- Email: [email protected]
M arrest through ROS generation and ATM pathway in human cervical carcinoma cells
Biochemical and Biophysical Research Communications 409 (2011) 489–493
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Asperlin induces G2/M arrest through ROS generation and ATM pathway in human cervical carcinoma cells Long He a,1, Mei-Hua Nan a,1, Hyun Cheol Oh b, Young Ho Kim c, Jae Hyuk Jang a, Raymond Leo Erikson d, Jong Seog Ahn a,⇑, Bo Yeon Kim a,e,⇑ a Chemical Biology Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 30 Yeongudanji-ro, Ochang-eup, Cheongwon-gun, Chungbuk 363-883, Republic of Korea b College of Medical and Life Sciences, Silla University, 100 Silladaehak-gil, Sasang-gu, Busan 617-736, Republic of Korea c College of Pharmacy, ChungNam National University, Yuseong, Daejeon, 305-764, Republic of Korea d Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA e World Class Institute, KRIBB, 30 Yeongudanji-ro, Ochang-eup, Cheongwon-gun, Chungbuk 363-883, Republic of Korea
a r t i c l e
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Article history: Received 4 May 2011 Available online 12 May 2011 Keywords: Asperlin ROS ATM
a b s t r a c t We exploited the biological activity of an antibiotic agent asperlin isolated from Aspergillus nidulans against human cervical carcinoma cells. We found that asperlin dramatically increased reactive oxygen species (ROS) generation accompanied by a significant reduction in cell proliferation. Cleavage of caspase-3 and PARP and reduction of Bcl-2 could also be detected after asperlin treatment to the cells. An anti-oxidant N-acetyl-L-cysteine (NAC), however, blocked all the apoptotic effects of asperlin. The involvement of oxidative stress in asperlin induced apoptosis could be supported by the findings that ROS- and DNA damage-associated G2/M phase arrest and ATM phosphorylation were increased by asperlin. In addition, expression and phosphorylation of cell cycle proteins as well as G2/M phase arrest in response to asperlin were significantly blocked by NAC or an ATM inhibitor KU-55933 pretreatment. Collectively, our study proved for the first time that asperlin could be developed as a potential anti-cancer therapeutics through ROS generation in HeLa cells. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction Reactive oxygen species (ROS) generation causes oxidative stress and has been shown to function to eliminate cancer cells [1]. Oxidation of all four DNA bases and induction of DNA strand breaks may occur as a result of oxidative DNA damage, and some of these lesions are converted to DNA double-strand breaks (DSBs) [2–4]. It has been well established that DNA damage causes phosphorylation and activation of ataxia telangiectasia mutated protein kinase (ATM) and initiates phosphorylation of substrate proteins including Chk2 [5,6]. Chk2 activation, followed by phosphorylation of cdc25C, plays a major role in arresting the cell cycle at G2/M phase in response to DNA damage [7,8]. Phosphorylation of cdc25C by Chk2 prevents the subsequent dephosphorylation of cyclin dependent kinases (CDKs), especially Cdc2, an event essential for G2/M transitions [9,10]. cdc2 is highly regulated during cell cycle progression. Cdc2 activation depends on the dephosphorylation of Tyr15 by cdc25C at G2/M transition [11]. In addition, Cdc2 binds ⇑ Corresponding author. Address: KRIBB, Ochang, Cheonwon, 363-883, Republic of Korea (B.Y. Kim). E-mail addresses: [email protected] (J.S. Ahn), [email protected] (B.Y. Kim). 1 These authors contributed equally to this work. 0006-291X/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2011.05.032
to cyclin A2 and cyclin B1 for further function in cell cycle [12]. Thus, ROS accumulation can severely affect DNA damage and induce G2/M arrest through ATM related pathway, leading to the apoptotic death of mitotic arrested cells [13]. Asperlin, [2-(3-methyloxiran-2-yl)-6-oxo-2,3-dihydropyran-3yl]-acetate, is a natural compound, known as antibiotic agent [14]. However, biological activity of this compound against tumor cells has not yet been reported. In the present study, we investigated the anti-cancer mechanism of asperlin in cancer cells and provided for the first time showing that asperlin induced G2/M arrest and apoptosis via ROS generation and ATM activation signaling. 2. Materials and methods 2.1. Cell culture and viability assay HeLa human cervical carcinoma cells were maintained in DMEM medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 mg/ml streptomycin and were incubated at 37 °C in a 5% CO2 incubator. Cells (4 104 cells/ml) in 96 well plates (100 ll/well) were treated with appropriate concentrations of asperlin with or without 5 lM NAC pretreatment for 24 h or 48 h. Ten microliters of CCK solution was added to each
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L. He et al. / Biochemical and Biophysical Research Communications 409 (2011) 489–493
well and the plates were further incubated at 37 °C for 1 h. Utilizing Dojindo’s highly water-soluble tetrazolium salt, the absorbance was measured at 450 nm with a reference wavelength at 650 nm using a microplate reader MR700 (Dynatech Inc., Chantilly, VA, USA).
