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M phase arrest and apoptosis in human hepatoma cancer cells through ROS generation
Cancer Letters 288 (2010) 204–213
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Cancer Letters journal homepage: www.elsevier.com/locate/canlet
Butein induces G2/M phase arrest and apoptosis in human hepatoma cancer cells through ROS generation Dong-Oh Moon a,b, Mun-Ock Kim a,b, Yung Hyun Choi c, Jin Won Hyun b,d, Weon Young Chang b,d, Gi-Young Kim a,b,* a
Department of Marine Life Science, Jeju National University, Republic of Korea Laboratory of Immunobiology, Department of Marine Life Sciences, Jeju National University, Jeju 690-756, Republic of Korea c Department of Biochemistry, Dongeui University College of Oriental Medicine, Busan 614-054, Republic of Korea d School of Medicine, Jeju National University, Jeju 690-756, Republic of Korea b
a r t i c l e
i n f o
Article history: Received 4 May 2009 Accepted 5 July 2009
Keywords: Butein G2/M phase arrest Apoptosis ROS JNK
a b s t r a c t We investigated the molecular effects of 3,4,20 ,40 -tetrahydroxychalcone (butein) treatment in two human hepatoma cancer cell lines–HepG2 and Hep3B. Butein treatment inhibited cancer cell growth by inducing G2/M phase arrest and apoptosis. Butein-induced G2/M phase arrest was associated with increased ATM, Chk1, and Chk2 phosphorylations and reduced cdc25C levels. Additionally, butein treatment enhanced inactivated phosphoCdc2 levels, reduced Cdc2 kinase activity, and generated reactive oxygen species (ROS) that was accompanied by JNK activation. The extent of butein-induced G2/M phase arrest significantly decreased following pretreatment with N-acetyl-L-cysteine or glutathione and following JNK phosphorylation reduction by SP600125. Both N-acetyl-L-cysteine and glutathione also decreased butein-mediated apoptosis. Taken together, these results imply a critical role of ROS and JNK in the anticancer effects of butein. Ó 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Hepatocellular carcinoma (HCC) is one of the most common human malignancies and the third leading cause of cancer death worldwide. It accounts for about 6% of all human cancers annually [1]. HCC commonly develops from liver cirrhosis, in which there is continuous hepatocyte regeneration and inflammation; this suggests that DNA damage and reactive oxygen species (ROS) are involved in the process of hepatocarcinogenesis [2]. Although substantial progress has been made in developing chemotherapeutic treatments for HCC, drug efficacy is often outweighed by undesirable side effects. Thus, there is a need to develop new therapies against HCC.
* Corresponding author. Address: Department of Marine Life Science, Jeju National University, Republic of Korea. Tel.: +82 64 754 3427; fax: +82 64 756 3493. E-mail address: [email protected] (G.-Y. Kim). 0304-3835/$ - see front matter Ó 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2009.07.002
All aerobic organisms experience physiological oxidant stress as a consequence of aerobic metabolism. In generating ATP, aerobic respiration releases the superoxide anion radical (O 2 ) as a byproduct. Once produced, O2 can form other ROS, such as hydrogen peroxide (H2O2) or the highly reactive hydroxyl radical (OH) [3,4]. In normal physiological states, these are produced through electron leakage and uncoupling when molecular oxygen is consumed in the electron transport chain [5], although ROS can also be generated in response to various exogenous stimuli, such as radiation, surgery, or chemicals. It is well established that ROS are mediators of intracellular signaling cascades. Excessive ROS generation can induce redox-signaling pathways, including those involved in oxidative stress, loss of cell function, cell cycle arrest, and apoptosis [6]. An accumulation of ROS can severely damage cellular macromolecules, especially DNA, and induce G2/M phase arrest through ataxia telangiectasia mutant (ATM) activation [7]. Cytotoxic ROS signaling also induces mitochondrial-
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dependent apoptosis; this occurs through the activation of apoptosis signal-regulating kinase 1 (ASK1)/mitogen-activated protein kinase (MAPK) pathways, and of the proapoptotic Bcl-2 proteins Bax or Bak, with the subsequent effects of mitochondrial membrane permeabilization and cell death [8–12]. 3,4,20 ,40 -Tetrahydroxychalcone (butein) is a polyphenolic compound extracted from the stembark of cashews and Rhus verniciflua Stokes, and has been used as a traditional herbal medicine. Previous studies have suggested that butein has anticancer activity against several human cancers, including leukemia, melanoma, breast carcinoma, colon carcinoma, osteosarcoma, and hepatic stellate cells [13– 19]. However, the role of ROS generation with respect to the anticancer effects of butein is not fully understood. The present study was designed to investigate the underlying mechanism involved in the induction of G2/M phase arrest and apoptosis by butein in human hepatoma cancer cells. Our data provide the first evidence that butein induces G2/M phase arrest and apoptosis in Hep3B and HepG2 cells via a mechanism involving the generation of ROS, the activation of JNK, and the subsequent induction of the mitochondrial apoptotic pathway.
