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 induced G2/M arrest and apoptosis.(A) A549 and H1299 cells were treated with various doses (0, 1, 3, 10, 30 and 100 nM) of austrobailignan-1 for 24 and 48 h. Cell number was measured by a Trypan-blue dye exclusion method. Data are expressed as mean ± S.D. from 3 independent experiments. (*P <0.05,** P <0.01, *** P < 0.001 v.s. control). (B) Cells were treated with varied doses (0, 3, 10, 30 and 100 nM) of austrobailignan-1 for 24 and 48 h, and then stained with propidium iodide, and flow cytometry was performed to examine the cell cycle distribution. (C) Cells were treated without or with 100 nM austrobailignan-1 for 48 h,a TUNEL assay was then performed to detect apoptotic cells (green) and the nuclear DNA was stained with DAPI (blue). The stained cells were investigated by fluorescence microscopy. Magnification x 400; scale bar, 50 μm.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0132052.g002: Austrobailignan-1 induced G2/M arrest and apoptosis.(A) A549 and H1299 cells were treated with various doses (0, 1, 3, 10, 30 and 100 nM) of austrobailignan-1 for 24 and 48 h. Cell number was measured by a Trypan-blue dye exclusion method. Data are expressed as mean ± S.D. from 3 independent experiments. (*P <0.05,** P <0.01, *** P < 0.001 v.s. control). (B) Cells were treated with varied doses (0, 3, 10, 30 and 100 nM) of austrobailignan-1 for 24 and 48 h, and then stained with propidium iodide, and flow cytometry was performed to examine the cell cycle distribution. (C) Cells were treated without or with 100 nM austrobailignan-1 for 48 h,a TUNEL assay was then performed to detect apoptotic cells (green) and the nuclear DNA was stained with DAPI (blue). The stained cells were investigated by fluorescence microscopy. Magnification x 400; scale bar, 50 μm.

Mentions: The loss of normal function of p53 had been finding in over half of all human tumors [29]. Literature shows that p53 is one of the most important regulators in mediating growth arrest and apoptosis induced by various intrinsic or extrinsic stresses, including chemotherapeutic agents [30]. Besides, the p53 is also an important connector and switcher between cell cycle arrest and apoptotic process. Once the damages are unable to be repaired, p53 activates the transcription of various pro-apoptotic genes, including Bax, Noxa, PUMA, Fas, and DR5 [31, 32] to execute the apoptotic process. Alternatively, p53 triggers apoptosis by repression of anti-apoptotic genes, such as Bcl-2, thus inducing the release of cytochrome c followed by the caspase-3 and -9 activation [31]. It is well documented that the status of p53 can affect the response of cancer cells to some chemotherapeutic drugs [33]. We firstly examined the antiproliferative effects of austrobailignan-1 purified from the leaves of K. henryi (Fig 1 and [12]) in human NSCLC A549 (+p53, which harvest a wild-type p53) and H1299 (-p53, which is p53-) cell lines. As shown in Fig 2A, treatment with austrobailignan-1 significantly inhibited the growth of A549 and H1299 cells in both concentration- and time-dependent manners with IC50 values of 41 and 22 nM after 48-h administration, respectively. The results also showed that treatment of lung cancer cells with low concentrations (lower than 10 nM) of austrobailignan-1 caused a cytostatic effect, only inhibited cell proliferation but no cytotoxic effect observed under microscopic investigation. However, treatment with high concentration (30 and 100 nM) of austrobailignan-1 exhibited morphological features of apoptotic cell death, floating and blebbing cells were observed (data not shown). To address the precise action responsible for the austrobailignan-1-mediated antiproliferative effect, the cell cycle distribution profile was examined. As indicated in Fig 2B, exposure of A549 and H1299 cells to 30 and 100 nM of austrobailignan-1 for 24 h led to an accumulation of cells in the G2/M phase compared with control cells, coupled with a concomitant decrease in the proportion of cells in the G1 and S phases. Additionally, a hypodiploid DNA content peak (sub-G1 population), which is indicative of degraded DNA and a hallmark of apoptosis, was observed following 24 h of high-dose treatment and increased continuously after 48-h austrobailignan-1 incubation (Fig 2B). To further confirm the induction of apoptosis by austrobailignan-1 in A549 cells, the TUNEL assay and DAPI staining were performed. As indicated in Fig 2C, treatment with 100 nM austrobailignan-1 for 48 h significantly induced the apoptotic cell death with condensed nuclei and increase of TUNEL positive cells (green fluorescent colored cells), suggesting that the DNA fragmentation was occurring 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)