tained from Sigma (St. Louis, MO). Asperlin was kindly provided by Dr. H.C. Oh (Shilla University, Pusan, Korea).
2.2. Reagents and materials
Flow cytometry was used to analyze cell cycle distribution in HeLa cells. Cells were fixed in 80% ethanol overnight at 20 °C and washed in phosphate buffered saline (PBS) and then further incubated with 10 lg/ml of propidium iodide (PI) 1 h at room-temperature. In order to analyze the percentage of apoptotic cells, all cultural cells were harvested and washed twice with cold PBS. The collected cells were re-suspended in annexin-V binding Ca2+ buffer in annexin-V-FITC staining solution (1 lg/ml) and incubated for 15 min at room temperature in the dark. Flow cytometric analysis was performed using a FACSCalibur (BectonDickinson, San Jose, CA).
Antibody to caspase-3 was obtained from Imgenex (San Diego, CA). Antibodies p-ATM, ATM, p-Chk2, p-cdc2 (Try15), cdc2 and cyclin B1 were purchased from Cell Signaling (Beverley, MA). Antibody to b-actin was from Sigma (St. Louis, MO). Antibodies to cdc25C, cyclin A2, PARP and p21 were obtained from SantaCruz Biotechnology (Santa Cruz, CA, USA). DMEM and fetal bovine serum were purchased from GIBCO-BRL (Grand Island, NY). Polyvinylidene difluoride (PVDF, 0.22 lm) membrane was from Bio-Rad (Hercules, CA). KU-55933 and N-acetyl-L-cysteine (NAC) were ob-
2.3. FACs analysis
Fig. 1. Asperlin induces ROS accumulation and suppresses the viability of HeLa cell. (A) Chemical structure of asperlin. (B) FACS analysis for ROS generation. Cells were seeded at 5 104 cells/ml in 96 well plate and treated with the indicated concentrations of asperlin for 1 h with or without the pretreatment of NAC at 5 lM for 1 h, followed by incubation with DFFDA treatment at 37 °C for 30 min. (C) Cell viability assay. After treating the cells with asperlin at the indicated concentrations for 24 and 48 h with or without NAC pretreatment (5 lM, 1 h), cell viability was determined by CCK-8 cell counting kit. (D) Apoptotic protein expressions. Cells treated with asperlin at 25 lM for 24 h or 48 h with or without NAC were lysed and equal amounts of cell lysate were subjected to western blotting analysis with antibodies to caspase-3, PARP and Bcl2. b-actin was used as a loading control. Arrow heads represent the fragments of caspase-3 and PARP cleavage. (E) In vitro caspase-3 assay expressed in O.D. Cells treated with the indicated concentration of asperlin for 48 h with or without NAC pretreatment (5 lM, 1 h) were lysed for the determination of caspase-3 activity in vitro using an caspase-3 assay kit. Each bar represents mean ± SE of triplicate experiments. The significance was determined by student’s t-test (⁄p < 0.05 vs. negative control; ⁄⁄p < 0.01 vs. negative control).
L. He et al. / Biochemical and Biophysical Research Communications 409 (2011) 489–493
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Fig. 1 (continued)
taining freshly added protease inhibitor mixture (Protease Inhibitor Cocktail Set III; Calbiochem, La Jolla, CA) on ice for 30 min. Whole cell lysates were centrifuged at 14,000 rpm for 15 min, and then the upper part of the solution was transferred into a new tube. For western blot analysis, appropriate amount of cell lysate was subjected to 10–14% SDS-PAGE, then the proteins were transferred onto a PVDF membrane (Millipore, Bedford, MA) for immune-blotting with specific antibodies and detected with chemiluminescence solution (ECL; Amersham Life Sciences Inc.). 2.5. ROS measurement Accumulation of intracellular ROS was examined by flow cytometry using DFFDA. Briefly, cells were plated in six well plates and incubated overnight. Cells were treated with asperlin for 1 h with or without 5 lM NAC and then stained with 5 lM DFFDA for 30 min at 37 °C. Cells were collected and fluorescence was analyzed using a flow cytometer. 2.6. In vitro caspase-3 assay
Fig. 2. Asperlin induces G2/M phase arrest in HeLa cells. (A) Cell cycle analysis. Cells were treated with varying concentrations of asperlin for 24 h, harvested and stained with propidium iodide (PI) for 30 min. (B, C) Western blot analysis. Cells treated with asperlin as above were lysed for subjection to western blotting analysis with antibodies to p-Bcl2, Bcl2, p-cdc2, cdc2, cdc25C, cyclin A2, cyclin B1, p-ATM, ATM, p-Chk2, Chk2 and p21. b-actin was used as a loading control.