2. Materials and methods 2.1. Reagents Antibodies against PARP, Bcl-2, Bax, cyclin B1, Cdc2, and cdc25C were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against phospho (p)-Cdc2 (Thr-14/Tyr-15), p-cdc25C (Ser-216), p-ATM (Ser-1981), p-Chk1 (Ser-345), p-Chk2 (Thr-68), JNK, p-JNK, c-jun, and p-c-jun were purchased from Cell Signal (Beverly, MA). Antibody against b-actin, and antioxidants glutathione (Glu) and N-acetyl-L-cysteine (NAC) were purchased from Sigma (St. Louis, MO). Butein was purchased from Sigma and dissolved in DMSO (vehicle). 2.2. Cell culture and viability assay Human hepatoma cancer cell lines HepG2 and Hep3B were cultured in RPMI medium (Life Technologies; Grand Island, NY) supplemented with 10% fetal bovine serum (FBS) and antibiotics (Sigma). Cells were seeded at 5 104 cells/ml, incubated for 24 h, and then treated with the indicated concentrations of butein for the indicated times. Cell viability was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and trypan blue exclusion assays.
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analyze the cells. Level of apoptotic cells containing subG1 DNA content was determined as a percentage of the total number of cells. For annexin-V staining, live cells were washed with PBS and then incubated with annexin-V fluorescein isothiocyanate (R&D Systems; Minneapolis, MN). Cells were analyzed using flow cytometry. 2.4. Western blot analysis Total cell extracts were prepared using PRO-PREP protein extraction solution (iNtRON Biotechnology; Sungnam, Republic of Korea). Cell extracts were separated on 10% polyacrylamide gels and then transferred to nitrocellulose membranes using standard procedures. The membranes were developed using an ECL reagent (Amersham; Arlington Heights, IL). 2.5. ROS measurement Generation of intracellular ROS was examined by flow cytometry using hydroethidine (HE) for O 2 and 6-carboxy-20 ,70 -dichlorofluorescein diacetate (H2DCFDA) for H2O2 (Molecular Probes; Eugene, OR) [20]. The HE is oxidized to ethidium bromide, whereas H2DCFDA is cleaved by nonspecific cellular esterases and oxidized in the presence of H2O2 and peroxidases to yield fluorescent 20 ,70 dichlorofluorescein (DCF). Briefly, 5 104 cells were plated in 60-mm dishes, allowed to attach overnight, and exposed to different concentrations of butein for 1 h. Cells were stained with 2 lM HE or 5 lM H2DCFDA for 30 min at 37 °C. Cells were collected and the fluorescence was analyzed using a flow cytometer. In some experiments, cells were pretreated with 10 mM NAC or 5 mM Glu prior to butein exposure and analysis of ROS generation. 2.6. In vitro caspase-3 activity assay Activity of caspase-like protease was measured using a caspase activation kit according to the manufacturer’s protocol (R&D systems; Minneapolis, MN). This assay is based on spectrophotometric detection of the color reporter molecule p-nitroaniline (pNA), which is linked to the end of the caspase-specific substrate. The caspase cleaves the peptide and releases the chromophore pNA, which can be quantified spectrophotometrically at a wavelength of 405 nm. 2.7. Cdc2 kinase activity Cdc2 kinase activity was measured by MESACUP Cdc2 kinase assay kit (MBL; Woburn, MA) according to the manufacturer’s instructions.
