Limits...
The Topoisomerase 1 Inhibitor Austrobailignan-1 Isolated from Koelreuteria henryi Induces a G2/M-Phase Arrest and Cell Death Independently of p53 in Non-Small Cell Lung Cancer Cells.

Wu CC, Huang KF, Yang TY, Li YL, Wen CL, Hsu SL, Chen TH - PLoS ONE (2015)

Bottom Line: The molecular event of austrobailignan-1-mediated G2/M phase arrest was associated with the increase of p21Waf1/Cip1 and p27Kip1 expression, and decrease of Cdc25C expression.These events were accompanied by the increase of PUMA and Bax, and the decrease of Mcl-1 and Bcl-2.Overall, our results suggest that austrobailignan-1 is a novel DNA damaging agent and displays a topoisomerase I inhibitory activity, causes DNA strand breaks, and consequently induces DNA damage response signaling for cell cycle G2/M arrest and apoptosis in a p53 independent manner.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medicine, Chung Shan Medical University, Taichung, Taiwan, ROC; Department of Medical Research, Chung-Shan Medical University Hospital, Taichung, Taiwan, ROC.

ABSTRACT
Koelreuteria henryi Dummer, an endemic plant of Taiwan, has been used as a folk medicine for the treatment of hepatitis, enteritis, cough, pharyngitis, allergy, hypertension, hyperlipidemia, and cancer. Austrobailignan-1, a natural lignan derivative isolated from Koelreuteria henryi Dummer, has anti-oxidative and anti-cancer properties. However, the effects of austrobailignan-1 on human cancer cells have not been studied yet. Here, we showed that austrobailignan-1 inhibited cell growth of human non-small cell lung cancer A549 and H1299 cell lines in both dose- and time-dependent manners, the IC50 value (48 h) of austrobailignan-1 were 41 and 22 nM, respectively. Data from flow cytometric analysis indicated that treatment with austrobailignan-1 for 24 h retarded the cell cycle at the G2/M phase. The molecular event of austrobailignan-1-mediated G2/M phase arrest was associated with the increase of p21Waf1/Cip1 and p27Kip1 expression, and decrease of Cdc25C expression. Moreover, treatment with 100 nM austrobailignan-1 for 48 h resulted in a pronounced release of cytochrome c followed by the activation of caspase-2, -3, and -9, and consequently induced apoptosis. These events were accompanied by the increase of PUMA and Bax, and the decrease of Mcl-1 and Bcl-2. Furthermore, our study also showed that austrobailignan-1 was a topoisomerase 1 inhibitor, as evidenced by a relaxation assay and induction of a DNA damage response signaling pathway, including ATM, and Chk1, Chk2, γH2AX phosphorylated activation. Overall, our results suggest that austrobailignan-1 is a novel DNA damaging agent and displays a topoisomerase I inhibitory activity, causes DNA strand breaks, and consequently induces DNA damage response signaling for cell cycle G2/M arrest and apoptosis in a p53 independent manner.

No MeSH data available.


Related in: MedlinePlus

Austrobailignan-1 inhibited topoisomerase 1 activity and induced a DNA damage signaling pathway.(A) Inhibition of DNA topoisomerase 1 activity. pHOT-1 DNA plasmid was incubated with various concentrations of austrobailignan-1 (0, 10, 30, and 100 nM) and topoisomerase 1 at 37°C for 30 min. The reaction products were separated by 1% agarose gel and stained by ethidium bromide. The fluorescence image was recorded by microphotography. Camptothecin (CPT) was used as a positive control. S. C. DNA: super coiled DNA, Relax DNA: unwind closed circular DNA. (B) DNA damage response. A549 and H1299 cells were treated without or with 30, 100 nM austrobailignan-1 for 24 h, and DNA damage on per cell basis was examined by a comet assay. Representative comet images from the cells exposed to austrobailignan-1 at various concentrations are shown (upper panel). The degree of DNA damage was scored by tail moment (% DNA in tail x tail length) from at least 100 cells in each treatment group (lower panel). Data are mean ± SD for three independent experiments. ** p < 0.01, *** p < 0.001. (C) Activation of ATM signaling pathway by austrobailignan-1. A549 and H1299 cells were treated with various concentrations of austrobailignan-1 for 24 h, the expressed levels of phosphorylated ATM, Chk1, Chk2, H2AX, and p53 proteins were investigated by Western blot analysis. β-actin was used as an internal loading control.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4492957&req=5