In vitro caspase-3 protease activity was measured using a caspase activation kit according to the manufacturer’s protocol (R&D systems: Minneapolis, MN). Active caspase cleaves the peptide and releases the chromophore pNA that can be detected spectrophotometrically at a wavelength of 405 nm. 3. Results
2.4. Western blotting
3.1. Asperlin induces apoptotic death of HeLa cells through ROS generation
After washing with cold PBS buffer (pH 7.4), cells were lysed with ice-cold lysis buffer [50 mM Tris–HCl, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 20 mM NaF, 100 mM Na3VO4, 0.5% NP40, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride (pH 7.4)] con-
To determine the biological activity of asperlin, ROS generation was measured in HeLa cells. Cells were exposed to asperlin (Fig. 1A) at varying concentrations for 1 h with or without 5 lM NAC, a ROS inhibitor. Flow cytometric analysis after 5-(and-6)-car-
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Fig. 3. ATM and ROS generation are involved in asperlin-induced apoptosis of HeLa cells. (A) Effect of KU-99533 and NAC on asperlin-induced cell cycle. Cells were pretreated with either 5 lM NAC or 5 lM KU-55933 for 1 h prior to the exposure to asperlin at 25 lM for 24 h. All the other procedures for FACS analysis were done according to Section 2. (B, C) Effect of KU-55933 and NAC on protein expression associated with cell cycle. Equal amounts of total lysates obtained from the cells treated with asperlin, NAC and KU55933 as in (A) were subjected to western blotting analysis with specific antibodies to p-Chk2, Chk2, p-cdc2 and cyclin B1. b-actin was used as a loading control.
boxy-20 ,70 -dihydrodifluorofluorescein diacetate (DFFDA) staining showed that asperlin treatment increased ROS generation while little increase could be seen in the presence of NAC (Fig. 1B). Because oxidative stress is usually associated with cell growth, cell viability was determined following the treatment with asperlin and NAC. It was found that cell growth was dose dependently inhibited by asperlin but was restored in the presence of NAC (Fig. 1C). Consistent with these results, caspase-3 activation and PARP cleavage as well as Bcl2 reduction in response to asperlin were completely blocked by NAC pretreatment to the cells (Fig. 1D). Measurement of caspase-3 activity using the total lysates from the cells treated with asperlin and NAC further confirmed that asperlin-induced caspase-3 activity was reduced by NAC treatment (Fig. 1E). 3.2. Asperlin induces G2/M phase arrest through ATM-Chk2 pathway in HeLa cells ROS generation has an important role for cell cycle transition by a number of anti-cancer agents [15]. Flow cytometric analysis for measurement of DNA content showed that asperlin treatment significantly increased G2/M phase cells (Fig. 2A). Investigation of the pivotal proteins involved in G2/M transition by asperlin showed that expression of cyclin A2 and cyclin B1 was increased by asperlin (Fig. 2B). Phosphorylation of Bcl-2 [16] and of cdc2 which is inactive when phosphorylated at residues Thr-14 and Tyr-15, was increased upon asperlin treatment. On the other hand, cdc25C expression was reduced by asperlin, possibly through proteasomic degradation as already reported [17]. ROS accumulation induces DNA damage and cell cycle arrest, the regulation being dependent on ATM and Chk2 [18]. Western blot analysis revealed that asperlin induced the phosphorylation of both ATM and Chk2 in the cells without having any effect on p21 (Fig. 2C). 3.3. Cell cycle arrest by asperlin is mediated through ROS and ATM signaling To confirm that asperlin-induced G2/M arrest is associated with ROS generation and ATM-Chk2 signaling pathway, cells were trea-