2.3. Flow cytometric analysis
2.8. Superoxide dismutase (SOD) activity
Flow cytometry was used to analyze cell cycle distribution. Cells (1 106) were fixed in 70% ethanol overnight at 4 °C and washed in phosphate-buffered saline (PBS) with 0.1% BSA. Cells were then incubated with 1 U/ml of RNase A (DNase free) and 10 lg/ml of propidium iodide (PI; Sigma) 1 h at room temperature in the dark. FACSCalibur flow cytometer (Becton Dickenson; San Jose, CA) was used to
HepG2 and Hep3B cells (5 105) were plated, allowed to attach overnight, and exposed to butein for 1 h at 37 °C. The cells were collected, washed with PBS, and pelleted by centrifugation at 500g for 6 min. The cell pellet was suspended in ice-cold 50 mM potassium phosphate (pH 7.0) containing 1 mM EDTA, sonicated and centrifuged at 10,000g for 15 min at 4 °C. The supernatant fraction was
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used for determination of SOD activity using kits from Cayman Chemical (Ann Arbor, MI) according to the manufacturer’s instructions. 2.9. Statistical analysis All data were derived from at least three independent experiments. The blots were visualized with the ChemiSmart 2000 (Vilber Lourmat; Marine, Cedex, France). Images were captured using Chemi-Capt software (Vilber Lourmat) and imported into Adobe Photoshop (Adobe Systems; San Jose, CA). Statistical analyses were conducted using SigmaPlot software (Aspire Software International; Ashburn, VA), and values are presented as mean ± standard deviation (SD). Significant differences between the groups were determined using the unpaired Student’s t-test. A value of P < 0.05 was accepted to be statistically significant. 3. Results 3.1. Butein suppresses the viability and growth of hepatoma cells through G2/ M phase arrest To determine the effect of butein on cell viability and growth, human hepatoma cells (HepG2 and Hep3B) were treated with one of various concentrations of butein for 24 h. Cell viability and growth were determined
by MTT and trypan blue exclusion assays, respectively. Treatment with butein resulted in a dose-dependent inhibition of HepG2 and Hep3B cell viability and growth (Fig. 1A and B). To further characterize the inhibitory effect of butein on cell proliferation, we monitored cell cycle progression using flow cytometry. Exposure to butein resulted in an increase of G2/M phase cells, accompanied by a decrease in G1 phase cells (Fig. 1C). The effect observed at 30 lM butein was the greatest, with approximately 58% of cells being in the G2/M phase, compared to approximately 20% in the control condition. Consequently, our results demonstrate that treatment with butein inhibits the proliferation of hepatoma cancer cells by inducing G2/M transition. 3.2. Butein controls the expression level of proteins that regulate G2/M transition The generation of ROS has an important role in the effects of various anticancer agents on cell cycle transition [6]. We thus investigated the possibility that butein induces G2/M phase arrest by allowing for the accumulation of ROS. Intracellular ROS generation in control and butein-treated Hep3B cells was assessed by flow cytometry after staining with HE and H2DCFDA. Butein treatment induced a dose-dependent increase in mean HE and DCF fluorescence, when compared with control cells (Fig. 2A). We then determined the effect of butein treatment on SOD enzyme activity. Treatment with butein for 1 h resulted in a modest, but statistically significant, increase of SOD activity, as compared with controls (Fig. 2B). To elucidate the mechanism of G2/M phase arrest in butein-treated cells, we investigated effects associated with the expression of pivotal protein for G2/M transition. This was done after treating cells with one of various concentrations of butein for 24 h. Butein treatment downregulated both cdc25C and Cdc2 in HepG2 and Hep3B cells
Fig. 1. Butein dose-dependently triggers G2/M phase arrest in hepatoma cells. Cells were seeded at 5 104 cells/ml and treated with the indicated concentrations of butein for 24 h. Cell viability (A) and number (B) were determined by MTT assay and hemocytometer counts of trypan blue-excluding cells, respectively. (C) In a parallel experiment, cells were harvested and stained with PI for 1 h; 10,000 events were analyzed for each sample. DNA content is on the x-axis; the number of cells counted is on the y-axis. Each point represents mean ± SD of three independent experiments. Statistically significant differences were determined using Student’s t-test (, P < 0.05 vs. vehicle control).
