) for 24 h. Protein from cell was subjected to western blot analysis. β-actin expression was served as a loading control. (f) Total RNAs from NCI-H460 cells incubated with EGCG (20–40 μM) for 24 h were extracted and subjected to real-time PCR using primer for topoisomerase IIα. GAPDH was used as an internal control. (g) A549 cells were treated with EGCG (20–40 μM) for 24 h and then protein from cell lysate was subjected to western blot analysis. β-actin expression was served as a loading control. (h) A549 cells were incubated in the absence or presence of EGCG (20–40 μM) for 24 h. Then cells were lysed for the detection expression of topoisomerase IIα mRNA by real-time PCR. GAPDH was used as an internal control. The A549 xenograft nude mice model was established and treated with EGCG or normal saline. (i) Tumor size was checked twice per week. (j) Protein obtained from tumor tissues was subjected to western blot. β-tubulin expression was served as a loading control. Error bars represent the mean ± SD of triplicate experiments. Statistical differences to the controls were shown as *p < 0.05, **p < 0.01.

Mentions: Firstly, the effect of EGCG on JWA expression was investigated in NSCLC cell lines. Total RNA or protein from A549 and NCI-H460 cells treated with indicated concentration of EGCG was isolated respectively. Western blot analysis was used to detect endogenous and exogenous JWA protein level. As shown in Fig. 1a, EGCG up-regulated endogenous JWA protein level in NCI-H460 cells in a dose-dependent manner. When the same concentrations of EGCG were treated to the cells transfected with Flag-JWA plasmid, the exogenous JWA protein level, as tested by anti-Flag antibody, also increased. Then, real-time PCR was preformed to examine JWA mRNA expression. As shown in Fig. 1b, EGCG increased JWA messenger RNA (mRNA) level as well in NCI-H460 cells. Meanwhile, the same effect of EGCG on JWA expression was observed in A549 cells (Fig. 1c,d).


EGCG regulates the cross-talk between JWA and topoisomerase IIα in non-small-cell lung cancer (NSCLC) cells.

Li Y, Shen X, Wang X, Li A, Wang P, Jiang P, Zhou J, Feng Q - Sci Rep (2015)

EGCG regulated the expression of JWA and topoisomerase IIα (topo IIα) in vitro (NCI-H460 and A549 cells) and in vivo.(a) Cells transfected with Flag-JWA (4 μg) or without transfection were incubated with EGCG (20–40 μM) for 24 h. JWA protein expression was assessed by Western blot analysis. β-actin expression served as a loading control. (b) NCI-H460 cells were treated with EGCG (20–40 μM) for 24 h and total cellular RNA was extracted. mRNA level of JWA was detected by real-time PCR. GAPDH was used as an internal control. (c) Western blot analysis of the protein level of JWA in EGCG-treated A549 cells for 24 h. β-actin expression served as a loading control. (d) After A549 cells were incubated with EGCG (20–40 μM) for 24 h, total RNAs were prepared and real-time PCR was applied to measure the JWA mRNA level. GAPDH was used as an internal control. (e) NCI-H460 cells were transfected with or without Flag-topoisomerase IIα plasmid (4 μg) and then treated with EGCG (20–40 μM) for 24 h. Protein from cell was subjected to western blot analysis. β-actin expression was served as a loading control. (f) Total RNAs from NCI-H460 cells incubated with EGCG (20–40 μM) for 24 h were extracted and subjected to real-time PCR using primer for topoisomerase IIα. GAPDH was used as an internal control. (g) A549 cells were treated with EGCG (20–40 μM) for 24 h and then protein from cell lysate was subjected to western blot analysis. β-actin expression was served as a loading control. (h) A549 cells were incubated in the absence or presence of EGCG (20–40 μM) for 24 h. Then cells were lysed for the detection expression of topoisomerase IIα mRNA by real-time PCR. GAPDH was used as an internal control. The A549 xenograft nude mice model was established and treated with EGCG or normal saline. (i) Tumor size was checked twice per week. (j) Protein obtained from tumor tissues was subjected to western blot. β-tubulin expression was served as a loading control. Error bars represent the mean ± SD of triplicate experiments. Statistical differences to the controls were shown as *p < 0.05, **p < 0.01.
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Related In: Results  -  Collection