ted with NAC and KU-55933, an anti-oxidant and an ATM inhibitor, respectively, before asperlin treatment. Pretreatment with either NAC or KU-55933 significantly abrogated the G2/M arresting effect of asperlin (Fig. 3A). Phosphorylation of Chk2, cdc2 and cyclinB1 by asperlin was also found to be reduced by pretreatment of NAC and KU-99533 (Fig. 3B and C). 4. Discussion Although isolated from Aspergillus nidulans in 1960s, little is known about the biological activity of asperlin [19]. Intracellular ROS accumulation generally causes DNA damage and leads to the induction of signaling cascades including ATM [20]. Activated ATM further phosphorylates other DNA damage-associated cell cycle proteins such as Chk1 and Chk2 [7,20]. Chk2 can in turn phosphorylate and inactivate cdc25C, resulting in the inactivation of cdc2-cyclin complex and cell cycle arrest in G2/M phase [21]. Our data demonstrated that asperlin significantly decreased the viability of HeLa cells through G2/M arrest which involved ROS generation and ATM-Chk2 activation pathway as evidenced by using NAC and KU-99533. It is, however, interesting to note that there was no obvious change in the level of p21 although p21 had already been shown to be downstream of Chk2, binding to cdc2-cyclin complex and regulating G2/M phase [22]. Given the inhibitory effect of KU-55933 [23] on asperlin-induced ATM signaling and mitochondrial anti-apoptotic protein expressions, asperlin seems to evoke cell death by ATM-mediated G2/M arrest. Given the reports demonstrating that ATM inhibition enhanced sensitization of cancer cells to apoptosis [24], and that ROS generation by chemotherapeutics is involved in apoptosis through the mitochondrial- or death receptor-associated signaling pathway [13,25], we are currently investigating the combined treatment of asperlin with TRAIL to other cancer cell types. Although asperlin was documented to inhibit the growth of various gram-positive and gram-negative bacteria, little is known about its anti-cancer activity in mammalian cells. Our study showed for the first time that asperlin has an anti-cancer activity that could be mediated through oxidative stress and ATM pathway. Since ROS is closely associated with cancer cell apoptosis and considering the resistance of cancer cells to anti-cancer drugs when
L. He et al. / Biochemical and Biophysical Research Communications 409 (2011) 489–493
used repeatedly, asperlin could be expected to be developed as an efficient anti-cancer therapeutics for cervical cancer treatment. Acknowledgments This work was supported by the World Class Institute (WCI) Program of the National Research Foundation of Korea (NRF), Global Partnership Program of Korea Foundation for International Cooperation of Science and Technology (M6060200000106E0200-00100), KRIBB Research Initiative Program, all grants from the Ministry of Education, Science and Technology (MEST), National R&D Program for Cancer Control (0820260) from the Ministry of Health & Welfare, Korea. References [1] S. Ueda, H. Nakamura, H. Masutani, T. Sasada, A. Takabayashi, Y. Yamaoka, J. Yodoi, Baicalin induces apoptosis via mitochondrial pathway as prooxidant, Mol. Immunol. 38 (2002) 781–791. [2] K.B. Beckman, B.N. Ames, Oxidative decay of DNA, J. Biol. Chem. 272 (1997) 19633–19636. [3] J. Cadet, T. Delatour, T. Douki, D. Gasparutto, J.P. Pouget, J.L. Ravanat, S. Sauvaigo, Hydroxyl radicals and DNA base damage, Mutat. Res. 424 (1999) 9– 21. [4] M.M. Vilenchik, A.G. Knudson, Endogenous DNA double-strand breaks: fidelity of production, repair, and induction of cancer, Proc. Natl. Acad. Sci. USA 100 (2003) 12871–12876. [5] J.H. Lee, T.T. Paull, ATM activation by DNA double-strand breaks through the Mre11-Rad50-Nbs1 complex, Science 308 (2005) 551–554. [6] T.T. Paull, J.H. Lee, The Mre11/Rad50/Nbs1 complex and its role as a DNA double-strand break sensor for ATM, Cell Cycle 4 (2005) 737–740. [7] B.B. Zhou, S.J. Elledge, The DNA damage response: putting checkpoints in perspective, Nature 408 (2000) 433–439. [8] J. Ahn, M. Urist, C. Prives, The Chk2 protein kinase, DNA Repair (Amst) 3 (2004) 1039–1047. [9] C.Y. Peng, P.R. Graves, R.S. Thoma, Z. Wu, A.S. Shaw, H. Piwnica-Worms, Mitotic and G2 checkpoint control: regulation of 14-3-3 protein binding by phosphorylation of Cdc25C on serine-216, Science 277 (1997) 1501–1505.