D.-O. Moon et al. / Cancer Letters 288 (2010) 204–213 (Fig. 2C). In contrast, butein did not influence the expression levels of cyclin B1. Further, butein treatment decreased Cdc2 kinase activity (Fig. 2D). Cdc2 is inactive when phosphorylated at the Thr-14 and Tyr-15 site. However, activated cdc25C dephosphorylates Cdc2 at these residues, leading to activation of the cyclin B1/Cdc2 complex [21]. We thus hypothesized that treatment with butein might result in an accumulation of Thr14/Tyr-15-phosphorylated Cdc2. We examined this possibility by Western blot analysis, using an antibody specific for p-Cdc2 (Thr-14/Tyr15). Compared with untreated control cells, treatment with butein was associated with a dose-dependent increase in the Thr-14/Tyr-15-phosphorylation of Cdc2 (Fig. 2C). After Chk1 and Chk2 phosphorylate cdc25C on Ser-216, thereby cdc25C is undergone to degradation. Next, we examined whether treatment with butein lead to an accumulation of phosphorylated Chk1 (Ser345) or Chk2 (Thr-68). Western blot analysis revealed the phosphorylated forms of Chk1 and Chk2 to be more abundant after 24 h of treatment with
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butein, as compared to a control condition (Fig. 2C). ATM is an upstream kinase implicated in the phosphorylation/activation of Chk1 and Chk2, and is known to be activated in response to DNA damage/genomic stress in eukaryotic cells [22]. Western blot analysis using an antibody specific for p-ATM (Ser-1981) revealed that treatment with butein increased the phosphorylation of ATM (Fig. 2C). These findings indicate that butein induces G2/M phase arrest, and that this coincides with the activation of ATM, Chk1, and Chk2, and with the inactivation of Cdc2. 3.3. Antioxidants NAC and Glu prevent butein-induced G2/M phase cell cycle arrest NAC and Glu scavenge ROS in cells by interacting with OH and H2O2 [3,4], and thus affect ROS-mediated signaling pathways. Because of this, we investigated whether these antioxidants affect butein-induced G2/M phase arrest. Pretreatment with NAC or Glu significantly blocked the
Fig. 2. Butein affects the expression of proteins involved in regulating the G2/M transition associated with the generation of ROS. Cells were treated with one of various concentrations of butein for 1 h, or for one of various times up to 24 h (C and D). (A) Hep3B cells were treated with the indicated concentration of butein, and then with either 2 lM HE or 5 lM H2DCFDA for 30 min at 37 °C and subsequent FACS analysis was used to assess the intracellular accumulation of ROS. (B) SOD activity in the lysates from HepG2 and Hep3B cells treated for 1 h with the indicated concentrations of butein. (C) The cells were lysed for protein extraction. Samples (50 lg) were subjected to 10% SDS–PAGE and Western blotting for the detection of specific proteins, as indicated [cyclin B1, Cdc2, cdc25C, p-Cdc2 (Thr-14/Tyr-15), p-ATM (Ser-1981), p-Chk1 (Ser-345), and p-Chk2 (Thr-68)]. b-Actin was used as a loading control. (D) Cdc2 kinase activity was determined as per the manufacturer’s protocol. Each point represents the mean ± SD of three independent experiments. Statistically significant differences were determined using Student’s t-test (, P < 0.05 vs. vehicle control).
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Fig. 3. The butein-induced generation of ROS is correlated with G2/M phase arrest. (A) Hep3B cells were pretreated with either 10 mM NAC or 5 mM Glu for 1 h before being exposed to 30 lM butein for 1 h following which either 2 lM HE or 5 lM H2DCFDA was administered for 30 min at 37 °C. FACS analysis was used to assess the intracellular accumulation of ROS. (B) In a parallel experiment, cell cycle distribution was analyzed by flow cytometry after 24 h. Cells were harvested and 10,000 events analyzed for each sample. Fluorescence intensity is plotted on the x-axis, and the relative number of cells plotted on the y-axis. (C) Equal amounts of cell lysates (50 lg) were resolved by SDS–PAGE, transferred to nitrocellulose, and probed with specific antibodies against pChk2 (Thr-68), cdc25C, Cdc2, and p-Cdc2. b-Actin was used as a loading control. (D) Cdc2 kinase activity was determined as per the manufacturer’s protocol. Each point represents the mean ± SD of three independent experiments. Statistically significant differences were determined using Student’s t-test (, P < 0.05 vs. vehicle control).