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f1: EGCG regulated the expression of JWA and topoisomerase IIα (topo IIα) in vitro (NCI-H460 and A549 cells) and in vivo.(a) Cells transfected with Flag-JWA (4 μg) or without transfection were incubated with EGCG (20–40 μM) for 24 h. JWA protein expression was assessed by Western blot analysis. β-actin expression served as a loading control. (b) NCI-H460 cells were treated with EGCG (20–40 μM) for 24 h and total cellular RNA was extracted. mRNA level of JWA was detected by real-time PCR. GAPDH was used as an internal control. (c) Western blot analysis of the protein level of JWA in EGCG-treated A549 cells for 24 h. β-actin expression served as a loading control. (d) After A549 cells were incubated with EGCG (20–40 μM) for 24 h, total RNAs were prepared and real-time PCR was applied to measure the JWA mRNA level. GAPDH was used as an internal control. (e) NCI-H460 cells were transfected with or without Flag-topoisomerase IIα plasmid (4 μg) and then treated with EGCG (20–40 μM) for 24 h. Protein from cell was subjected to western blot analysis. β-actin expression was served as a loading control. (f) Total RNAs from NCI-H460 cells incubated with EGCG (20–40 μM) for 24 h were extracted and subjected to real-time PCR using primer for topoisomerase IIα. GAPDH was used as an internal control. (g) A549 cells were treated with EGCG (20–40 μM) for 24 h and then protein from cell lysate was subjected to western blot analysis. β-actin expression was served as a loading control. (h) A549 cells were incubated in the absence or presence of EGCG (20–40 μM) for 24 h. Then cells were lysed for the detection expression of topoisomerase IIα mRNA by real-time PCR. GAPDH was used as an internal control. The A549 xenograft nude mice model was established and treated with EGCG or normal saline. (i) Tumor size was checked twice per week. (j) Protein obtained from tumor tissues was subjected to western blot. β-tubulin expression was served as a loading control. Error bars represent the mean ± SD of triplicate experiments. Statistical differences to the controls were shown as *p < 0.05, **p < 0.01.
Mentions: Firstly, the effect of EGCG on JWA expression was investigated in NSCLC cell lines. Total RNA or protein from A549 and NCI-H460 cells treated with indicated concentration of EGCG was isolated respectively. Western blot analysis was used to detect endogenous and exogenous JWA protein level. As shown in Fig. 1a, EGCG up-regulated endogenous JWA protein level in NCI-H460 cells in a dose-dependent manner. When the same concentrations of EGCG were treated to the cells transfected with Flag-JWA plasmid, the exogenous JWA protein level, as tested by anti-Flag antibody, also increased. Then, real-time PCR was preformed to examine JWA mRNA expression. As shown in Fig. 1b, EGCG increased JWA messenger RNA (mRNA) level as well in NCI-H460 cells. Meanwhile, the same effect of EGCG on JWA expression was observed in A549 cells (Fig. 1c,d).

Bottom Line: The results revealed that EGCG up-regulated JWA while decreased topoisomerase IIα expression in both human non-small cell lung cancer (NSCLC) cells and an NSCLC xenograft mice model.Topoisomerase IIα overexpression reduced JWA at the translational level.These results may serve a novel mechanism for cancer prevention.

View Article: PubMed Central - PubMed

Affiliation: Department of Nutrition and Food Hygiene, School of Public Health, Nanjing Medical University, Nanjing, Jiangsu, 211166, China.

ABSTRACT
(-)-epigallocatechin-3-gallate (EGCG) is a well-known cancer chemopreventive agent. The potential mechanisms include regulation of multiple molecules. Carcinogenesis in lung cancer is related to the imbalance of tumor suppressor and oncogene. JWA is a structurally novel microtubule-binding protein and is a potential tumor suppressor. DNA topoisomerase IIα is a nuclear enzyme that governs DNA topology and is usually highly expressed in many types of cancer. It serves as a target of anticancer drugs. In the current study, the regulation of JWA and topoisomerase IIα by EGCG, and thereafter the mutual interaction between them was investigated. The results revealed that EGCG up-regulated JWA while decreased topoisomerase IIα expression in both human non-small cell lung cancer (NSCLC) cells and an NSCLC xenograft mice model. There was a negative correlation between JWA and topoisomerase IIα in NSCLC as well as in human NSCLC tissue specimens. Topoisomerase IIα overexpression reduced JWA at the translational level. Meanwhile, JWA-induced topoisomerase IIα degradation was regulated both in the transcriptional and post-translational level. Interestingly, JWA and topoisomerase IIα regulated each other in the cells arrested in G2/M. Furthermore, JWA and topoisomerase IIα synergistically affected NCI-H460 cells invasion. These results may serve a novel mechanism for cancer prevention.

No MeSH data available.


Related in: MedlinePlus