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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

p53 was not necessarily required for austrobailignan-1-induced cell cycle G2/M arrest and cell death.(A) The A549-shRNA or A549-p53shRNA cells were treated with austrobailignan-1 (0, 10, 30, and 100 nM) for 48 h. The cell cycle distribution was determined by flow cyotmetry. (B) A549-shRNA and A549-p53shRNA cells were treated without or with 100 nM austrobailignan-1 for 48 h. The levels of p53 protein were detected by Western blot (upper panel). The cell viability was determined by a trypan blue exclusion method (lower panel).
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pone.0132052.g006: p53 was not necessarily required for austrobailignan-1-induced cell cycle G2/M arrest and cell death.(A) The A549-shRNA or A549-p53shRNA cells were treated with austrobailignan-1 (0, 10, 30, and 100 nM) for 48 h. The cell cycle distribution was determined by flow cyotmetry. (B) A549-shRNA and A549-p53shRNA cells were treated without or with 100 nM austrobailignan-1 for 48 h. The levels of p53 protein were detected by Western blot (upper panel). The cell viability was determined by a trypan blue exclusion method (lower panel).

Mentions: Our results have showed that autrobailignan-1 induced cell death in both A549 and H1299 cell lines (Fig 2A and 2B). The phosphorylation of p53 at ser15 site usually represents apoptotic activation [46]. Figs 4A and 5E showed that austrobailignan-1 treatment increased the levels of total p53 and phosphorylated p53-ser15 in A549 cells, implying a role of p53 in austrobailigan-1-induced cell death. To characterize whether p53 indeed plays a role in austrobailignan-1-mediated cell cycle arrest and apoptosis, we next examined the effect of austrobailignan-1 in p53-knockdown A549 (A549-p53-shRNA) cells, which were stably transfected with a p53-specific shRNA [47]. There was no significant difference between A549-vector control cells (A549 (-shRNA) and A549-p53-shRNA cells in cell cycle arrest (Fig 6A) and in cell viability after 48 h austrobailignan-1 treatment (Fig 6B). We therefore conclude that austrobailignan-1-mediated G2/M arrest and cell death does not necessarily require p53.


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)

p53 was not necessarily required for austrobailignan-1-induced cell cycle G2/M arrest and cell death.(A) The A549-shRNA or A549-p53shRNA cells were treated with austrobailignan-1 (0, 10, 30, and 100 nM) for 48 h. The cell cycle distribution was determined by flow cyotmetry. (B) A549-shRNA and A549-p53shRNA cells were treated without or with 100 nM austrobailignan-1 for 48 h. The levels of p53 protein were detected by Western blot (upper panel). The cell viability was determined by a trypan blue exclusion method (lower panel).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0132052.g006: p53 was not necessarily required for austrobailignan-1-induced cell cycle G2/M arrest and cell death.(A) The A549-shRNA or A549-p53shRNA cells were treated with austrobailignan-1 (0, 10, 30, and 100 nM) for 48 h. The cell cycle distribution was determined by flow cyotmetry. (B) A549-shRNA and A549-p53shRNA cells were treated without or with 100 nM austrobailignan-1 for 48 h. The levels of p53 protein were detected by Western blot (upper panel). The cell viability was determined by a trypan blue exclusion method (lower panel).
Mentions: Our results have showed that autrobailignan-1 induced cell death in both A549 and H1299 cell lines (Fig 2A and 2B). The phosphorylation of p53 at ser15 site usually represents apoptotic activation [46]. Figs 4A and 5E showed that austrobailignan-1 treatment increased the levels of total p53 and phosphorylated p53-ser15 in A549 cells, implying a role of p53 in austrobailigan-1-induced cell death. To characterize whether p53 indeed plays a role in austrobailignan-1-mediated cell cycle arrest and apoptosis, we next examined the effect of austrobailignan-1 in p53-knockdown A549 (A549-p53-shRNA) cells, which were stably transfected with a p53-specific shRNA [47]. There was no significant difference between A549-vector control cells (A549 (-shRNA) and A549-p53-shRNA cells in cell cycle arrest (Fig 6A) and in cell viability after 48 h austrobailignan-1 treatment (Fig 6B). We therefore conclude that austrobailignan-1-mediated G2/M arrest and cell death does not necessarily require p53.

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