D.-O. Moon et al. / Cancer Letters 288 (2010) 204–213 butein-mediated generation of ROS, including O 2 and H2O2, (Fig. 3A), and completely abrogated butein-induced G2/M phase arrest (Fig. 3B). We used Western blot analysis to further examine the role of ROS in butein-induced G2/M phase arrest. Pretreatment with NAC or Glu suppressed the phosphorylation of Chk2 and Cdc2 proteins induced by butein (Fig. 3C). The phosphorylation status of these proteins was not affected by NAC or Glu administered in isolation of butein as compared with a control condition. Indeed, pretreatment with these antioxidants produced a significant restoration of cdc25C and Cdc2 expression levels (Fig. 3C), and Cdc2 kinase activity (Fig. 3D). Therefore, these data indicate that cdc25C and Cdc2 have important roles in regulating butein-mediated G2/M phase arrest.
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3.4. The role of JNK activation in butein-induced cell cycle arrest It is well known that ROS-mediated DNA damage induces JNK activation, and subsequently induces cell death [12,23]. Accordingly, we assessed the status of JNK at 24 h after treatment with butein. Treatment with butein resulted in the activation of JNK (Fig. 4A). On the other hand, the total amount of JNK (unphosphorylated form) was not altered by treatment with butein. We performed Western blot analysis to investigate whether butein-mediated JNK activation depends on the intracellular level of ROS. Pretreatment with NAC significantly blocked butein-induced JNK activation (Fig. 4B). We next investigated the possible roles of these alterations of JNK activity in butein-induced G2/M phase arrest. As shown
Fig. 4. Butein induces G2/M phase arrest through JNK activation. (A) HepG2 and Hep3B cells were treated with the indicated concentration of butein for 24 h. Equal amounts of cell lysates (50 lg) were resolved on SDS–polyacrylamide gels, transferred to nitrocellulose membranes, and probed with antibodies against anti-p-JNK and anti-JNK. (B) Hep3B cells were pretreated with the indicated concentration of NAC for 1 h before exposure to 30 lM butein for 24 h. Western blot analysis was performed to detect p-JNK and JNK. (C) HepG2 and Hep3B cells were stimulated with 30 lM butein for 24 h after pretreatment with SP600125 (20 or 40 lM) for 1 h. The cells were stained with PI and analyzed by flow cytometry after 24 h. (D) Portions of the G2/M phase are presented. (E) Equal amounts of cell lysates (50 lg) were resolved by SDS–PAGE, transferred to nitrocellulose, and probed with specific antibodies (anti-p-cjun, anti-c-jun, anti-p-Chk2, and anti-cdc25C). b-Actin was used as a loading control. SP, SP600125.
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by flow cytometry analysis of HepG2 cells (Fig. 4C and D), pretreatment with SP600125 (an inhibitor of JNK) resulted in a greater decrease in the number of G2/M cells (32 ± 3%) than treatment with butein alone (60 ± 3%). We found similar results for Hep3B cells. In order to confirm that this effect was specific for the JNK pathway, the phosphorylation status of c-jun (a downstream of JNK) was assessed by Western blot analysis after 24 h. Butein-induced c-jun phosphorylation was significantly attenuated by treatment with JNK inhibitors (Fig. 4E). We also investigated whether SP600125 modulates protein expression associated with butein-induced G2/M phase arrest. Treatment with SP600125 significantly blocked the phosphorylation of the Chk2 and Cdc2 proteins induced by butein (Fig. 4E). Indeed, pretreatment with SP600125 restored the levels to which cdc25C was expressed (Fig. 4E). These data suggest that the phosphorylation of JNK regulates butein-induced G2/M phase arrest. 3.5. Butein induces apoptotic cell death by triggering mitochondrial apoptosis pathway In order to investigate whether treatment with butein affects apoptosis, HepG2 and Hep3B cells were stimulated with various concentrations of butein (0–40 lM) for 48 h. Treatment with butein significantly increased the proportion of annexin-V in both hepatoma cell lines (Fig. 5A). The Bcl-2 protein family contains both antiapoptotic and proapoptotic members. These proteins play pivotal regulatory roles in deciding cell fate, through interactions that integrate a wide array of diverse upstream survival and distress signals [24]. In order to examine the role of the Bcl-2 family in butein-induced apoptosis, we used Western blot analysis to assess the effect of butein on the levels of anti-apoptotic Bcl-2 and pro-apoptotic Bax. Treatment with butein induced a dose-
dependent increase in Bax, and a dose-dependent decrease in Bcl-2 (Fig. 5B). Recent studies have demonstrated that caspases are important regulators of apoptosis [25]. Therefore, we investigated the involvement of caspase-3 in butein-induced apoptosis. Treatment with butein resulted in the activation of caspase-3 in HepG2 and Hep3B cells (Fig. 5C). There data suggest that treatment with butein induces apoptosis via increases in the Bax/Bcl-2 ratio and caspase-3 activity. 3.6. ROS are involved in butein-induced apoptosis We investigated whether the ROS generation is directly associated with butein-induced apoptosis, by assessing these outcomes in hepatoma cells pretreated for 1 h with either 10 lM NAC or 5 lM Glu followed by treatment with 30 lM butein. Pretreatment with NAC or Glu prevented the butein-induced increases in sub-G1 phase DNA content (Fig. 6A) and annexin-V (Fig. 6B). Additionally, these antioxidants decreased the butein-mediated upregulation of Bax and cleavage of PARP, and abrogated the butein-induced downregulation of Bcl-2 (Fig. 6C). Furthermore, the butein-induced activation of caspase-3 was also attenuated by pretreatment with NAC or Glu (data not shown). These results suggest that the generation of ROS plays an upstream role in butein-mediated mitochondrial apoptotic pathways.