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Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Asperlin induces G2/M arrest through ROS generation and ATM pathway in human cervical carcinoma cells Long He a,1, Mei-Hua Nan a,1, Hyun Cheol Oh b, Young Ho Kim c, Jae Hyuk Jang a, Raymond Leo Erikson d, Jong Seog Ahn a,⇑, Bo Yeon Kim a,e,⇑ a Chemical Biology Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 30 Yeongudanji-ro, Ochang-eup, Cheongwon-gun, Chungbuk 363-883, Republic of Korea b College of Medical and Life Sciences, Silla University, 100 Silladaehak-gil, Sasang-gu, Busan 617-736, Republic of Korea c College of Pharmacy, ChungNam National University, Yuseong, Daejeon, 305-764, Republic of Korea d Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA e World Class Institute, KRIBB, 30 Yeongudanji-ro, Ochang-eup, Cheongwon-gun, Chungbuk 363-883, Republic of Korea
a r t i c l e
i n f o
Article history: Received 4 May 2011 Available online 12 May 2011 Keywords: Asperlin ROS ATM
a b s t r a c t We exploited the biological activity of an antibiotic agent asperlin isolated from Aspergillus nidulans against human cervical carcinoma cells. We found that asperlin dramatically increased reactive oxygen species (ROS) generation accompanied by a significant reduction in cell proliferation. Cleavage of caspase-3 and PARP and reduction of Bcl-2 could also be detected after asperlin treatment to the cells. An anti-oxidant N-acetyl-L-cysteine (NAC), however, blocked all the apoptotic effects of asperlin. The involvement of oxidative stress in asperlin induced apoptosis could be supported by the findings that ROS- and DNA damage-associated G2/M phase arrest and ATM phosphorylation were increased by asperlin. In addition, expression and phosphorylation of cell cycle proteins as well as G2/M phase arrest in response to asperlin were significantly blocked by NAC or an ATM inhibitor KU-55933 pretreatment. Collectively, our study proved for the first time that asperlin could be developed as a potential anti-cancer therapeutics through ROS generation in HeLa cells. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction Reactive oxygen species (ROS) generation causes oxidative stress and has been shown to function to eliminate cancer cells [1]. Oxidation of all four DNA bases and induction of DNA strand breaks may occur as a result of oxidative DNA damage, and some of these lesions are converted to DNA double-strand breaks (DSBs) [2–4]. It has been well established that DNA damage causes phosphorylation and activation of ataxia telangiectasia mutated protein kinase (ATM) and initiates phosphorylation of substrate proteins including Chk2 [5,6]. Chk2 activation, followed by phosphorylation of cdc25C, plays a major role in arresting the cell cycle at G2/M phase in response to DNA damage [7,8]. Phosphorylation of cdc25C by Chk2 prevents the subsequent dephosphorylation of cyclin dependent kinases (CDKs), especially Cdc2, an event essential for G2/M transitions [9,10]. cdc2 is highly regulated during cell cycle progression. Cdc2 activation depends on the dephosphorylation of Tyr15 by cdc25C at G2/M transition [11]. In addition, Cdc2 binds ⇑ Corresponding author. Address: KRIBB, Ochang, Cheonwon, 363-883, Republic of Korea (B.Y. Kim). E-mail addresses: [email protected] (J.S. Ahn), [email protected] (B.Y. Kim). 1 These authors contributed equally to this work. 0006-291X/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2011.05.032
to cyclin A2 and cyclin B1 for further function in cell cycle [12]. Thus, ROS accumulation can severely affect DNA damage and induce G2/M arrest through ATM related pathway, leading to the apoptotic death of mitotic arrested cells [13]. Asperlin, [2-(3-methyloxiran-2-yl)-6-oxo-2,3-dihydropyran-3yl]-acetate, is a natural compound, known as antibiotic agent [14]. However, biological activity of this compound against tumor cells has not yet been reported. In the present study, we investigated the anti-cancer mechanism of asperlin in cancer cells and provided for the first time showing that asperlin induced G2/M arrest and apoptosis via ROS generation and ATM activation signaling. 2. Materials and methods 2.1. Cell culture and viability assay HeLa human cervical carcinoma cells were maintained in DMEM medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 mg/ml streptomycin and were incubated at 37 °C in a 5% CO2 incubator. Cells (4 104 cells/ml) in 96 well plates (100 ll/well) were treated with appropriate concentrations of asperlin with or without 5 lM NAC pretreatment for 24 h or 48 h. Ten microliters of CCK solution was added to each
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well and the plates were further incubated at 37 °C for 1 h. Utilizing Dojindo’s highly water-soluble tetrazolium salt, the absorbance was measured at 450 nm with a reference wavelength at 650 nm using a microplate reader MR700 (Dynatech Inc., Chantilly, VA, USA).