Austrobailignan-1 induced G2/M arrest and apoptosis.(A) A549 and H1299 cells were treated with various doses (0, 1, 3, 10, 30 and 100 nM) of austrobailignan-1 for 24 and 48 h. Cell number was measured by a Trypan-blue dye exclusion method. Data are expressed as mean ± S.D. from 3 independent experiments. (*P <0.05,** P <0.01, *** P < 0.001 v.s. control). (B) Cells were treated with varied doses (0, 3, 10, 30 and 100 nM) of austrobailignan-1 for 24 and 48 h, and then stained with propidium iodide, and flow cytometry was performed to examine the cell cycle distribution. (C) Cells were treated without or with 100 nM austrobailignan-1 for 48 h,a TUNEL assay was then performed to detect apoptotic cells (green) and the nuclear DNA was stained with DAPI (blue). The stained cells were investigated by fluorescence microscopy. Magnification x 400; scale bar, 50 μm.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0132052.g002: Austrobailignan-1 induced G2/M arrest and apoptosis.(A) A549 and H1299 cells were treated with various doses (0, 1, 3, 10, 30 and 100 nM) of austrobailignan-1 for 24 and 48 h. Cell number was measured by a Trypan-blue dye exclusion method. Data are expressed as mean ± S.D. from 3 independent experiments. (*P <0.05,** P <0.01, *** P < 0.001 v.s. control). (B) Cells were treated with varied doses (0, 3, 10, 30 and 100 nM) of austrobailignan-1 for 24 and 48 h, and then stained with propidium iodide, and flow cytometry was performed to examine the cell cycle distribution. (C) Cells were treated without or with 100 nM austrobailignan-1 for 48 h,a TUNEL assay was then performed to detect apoptotic cells (green) and the nuclear DNA was stained with DAPI (blue). The stained cells were investigated by fluorescence microscopy. Magnification x 400; scale bar, 50 μm.
Mentions: The loss of normal function of p53 had been finding in over half of all human tumors [29]. Literature shows that p53 is one of the most important regulators in mediating growth arrest and apoptosis induced by various intrinsic or extrinsic stresses, including chemotherapeutic agents [30]. Besides, the p53 is also an important connector and switcher between cell cycle arrest and apoptotic process. Once the damages are unable to be repaired, p53 activates the transcription of various pro-apoptotic genes, including Bax, Noxa, PUMA, Fas, and DR5 [31, 32] to execute the apoptotic process. Alternatively, p53 triggers apoptosis by repression of anti-apoptotic genes, such as Bcl-2, thus inducing the release of cytochrome c followed by the caspase-3 and -9 activation [31]. It is well documented that the status of p53 can affect the response of cancer cells to some chemotherapeutic drugs [33]. We firstly examined the antiproliferative effects of austrobailignan-1 purified from the leaves of K. henryi (Fig 1 and [12]) in human NSCLC A549 (+p53, which harvest a wild-type p53) and H1299 (-p53, which is p53-) cell lines. As shown in Fig 2A, treatment with austrobailignan-1 significantly inhibited the growth of A549 and H1299 cells in both concentration- and time-dependent manners with IC50 values of 41 and 22 nM after 48-h administration, respectively. The results also showed that treatment of lung cancer cells with low concentrations (lower than 10 nM) of austrobailignan-1 caused a cytostatic effect, only inhibited cell proliferation but no cytotoxic effect observed under microscopic investigation. However, treatment with high concentration (30 and 100 nM) of austrobailignan-1 exhibited morphological features of apoptotic cell death, floating and blebbing cells were observed (data not shown). To address the precise action responsible for the austrobailignan-1-mediated antiproliferative effect, the cell cycle distribution profile was examined. As indicated in Fig 2B, exposure of A549 and H1299 cells to 30 and 100 nM of austrobailignan-1 for 24 h led to an accumulation of cells in the G2/M phase compared with control cells, coupled with a concomitant decrease in the proportion of cells in the G1 and S phases. Additionally, a hypodiploid DNA content peak (sub-G1 population), which is indicative of degraded DNA and a hallmark of apoptosis, was observed following 24 h of high-dose treatment and increased continuously after 48-h austrobailignan-1 incubation (Fig 2B). To further confirm the induction of apoptosis by austrobailignan-1 in A549 cells, the TUNEL assay and DAPI staining were performed. As indicated in Fig 2C, treatment with 100 nM austrobailignan-1 for 48 h significantly induced the apoptotic cell death with condensed nuclei and increase of TUNEL positive cells (green fluorescent colored cells), suggesting that the DNA fragmentation was occurring 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