pone.0132052.g003: Austrobailignan-1 inhibited topoisomerase 1 activity and induced a DNA damage signaling pathway.(A) Inhibition of DNA topoisomerase 1 activity. pHOT-1 DNA plasmid was incubated with various concentrations of austrobailignan-1 (0, 10, 30, and 100 nM) and topoisomerase 1 at 37°C for 30 min. The reaction products were separated by 1% agarose gel and stained by ethidium bromide. The fluorescence image was recorded by microphotography. Camptothecin (CPT) was used as a positive control. S. C. DNA: super coiled DNA, Relax DNA: unwind closed circular DNA. (B) DNA damage response. A549 and H1299 cells were treated without or with 30, 100 nM austrobailignan-1 for 24 h, and DNA damage on per cell basis was examined by a comet assay. Representative comet images from the cells exposed to austrobailignan-1 at various concentrations are shown (upper panel). The degree of DNA damage was scored by tail moment (% DNA in tail x tail length) from at least 100 cells in each treatment group (lower panel). Data are mean ± SD for three independent experiments. ** p < 0.01, *** p < 0.001. (C) Activation of ATM signaling pathway by austrobailignan-1. A549 and H1299 cells were treated with various concentrations of austrobailignan-1 for 24 h, the expressed levels of phosphorylated ATM, Chk1, Chk2, H2AX, and p53 proteins were investigated by Western blot analysis. β-actin was used as an internal loading control.

Mentions: Lignan family compounds have been found to be potent inhibitors of human DNA topoisomerase 1 [16, 17]. Next, we used a commercial DNA relaxation assay kit for in vitro measurement of topoisomerase 1 activity in the presence of austrobailignan-1. This kit is majorly to analyze the ability of topoisomerase-1 to unwind a supercoiled DNA. Fig 3A shows that austrobailignan-1 inhibited the DNA relaxation activity of topoisomerase 1 dose-dependently. Camptothecin, a known Topoisomerase 1 inhibitor, was used as the positive control. At 100 nM, austrobailignan-1 exhibited equipotent inhibitory activity to camptothecin (100 μM), indicating that austrobailignan-1 might be more effective than camptothecin.


The Topoisomerase 1 Inhibitor Austrobailignan-1 Isolated from Koelreuteria henryi Induces a G2/M-Phase Arrest and Cell Death Independently of p53 in Non-Small Cell Lung Cancer Cells.

Wu CC, Huang KF, Yang TY, Li YL, Wen CL, Hsu SL, Chen TH - PLoS ONE (2015)

Austrobailignan-1 inhibited topoisomerase 1 activity and induced a DNA damage signaling pathway.(A) Inhibition of DNA topoisomerase 1 activity. pHOT-1 DNA plasmid was incubated with various concentrations of austrobailignan-1 (0, 10, 30, and 100 nM) and topoisomerase 1 at 37°C for 30 min. The reaction products were separated by 1% agarose gel and stained by ethidium bromide. The fluorescence image was recorded by microphotography. Camptothecin (CPT) was used as a positive control. S. C. DNA: super coiled DNA, Relax DNA: unwind closed circular DNA. (B) DNA damage response. A549 and H1299 cells were treated without or with 30, 100 nM austrobailignan-1 for 24 h, and DNA damage on per cell basis was examined by a comet assay. Representative comet images from the cells exposed to austrobailignan-1 at various concentrations are shown (upper panel). The degree of DNA damage was scored by tail moment (% DNA in tail x tail length) from at least 100 cells in each treatment group (lower panel). Data are mean ± SD for three independent experiments. ** p < 0.01, *** p < 0.001. (C) Activation of ATM signaling pathway by austrobailignan-1. A549 and H1299 cells were treated with various concentrations of austrobailignan-1 for 24 h, the expressed levels of phosphorylated ATM, Chk1, Chk2, H2AX, and p53 proteins were investigated by Western blot analysis. β-actin was used as an internal loading control.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4492957&req=5