tained from Sigma (St. Louis, MO). Asperlin was kindly provided by Dr. H.C. Oh (Shilla University, Pusan, Korea).
2.2. Reagents and materials
Flow cytometry was used to analyze cell cycle distribution in HeLa cells. Cells were fixed in 80% ethanol overnight at 20 °C and washed in phosphate buffered saline (PBS) and then further incubated with 10 lg/ml of propidium iodide (PI) 1 h at room-temperature. In order to analyze the percentage of apoptotic cells, all cultural cells were harvested and washed twice with cold PBS. The collected cells were re-suspended in annexin-V binding Ca2+ buffer in annexin-V-FITC staining solution (1 lg/ml) and incubated for 15 min at room temperature in the dark. Flow cytometric analysis was performed using a FACSCalibur (BectonDickinson, San Jose, CA).
Antibody to caspase-3 was obtained from Imgenex (San Diego, CA). Antibodies p-ATM, ATM, p-Chk2, p-cdc2 (Try15), cdc2 and cyclin B1 were purchased from Cell Signaling (Beverley, MA). Antibody to b-actin was from Sigma (St. Louis, MO). Antibodies to cdc25C, cyclin A2, PARP and p21 were obtained from SantaCruz Biotechnology (Santa Cruz, CA, USA). DMEM and fetal bovine serum were purchased from GIBCO-BRL (Grand Island, NY). Polyvinylidene difluoride (PVDF, 0.22 lm) membrane was from Bio-Rad (Hercules, CA). KU-55933 and N-acetyl-L-cysteine (NAC) were ob-
2.3. FACs analysis
Fig. 1. Asperlin induces ROS accumulation and suppresses the viability of HeLa cell. (A) Chemical structure of asperlin. (B) FACS analysis for ROS generation. Cells were seeded at 5 104 cells/ml in 96 well plate and treated with the indicated concentrations of asperlin for 1 h with or without the pretreatment of NAC at 5 lM for 1 h, followed by incubation with DFFDA treatment at 37 °C for 30 min. (C) Cell viability assay. After treating the cells with asperlin at the indicated concentrations for 24 and 48 h with or without NAC pretreatment (5 lM, 1 h), cell viability was determined by CCK-8 cell counting kit. (D) Apoptotic protein expressions. Cells treated with asperlin at 25 lM for 24 h or 48 h with or without NAC were lysed and equal amounts of cell lysate were subjected to western blotting analysis with antibodies to caspase-3, PARP and Bcl2. b-actin was used as a loading control. Arrow heads represent the fragments of caspase-3 and PARP cleavage. (E) In vitro caspase-3 assay expressed in O.D. Cells treated with the indicated concentration of asperlin for 48 h with or without NAC pretreatment (5 lM, 1 h) were lysed for the determination of caspase-3 activity in vitro using an caspase-3 assay kit. Each bar represents mean ± SE of triplicate experiments. The significance was determined by student’s t-test (⁄p < 0.05 vs. negative control; ⁄⁄p < 0.01 vs. negative control).
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Fig. 1 (continued)
taining freshly added protease inhibitor mixture (Protease Inhibitor Cocktail Set III; Calbiochem, La Jolla, CA) on ice for 30 min. Whole cell lysates were centrifuged at 14,000 rpm for 15 min, and then the upper part of the solution was transferred into a new tube. For western blot analysis, appropriate amount of cell lysate was subjected to 10–14% SDS-PAGE, then the proteins were transferred onto a PVDF membrane (Millipore, Bedford, MA) for immune-blotting with specific antibodies and detected with chemiluminescence solution (ECL; Amersham Life Sciences Inc.). 2.5. ROS measurement Accumulation of intracellular ROS was examined by flow cytometry using DFFDA. Briefly, cells were plated in six well plates and incubated overnight. Cells were treated with asperlin for 1 h with or without 5 lM NAC and then stained with 5 lM DFFDA for 30 min at 37 °C. Cells were collected and fluorescence was analyzed using a flow cytometer. 2.6. In vitro caspase-3 assay
Fig. 2. Asperlin induces G2/M phase arrest in HeLa cells. (A) Cell cycle analysis. Cells were treated with varying concentrations of asperlin for 24 h, harvested and stained with propidium iodide (PI) for 30 min. (B, C) Western blot analysis. Cells treated with asperlin as above were lysed for subjection to western blotting analysis with antibodies to p-Bcl2, Bcl2, p-cdc2, cdc2, cdc25C, cyclin A2, cyclin B1, p-ATM, ATM, p-Chk2, Chk2 and p21. b-actin was used as a loading control.
