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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

Induction of mitochondrial apoptotic pathway by austrobailignan-1.(A) A549 cells were treated with 100 nM of austrobailignan-1 for indicated time periods (0, 12, 24, and 48 h). (B) H1299 cells were exposed to austrobailignan-1 for 48 h. The cell lysates were harvested, and the caspase activities were determined using fluorogen substrates. Data are expressed as mean ± S.D. from three independent experiments. (*P <0.05,** P <0.01, *** P < 0.001 v.s. vehicle-treated control). (C) Caspase inhibitors block austrobailignan-1-induced cell death. A549 and H1299 cells were pretreated with 50 μM indicated caspase inhibitors for 1 h, and then treated with 100 nM austrobailignan-1 for another 48 h. The cell viability was measured by a Trypan blue dye exclusion method. (D) Regulation of Bcl-2 family proteins. A549 and H1299 cells were treated with 0, 30, 100 nM austrobailignan-1 for 48 h. The levels of indicated Bcl-2 family proteins were examined by Western blot. (E) Release of cytochrome c from mitochondria to cytosol. A549 and H1299 cells were treated without or with 100 nM austrobailignan-1 for 24 and 48 h. After treatment, particulate and cytosolic fractions were isolated, the level of cytochrome c protein was analyzed by Western blot. α-tubulin and cytochrome oxidase IV were used as loading control.
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pone.0132052.g005: Induction of mitochondrial apoptotic pathway by austrobailignan-1.(A) A549 cells were treated with 100 nM of austrobailignan-1 for indicated time periods (0, 12, 24, and 48 h). (B) H1299 cells were exposed to austrobailignan-1 for 48 h. The cell lysates were harvested, and the caspase activities were determined using fluorogen substrates. Data are expressed as mean ± S.D. from three independent experiments. (*P <0.05,** P <0.01, *** P < 0.001 v.s. vehicle-treated control). (C) Caspase inhibitors block austrobailignan-1-induced cell death. A549 and H1299 cells were pretreated with 50 μM indicated caspase inhibitors for 1 h, and then treated with 100 nM austrobailignan-1 for another 48 h. The cell viability was measured by a Trypan blue dye exclusion method. (D) Regulation of Bcl-2 family proteins. A549 and H1299 cells were treated with 0, 30, 100 nM austrobailignan-1 for 48 h. The levels of indicated Bcl-2 family proteins were examined by Western blot. (E) Release of cytochrome c from mitochondria to cytosol. A549 and H1299 cells were treated without or with 100 nM austrobailignan-1 for 24 and 48 h. After treatment, particulate and cytosolic fractions were isolated, the level of cytochrome c protein was analyzed by Western blot. α-tubulin and cytochrome oxidase IV were used as loading control.

Mentions: Because caspase activation plays a central role during the executional phase of apoptosis [44], to examine whether austrobailignan-1 induced apoptosis through activation of the caspase pathway, the activity of caspases-2, -3, -8, -9, and -12 was estimated using a caspase fluorogenic peptide substrate kit. As shown in Fig 5A and 5B, treatment of A549 and H1299 cells with 100 nM austrobailignan-1 resulted in the activation of mitochondria-related caspase-2, -9, and -3, but not death receptor-related caspase-8 and endoplasmic reticulum-associated caspase-12. To characterize the role of caspase activation in austrobailignan-1-induced apoptosis, A549 and H1299 cells were pretreated with inhibitors of caspase-2 (Z-VDDADFMK), caspase-3 (Z-DEVD-FMK), and caspase-9 (Z-LEHD-FMK) for 1 h, and then treated with 100 nM austrobailignan-1 for another 48 h. The inhibitors of caspase-2, -3, and -9 significantly protected A549 and H1299 cells against austrobailignan-1-mediated cell death (Fig 5C). These results suggested that the activated caspases might contribute to austrobailignan-1-triggered apoptosis in these cells.


