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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

Regulation of cell-cycle regulatory proteins by austrobailignan-1.(A) A549 cells were treated with 0, 3, 10, 30 and 100 nM of austrobailignan-1 for 24. After treatment, cell extract was collected and analyzed by Western blot. (B) H1299 cells were treated with 0, 10, 30, 100 nM austrobailignan-1 for 24 h, the levels of p21Waf1/Cip1, p27Kip1, and Cdc25C were detected by Western blot. β-Actin was used as a loading control.
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pone.0132052.g004: Regulation of cell-cycle regulatory proteins by austrobailignan-1.(A) A549 cells were treated with 0, 3, 10, 30 and 100 nM of austrobailignan-1 for 24. After treatment, cell extract was collected and analyzed by Western blot. (B) H1299 cells were treated with 0, 10, 30, 100 nM austrobailignan-1 for 24 h, the levels of p21Waf1/Cip1, p27Kip1, and Cdc25C were detected by Western blot. β-Actin was used as a loading control.

Mentions: We have showed that p53 can be phosphorylated by ATM/ATR kinases in the presence of austrabailignan-1 in A549 cells. The active p53 can transcriptionally increase the expression levels of p21Waf1/Cip1, p27Kip 1 [39], which both are breakers of cell cycle progression. Besides, the Cdc25 dual specificity phosphatase family (Cdc25A, Cdc25B and Cdc25C) is another common signal transducer downstream substrate of ATR/ATR/Chks. Phosphorylated inactivation of Cdc25C mediated by ATM/ATR/Chks plays a pivotal role in G2/M phase arrest and subsequently apoptosis induced by several antitumor agents [40–43]. To address the subsequent molecular event of the austrobailignan-1-mediated cell cycle retardation, the expression levels of G2/M-related molecules such as p53, p27Kip 1, p21waf1/Cip1, Cdk1, Cdk2, cyclin A, cyclin B1 and Cdc25C were examined after various doses of austrobailignan-1 (0, 10, 30, and 100 nM) treatment of A549 cells for 24 h. As expected, the expressions of p53, p21Waf1/Cip1, p27Kip1 and cyclin B1 were increased while cyclin A and Cdc25C were decreased (Fig 4A) in austrobailignan-1-treated cells compared to untreated control cells. The levels of Cdk1 and Cdk2 were not affected by austrobailignan-1. Limited by the compound availability, only p21Waf1/Cip1, p27Kip 1 and Cdc25C levels were examined in p53- H1299 cell line. Similarly, the up-regulation of p21Waf1/Cip1 and p27KIP 1 and down-regulation of Cdc25C were observed in austrobailignan-1-treated H1299 cells (Fig 4B). These results indicated that austrobailignan-1-mediated cellular and molecular events in the tested cell lines were p53 independently.


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)

Regulation of cell-cycle regulatory proteins by austrobailignan-1.(A) A549 cells were treated with 0, 3, 10, 30 and 100 nM of austrobailignan-1 for 24. After treatment, cell extract was collected and analyzed by Western blot. (B) H1299 cells were treated with 0, 10, 30, 100 nM austrobailignan-1 for 24 h, the levels of p21Waf1/Cip1, p27Kip1, and Cdc25C were detected by Western blot. β-Actin was used as a loading control.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0132052.g004: Regulation of cell-cycle regulatory proteins by austrobailignan-1.(A) A549 cells were treated with 0, 3, 10, 30 and 100 nM of austrobailignan-1 for 24. After treatment, cell extract was collected and analyzed by Western blot. (B) H1299 cells were treated with 0, 10, 30, 100 nM austrobailignan-1 for 24 h, the levels of p21Waf1/Cip1, p27Kip1, and Cdc25C were detected by Western blot. β-Actin was used as a loading control.
Mentions: We have showed that p53 can be phosphorylated by ATM/ATR kinases in the presence of austrabailignan-1 in A549 cells. The active p53 can transcriptionally increase the expression levels of p21Waf1/Cip1, p27Kip 1 [39], which both are breakers of cell cycle progression. Besides, the Cdc25 dual specificity phosphatase family (Cdc25A, Cdc25B and Cdc25C) is another common signal transducer downstream substrate of ATR/ATR/Chks. Phosphorylated inactivation of Cdc25C mediated by ATM/ATR/Chks plays a pivotal role in G2/M phase arrest and subsequently apoptosis induced by several antitumor agents [40–43]. To address the subsequent molecular event of the austrobailignan-1-mediated cell cycle retardation, the expression levels of G2/M-related molecules such as p53, p27Kip 1, p21waf1/Cip1, Cdk1, Cdk2, cyclin A, cyclin B1 and Cdc25C were examined after various doses of austrobailignan-1 (0, 10, 30, and 100 nM) treatment of A549 cells for 24 h. As expected, the expressions of p53, p21Waf1/Cip1, p27Kip1 and cyclin B1 were increased while cyclin A and Cdc25C were decreased (Fig 4A) in austrobailignan-1-treated cells compared to untreated control cells. The levels of Cdk1 and Cdk2 were not affected by austrobailignan-1. Limited by the compound availability, only p21Waf1/Cip1, p27Kip 1 and Cdc25C levels were examined in p53- H1299 cell line. Similarly, the up-regulation of p21Waf1/Cip1 and p27KIP 1 and down-regulation of Cdc25C were observed in austrobailignan-1-treated H1299 cells (Fig 4B). These results indicated that austrobailignan-1-mediated cellular and molecular events in the tested cell lines were p53 independently.

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