pone.0132052.g003: Austrobailignan-1 inhibited topoisomerase 1 activity and induced a DNA damage signaling pathway.(A) Inhibition of DNA topoisomerase 1 activity. pHOT-1 DNA plasmid was incubated with various concentrations of austrobailignan-1 (0, 10, 30, and 100 nM) and topoisomerase 1 at 37°C for 30 min. The reaction products were separated by 1% agarose gel and stained by ethidium bromide. The fluorescence image was recorded by microphotography. Camptothecin (CPT) was used as a positive control. S. C. DNA: super coiled DNA, Relax DNA: unwind closed circular DNA. (B) DNA damage response. A549 and H1299 cells were treated without or with 30, 100 nM austrobailignan-1 for 24 h, and DNA damage on per cell basis was examined by a comet assay. Representative comet images from the cells exposed to austrobailignan-1 at various concentrations are shown (upper panel). The degree of DNA damage was scored by tail moment (% DNA in tail x tail length) from at least 100 cells in each treatment group (lower panel). Data are mean ± SD for three independent experiments. ** p < 0.01, *** p < 0.001. (C) Activation of ATM signaling pathway by austrobailignan-1. A549 and H1299 cells were treated with various concentrations of austrobailignan-1 for 24 h, the expressed levels of phosphorylated ATM, Chk1, Chk2, H2AX, and p53 proteins were investigated by Western blot analysis. β-actin was used as an internal loading control.
Mentions: Lignan family compounds have been found to be potent inhibitors of human DNA topoisomerase 1 [16, 17]. Next, we used a commercial DNA relaxation assay kit for in vitro measurement of topoisomerase 1 activity in the presence of austrobailignan-1. This kit is majorly to analyze the ability of topoisomerase-1 to unwind a supercoiled DNA. Fig 3A shows that austrobailignan-1 inhibited the DNA relaxation activity of topoisomerase 1 dose-dependently. Camptothecin, a known Topoisomerase 1 inhibitor, was used as the positive control. At 100 nM, austrobailignan-1 exhibited equipotent inhibitory activity to camptothecin (100 μM), indicating that austrobailignan-1 might be more effective than camptothecin.

Bottom Line: The molecular event of austrobailignan-1-mediated G2/M phase arrest was associated with the increase of p21Waf1/Cip1 and p27Kip1 expression, and decrease of Cdc25C expression.These events were accompanied by the increase of PUMA and Bax, and the decrease of Mcl-1 and Bcl-2.Overall, our results suggest that austrobailignan-1 is a novel DNA damaging agent and displays a topoisomerase I inhibitory activity, causes DNA strand breaks, and consequently induces DNA damage response signaling for cell cycle G2/M arrest and apoptosis in a p53 independent manner.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medicine, Chung Shan Medical University, Taichung, Taiwan, ROC; Department of Medical Research, Chung-Shan Medical University Hospital, Taichung, Taiwan, ROC.

ABSTRACT
Koelreuteria henryi Dummer, an endemic plant of Taiwan, has been used as a folk medicine for the treatment of hepatitis, enteritis, cough, pharyngitis, allergy, hypertension, hyperlipidemia, and cancer. Austrobailignan-1, a natural lignan derivative isolated from Koelreuteria henryi Dummer, has anti-oxidative and anti-cancer properties. However, the effects of austrobailignan-1 on human cancer cells have not been studied yet. Here, we showed that austrobailignan-1 inhibited cell growth of human non-small cell lung cancer A549 and H1299 cell lines in both dose- and time-dependent manners, the IC50 value (48 h) of austrobailignan-1 were 41 and 22 nM, respectively. Data from flow cytometric analysis indicated that treatment with austrobailignan-1 for 24 h retarded the cell cycle at the G2/M phase. The molecular event of austrobailignan-1-mediated G2/M phase arrest was associated with the increase of p21Waf1/Cip1 and p27Kip1 expression, and decrease of Cdc25C expression. Moreover, treatment with 100 nM austrobailignan-1 for 48 h resulted in a pronounced release of cytochrome c followed by the activation of caspase-2, -3, and -9, and consequently induced apoptosis. These events were accompanied by the increase of PUMA and Bax, and the decrease of Mcl-1 and Bcl-2. Furthermore, our study also showed that austrobailignan-1 was a topoisomerase 1 inhibitor, as evidenced by a relaxation assay and induction of a DNA damage response signaling pathway, including ATM, and Chk1, Chk2, γH2AX phosphorylated activation. Overall, our results suggest that austrobailignan-1 is a novel DNA damaging agent and displays a topoisomerase I inhibitory activity, causes DNA strand breaks, and consequently induces DNA damage response signaling for cell cycle G2/M arrest and apoptosis in a p53 independent manner.

No MeSH data available.


Related in: MedlinePlus