4. Discussion It is well known that O 2 and H2O2 are the two major ROS in aerobic organisms. The functions of ROS include stimulating mitotic cell division and inducing cellular
Fig. 5. Butein induces apoptosis. HepG2 and Hep3B cells were seeded at 5 104 cells/ml and treated with the indicated concentrations of butein for up to 48 h. (A) Cells were treated with one of various concentrations of butein for 48 h, harvested, and 10,000 events analyzed for apoptotic annexin-V+ population. (B) Cells treated with the indicated concentrations of butein for 48 h were lysed for protein extraction. Samples were subjected to 10% SDS– PAGE and Western blotting for the detection of specific proteins, as indicated (anti-Bax and anti-Bcl-2). b-Actin was used as a loading control. (C) Caspase-3 activity was determined using a caspase-3 assay kit. Each point represents the mean ± SD of three independent experiments. Statistically significant differences were determined using Student’s t-test (, P < 0.05 vs. vehicle control).
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senescence. ROS are also important regulators of apoptosis. ROS production has been shown to be part of the signaling process, whereby many bioactive agents activate a cascade that functions to eliminate cancer cells [26,27]. Accordingly, excessive generation of ROS has been shown to cause cell damage and initiate various effects [28]. Recent reports have documented that many forms of apoptosis, as well as cell death in ischemia and neurodegenerative disease are associated with ROS production [29,30]. In the present study, we found that butein induces ROS-dependent cell cycle arrest at the G2/M phase, followed by a late apoptosis, in HepG2 and Hep3B cells. Butein arrested cell cycle progression by upregulating the phosphorylation of Cdc2, which is required for G2/M transition. It is well known that cdc25C is a specific tyrosine phosphatase that can directly
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induce Cdc2 activation [21], and the phosphorylation and degradation of cdc25C are important regulatory steps by which cells can delay or block mitotic entry under normal conditions. It is therefore not surprising that we also found a decreased accumulation of cdc25C as being associated with butein-induced G2/M arrest. Chk1 and Chk2 are known to be activated in response to DNA damage/genomic stress via phosphorylations at Ser-345/Ser-317 and Thr-68, respectively [31,32]. Upon activation, Chk1 and Chk2 can inactivate cdc25C via phosphorylation at Ser216, blocking downstream activation of Cdc2 and mitosis [33]. Our results show the abundance of the phosphorylated forms of Chk1 and Chk2 as dose-dependently increasing in cells treated with butein for 24 h. Chk1 and Chk2 are important intermediaries of DNA damage check-
Fig. 6. Antioxidants prevent butein-induced apoptosis. HepG2 and Hep3B cells were pretreated with either 10 mM NAC or 5 mM Glu for 1 h before exposure to 30 lM butein for 48 h. FACS analysis for DNA contents (A) and annexin-V (B). (C) In a parallel experiment, equal amounts of cell lysates (50 lg) were resolved by SDS–PAGE, transferred to nitrocellulose, and probed with specific antibodies against Bcl-2, Bax, and PARP. b-Actin was used as a loading control.