In vitro caspase-3 protease activity was measured using a caspase activation kit according to the manufacturer’s protocol (R&D systems: Minneapolis, MN). Active caspase cleaves the peptide and releases the chromophore pNA that can be detected spectrophotometrically at a wavelength of 405 nm. 3. Results
2.4. Western blotting
3.1. Asperlin induces apoptotic death of HeLa cells through ROS generation
After washing with cold PBS buffer (pH 7.4), cells were lysed with ice-cold lysis buffer [50 mM Tris–HCl, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 20 mM NaF, 100 mM Na3VO4, 0.5% NP40, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride (pH 7.4)] con-
To determine the biological activity of asperlin, ROS generation was measured in HeLa cells. Cells were exposed to asperlin (Fig. 1A) at varying concentrations for 1 h with or without 5 lM NAC, a ROS inhibitor. Flow cytometric analysis after 5-(and-6)-car-
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Fig. 3. ATM and ROS generation are involved in asperlin-induced apoptosis of HeLa cells. (A) Effect of KU-99533 and NAC on asperlin-induced cell cycle. Cells were pretreated with either 5 lM NAC or 5 lM KU-55933 for 1 h prior to the exposure to asperlin at 25 lM for 24 h. All the other procedures for FACS analysis were done according to Section 2. (B, C) Effect of KU-55933 and NAC on protein expression associated with cell cycle. Equal amounts of total lysates obtained from the cells treated with asperlin, NAC and KU55933 as in (A) were subjected to western blotting analysis with specific antibodies to p-Chk2, Chk2, p-cdc2 and cyclin B1. b-actin was used as a loading control.
boxy-20 ,70 -dihydrodifluorofluorescein diacetate (DFFDA) staining showed that asperlin treatment increased ROS generation while little increase could be seen in the presence of NAC (Fig. 1B). Because oxidative stress is usually associated with cell growth, cell viability was determined following the treatment with asperlin and NAC. It was found that cell growth was dose dependently inhibited by asperlin but was restored in the presence of NAC (Fig. 1C). Consistent with these results, caspase-3 activation and PARP cleavage as well as Bcl2 reduction in response to asperlin were completely blocked by NAC pretreatment to the cells (Fig. 1D). Measurement of caspase-3 activity using the total lysates from the cells treated with asperlin and NAC further confirmed that asperlin-induced caspase-3 activity was reduced by NAC treatment (Fig. 1E). 3.2. Asperlin induces G2/M phase arrest through ATM-Chk2 pathway in HeLa cells ROS generation has an important role for cell cycle transition by a number of anti-cancer agents [15]. Flow cytometric analysis for measurement of DNA content showed that asperlin treatment significantly increased G2/M phase cells (Fig. 2A). Investigation of the pivotal proteins involved in G2/M transition by asperlin showed that expression of cyclin A2 and cyclin B1 was increased by asperlin (Fig. 2B). Phosphorylation of Bcl-2 [16] and of cdc2 which is inactive when phosphorylated at residues Thr-14 and Tyr-15, was increased upon asperlin treatment. On the other hand, cdc25C expression was reduced by asperlin, possibly through proteasomic degradation as already reported [17]. ROS accumulation induces DNA damage and cell cycle arrest, the regulation being dependent on ATM and Chk2 [18]. Western blot analysis revealed that asperlin induced the phosphorylation of both ATM and Chk2 in the cells without having any effect on p21 (Fig. 2C). 3.3. Cell cycle arrest by asperlin is mediated through ROS and ATM signaling To confirm that asperlin-induced G2/M arrest is associated with ROS generation and ATM-Chk2 signaling pathway, cells were trea-