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)

Induction of mitochondrial apoptotic pathway by austrobailignan-1.(A) A549 cells were treated with 100 nM of austrobailignan-1 for indicated time periods (0, 12, 24, and 48 h). (B) H1299 cells were exposed to austrobailignan-1 for 48 h. The cell lysates were harvested, and the caspase activities were determined using fluorogen substrates. Data are expressed as mean ± S.D. from three independent experiments. (*P <0.05,** P <0.01, *** P < 0.001 v.s. vehicle-treated control). (C) Caspase inhibitors block austrobailignan-1-induced cell death. A549 and H1299 cells were pretreated with 50 μM indicated caspase inhibitors for 1 h, and then treated with 100 nM austrobailignan-1 for another 48 h. The cell viability was measured by a Trypan blue dye exclusion method. (D) Regulation of Bcl-2 family proteins. A549 and H1299 cells were treated with 0, 30, 100 nM austrobailignan-1 for 48 h. The levels of indicated Bcl-2 family proteins were examined by Western blot. (E) Release of cytochrome c from mitochondria to cytosol. A549 and H1299 cells were treated without or with 100 nM austrobailignan-1 for 24 and 48 h. After treatment, particulate and cytosolic fractions were isolated, the level of cytochrome c protein was analyzed by Western blot. α-tubulin and cytochrome oxidase IV were used as loading control.
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Related In: Results  -  Collection

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

pone.0132052.g005: Induction of mitochondrial apoptotic pathway by austrobailignan-1.(A) A549 cells were treated with 100 nM of austrobailignan-1 for indicated time periods (0, 12, 24, and 48 h). (B) H1299 cells were exposed to austrobailignan-1 for 48 h. The cell lysates were harvested, and the caspase activities were determined using fluorogen substrates. Data are expressed as mean ± S.D. from three independent experiments. (*P <0.05,** P <0.01, *** P < 0.001 v.s. vehicle-treated control). (C) Caspase inhibitors block austrobailignan-1-induced cell death. A549 and H1299 cells were pretreated with 50 μM indicated caspase inhibitors for 1 h, and then treated with 100 nM austrobailignan-1 for another 48 h. The cell viability was measured by a Trypan blue dye exclusion method. (D) Regulation of Bcl-2 family proteins. A549 and H1299 cells were treated with 0, 30, 100 nM austrobailignan-1 for 48 h. The levels of indicated Bcl-2 family proteins were examined by Western blot. (E) Release of cytochrome c from mitochondria to cytosol. A549 and H1299 cells were treated without or with 100 nM austrobailignan-1 for 24 and 48 h. After treatment, particulate and cytosolic fractions were isolated, the level of cytochrome c protein was analyzed by Western blot. α-tubulin and cytochrome oxidase IV were used as loading control.
Mentions: Because caspase activation plays a central role during the executional phase of apoptosis [44], to examine whether austrobailignan-1 induced apoptosis through activation of the caspase pathway, the activity of caspases-2, -3, -8, -9, and -12 was estimated using a caspase fluorogenic peptide substrate kit. As shown in Fig 5A and 5B, treatment of A549 and H1299 cells with 100 nM austrobailignan-1 resulted in the activation of mitochondria-related caspase-2, -9, and -3, but not death receptor-related caspase-8 and endoplasmic reticulum-associated caspase-12. To characterize the role of caspase activation in austrobailignan-1-induced apoptosis, A549 and H1299 cells were pretreated with inhibitors of caspase-2 (Z-VDDADFMK), caspase-3 (Z-DEVD-FMK), and caspase-9 (Z-LEHD-FMK) for 1 h, and then treated with 100 nM austrobailignan-1 for another 48 h. The inhibitors of caspase-2, -3, and -9 significantly protected A549 and H1299 cells against austrobailignan-1-mediated cell death (Fig 5C). These results suggested that the activated caspases might contribute to austrobailignan-1-triggered apoptosis in these cells.

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