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point pathways, and ATM is an upstream kinase implicated in the phosphorylation/activation of Chk1 and Chk2 [32]. Western blot analysis using an antibody specific for pATM (Ser-1981) revealed an increase of p-ATM in buteintreated cells. The results of the present study indicate that an increased degradation of cdc25C in butein-treated cells is associated with ATM-dependent activation of Chk1 and Chk2. Furthermore, Western blot analysis also revealed that the Bax/Bcl-2 ratio was affected by treatment with butein. Moreover, although the activity of caspase-3 was elevated in the presence of butein, cell death was not stopped by a caspase-3 specific inhibitor, Ac-DEVD-CHO, nor by the pan-caspase inhibitor z-VAD-fmk (data not shown). These findings suggest that butein-induced apoptosis involves ROS generation, but not involve a caspase-dependent pathway. However, further study is necessary to clarify if there is a role for caspases in butein-induced apoptosis, given that caspases are the main regulators of apoptosis in various types of cancer cells. JNKs have been implicated in the apoptotic response of cells exposed to UV irradiation, heat shock, chemotherapy, and proinflammatory cytokines [34,35]. A number of anticancer drugs have been reported to kill cancer cells via the JNK apoptotic pathway, so it is to be expected that a blockade of JNK activation could inhibit anticancer activity. In the present study, we have shown that treatment with butein resulted in the in vitro accumulation of p-JNK in HepG2 and Hep3B cancer cells. JNK activation was found to correlate well with the butein-mediated upregulation of JNK activity, as measured by the JNK substrate p-c-Jun. Furthermore, by administering the JNK specific inhibitor SP600125, we found that preventing the butein-induced activation of JNK blocks butein-mediated G2/M arrest, and that this occurs through a downregulation of the ATM-Chk1/2 signaling pathway induced by butein. However, the inhibition of JNK did not prevent butein-induced apoptosis (data not shown), suggesting that JNK does not cooperate with a mitochondrial apoptotic pathway in generating butein-induced apoptosis. In conclusion, our data indicate that human hepatoma cancer cells are highly sensitive to butein, which inhibits their growth and induces apoptosis. Underlying butein-induced cell cycle arrest is a generation of ROS and a subsequent activation of JNK. Conflict of interest None declared. Acknowledgment This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2009-0076654). References [1] A.K. Nowak, P.K.H. Chow, M. Findlay, Systemic therapy for advanced hepatocellular carcinoma: a review, Eur. J. Cancer 40 (2004) 1474– 1484.
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Cancer Letters journal homepage: www.elsevier.com/locate/canlet
Butein induces G2/M phase arrest and apoptosis in human hepatoma cancer cells through ROS generation Dong-Oh Moon a,b, Mun-Ock Kim a,b, Yung Hyun Choi c, Jin Won Hyun b,d, Weon Young Chang b,d, Gi-Young Kim a,b,* a
Department of Marine Life Science, Jeju National University, Republic of Korea Laboratory of Immunobiology, Department of Marine Life Sciences, Jeju National University, Jeju 690-756, Republic of Korea c Department of Biochemistry, Dongeui University College of Oriental Medicine, Busan 614-054, Republic of Korea d School of Medicine, Jeju National University, Jeju 690-756, Republic of Korea b
a r t i c l e
i n f o
Article history: Received 4 May 2009 Accepted 5 July 2009
Keywords: Butein G2/M phase arrest Apoptosis ROS JNK
a b s t r a c t We investigated the molecular effects of 3,4,20 ,40 -tetrahydroxychalcone (butein) treatment in two human hepatoma cancer cell lines–HepG2 and Hep3B. Butein treatment inhibited cancer cell growth by inducing G2/M phase arrest and apoptosis. Butein-induced G2/M phase arrest was associated with increased ATM, Chk1, and Chk2 phosphorylations and reduced cdc25C levels. Additionally, butein treatment enhanced inactivated phosphoCdc2 levels, reduced Cdc2 kinase activity, and generated reactive oxygen species (ROS) that was accompanied by JNK activation. The extent of butein-induced G2/M phase arrest significantly decreased following pretreatment with N-acetyl-L-cysteine or glutathione and following JNK phosphorylation reduction by SP600125. Both N-acetyl-L-cysteine and glutathione also decreased butein-mediated apoptosis. Taken together, these results imply a critical role of ROS and JNK in the anticancer effects of butein. Ó 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Hepatocellular carcinoma (HCC) is one of the most common human malignancies and the third leading cause of cancer death worldwide. It accounts for about 6% of all human cancers annually [1]. HCC commonly develops from liver cirrhosis, in which there is continuous hepatocyte regeneration and inflammation; this suggests that DNA damage and reactive oxygen species (ROS) are involved in the process of hepatocarcinogenesis [2]. Although substantial progress has been made in developing chemotherapeutic treatments for HCC, drug efficacy is often outweighed by undesirable side effects. Thus, there is a need to develop new therapies against HCC.