ted with NAC and KU-55933, an anti-oxidant and an ATM inhibitor, respectively, before asperlin treatment. Pretreatment with either NAC or KU-55933 significantly abrogated the G2/M arresting effect of asperlin (Fig. 3A). Phosphorylation of Chk2, cdc2 and cyclinB1 by asperlin was also found to be reduced by pretreatment of NAC and KU-99533 (Fig. 3B and C). 4. Discussion Although isolated from Aspergillus nidulans in 1960s, little is known about the biological activity of asperlin [19]. Intracellular ROS accumulation generally causes DNA damage and leads to the induction of signaling cascades including ATM [20]. Activated ATM further phosphorylates other DNA damage-associated cell cycle proteins such as Chk1 and Chk2 [7,20]. Chk2 can in turn phosphorylate and inactivate cdc25C, resulting in the inactivation of cdc2-cyclin complex and cell cycle arrest in G2/M phase [21]. Our data demonstrated that asperlin significantly decreased the viability of HeLa cells through G2/M arrest which involved ROS generation and ATM-Chk2 activation pathway as evidenced by using NAC and KU-99533. It is, however, interesting to note that there was no obvious change in the level of p21 although p21 had already been shown to be downstream of Chk2, binding to cdc2-cyclin complex and regulating G2/M phase [22]. Given the inhibitory effect of KU-55933 [23] on asperlin-induced ATM signaling and mitochondrial anti-apoptotic protein expressions, asperlin seems to evoke cell death by ATM-mediated G2/M arrest. Given the reports demonstrating that ATM inhibition enhanced sensitization of cancer cells to apoptosis [24], and that ROS generation by chemotherapeutics is involved in apoptosis through the mitochondrial- or death receptor-associated signaling pathway [13,25], we are currently investigating the combined treatment of asperlin with TRAIL to other cancer cell types. Although asperlin was documented to inhibit the growth of various gram-positive and gram-negative bacteria, little is known about its anti-cancer activity in mammalian cells. Our study showed for the first time that asperlin has an anti-cancer activity that could be mediated through oxidative stress and ATM pathway. Since ROS is closely associated with cancer cell apoptosis and considering the resistance of cancer cells to anti-cancer drugs when
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used repeatedly, asperlin could be expected to be developed as an efficient anti-cancer therapeutics for cervical cancer treatment. Acknowledgments This work was supported by the World Class Institute (WCI) Program of the National Research Foundation of Korea (NRF), Global Partnership Program of Korea Foundation for International Cooperation of Science and Technology (M6060200000106E0200-00100), KRIBB Research Initiative Program, all grants from the Ministry of Education, Science and Technology (MEST), National R&D Program for Cancer Control (0820260) from the Ministry of Health & Welfare, Korea. References [1] S. Ueda, H. Nakamura, H. Masutani, T. Sasada, A. Takabayashi, Y. Yamaoka, J. Yodoi, Baicalin induces apoptosis via mitochondrial pathway as prooxidant, Mol. Immunol. 38 (2002) 781–791. [2] K.B. Beckman, B.N. Ames, Oxidative decay of DNA, J. Biol. Chem. 272 (1997) 19633–19636. [3] J. Cadet, T. Delatour, T. Douki, D. Gasparutto, J.P. Pouget, J.L. Ravanat, S. Sauvaigo, Hydroxyl radicals and DNA base damage, Mutat. Res. 424 (1999) 9– 21. [4] M.M. Vilenchik, A.G. Knudson, Endogenous DNA double-strand breaks: fidelity of production, repair, and induction of cancer, Proc. Natl. Acad. Sci. USA 100 (2003) 12871–12876. [5] J.H. Lee, T.T. Paull, ATM activation by DNA double-strand breaks through the Mre11-Rad50-Nbs1 complex, Science 308 (2005) 551–554. [6] T.T. Paull, J.H. Lee, The Mre11/Rad50/Nbs1 complex and its role as a DNA double-strand break sensor for ATM, Cell Cycle 4 (2005) 737–740. [7] B.B. Zhou, S.J. Elledge, The DNA damage response: putting checkpoints in perspective, Nature 408 (2000) 433–439. [8] J. Ahn, M. Urist, C. Prives, The Chk2 protein kinase, DNA Repair (Amst) 3 (2004) 1039–1047. [9] C.Y. Peng, P.R. Graves, R.S. Thoma, Z. Wu, A.S. Shaw, H. Piwnica-Worms, Mitotic and G2 checkpoint control: regulation of 14-3-3 protein binding by phosphorylation of Cdc25C on serine-216, Science 277 (1997) 1501–1505.
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