* Corresponding author. Address: Department of Marine Life Science, Jeju National University, Republic of Korea. Tel.: +82 64 754 3427; fax: +82 64 756 3493. E-mail address: [email protected] (G.-Y. Kim). 0304-3835/$ - see front matter Ó 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2009.07.002
All aerobic organisms experience physiological oxidant stress as a consequence of aerobic metabolism. In generating ATP, aerobic respiration releases the superoxide anion radical (O 2 ) as a byproduct. Once produced, O2 can form other ROS, such as hydrogen peroxide (H2O2) or the highly reactive hydroxyl radical (OH) [3,4]. In normal physiological states, these are produced through electron leakage and uncoupling when molecular oxygen is consumed in the electron transport chain [5], although ROS can also be generated in response to various exogenous stimuli, such as radiation, surgery, or chemicals. It is well established that ROS are mediators of intracellular signaling cascades. Excessive ROS generation can induce redox-signaling pathways, including those involved in oxidative stress, loss of cell function, cell cycle arrest, and apoptosis [6]. An accumulation of ROS can severely damage cellular macromolecules, especially DNA, and induce G2/M phase arrest through ataxia telangiectasia mutant (ATM) activation [7]. Cytotoxic ROS signaling also induces mitochondrial-
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dependent apoptosis; this occurs through the activation of apoptosis signal-regulating kinase 1 (ASK1)/mitogen-activated protein kinase (MAPK) pathways, and of the proapoptotic Bcl-2 proteins Bax or Bak, with the subsequent effects of mitochondrial membrane permeabilization and cell death [8–12]. 3,4,20 ,40 -Tetrahydroxychalcone (butein) is a polyphenolic compound extracted from the stembark of cashews and Rhus verniciflua Stokes, and has been used as a traditional herbal medicine. Previous studies have suggested that butein has anticancer activity against several human cancers, including leukemia, melanoma, breast carcinoma, colon carcinoma, osteosarcoma, and hepatic stellate cells [13– 19]. However, the role of ROS generation with respect to the anticancer effects of butein is not fully understood. The present study was designed to investigate the underlying mechanism involved in the induction of G2/M phase arrest and apoptosis by butein in human hepatoma cancer cells. Our data provide the first evidence that butein induces G2/M phase arrest and apoptosis in Hep3B and HepG2 cells via a mechanism involving the generation of ROS, the activation of JNK, and the subsequent induction of the mitochondrial apoptotic pathway.
2. Materials and methods 2.1. Reagents Antibodies against PARP, Bcl-2, Bax, cyclin B1, Cdc2, and cdc25C were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against phospho (p)-Cdc2 (Thr-14/Tyr-15), p-cdc25C (Ser-216), p-ATM (Ser-1981), p-Chk1 (Ser-345), p-Chk2 (Thr-68), JNK, p-JNK, c-jun, and p-c-jun were purchased from Cell Signal (Beverly, MA). Antibody against b-actin, and antioxidants glutathione (Glu) and N-acetyl-L-cysteine (NAC) were purchased from Sigma (St. Louis, MO). Butein was purchased from Sigma and dissolved in DMSO (vehicle). 2.2. Cell culture and viability assay Human hepatoma cancer cell lines HepG2 and Hep3B were cultured in RPMI medium (Life Technologies; Grand Island, NY) supplemented with 10% fetal bovine serum (FBS) and antibiotics (Sigma). Cells were seeded at 5 104 cells/ml, incubated for 24 h, and then treated with the indicated concentrations of butein for the indicated times. Cell viability was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and trypan blue exclusion assays.
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analyze the cells. Level of apoptotic cells containing subG1 DNA content was determined as a percentage of the total number of cells. For annexin-V staining, live cells were washed with PBS and then incubated with annexin-V fluorescein isothiocyanate (R&D Systems; Minneapolis, MN). Cells were analyzed using flow cytometry. 2.4. Western blot analysis Total cell extracts were prepared using PRO-PREP protein extraction solution (iNtRON Biotechnology; Sungnam, Republic of Korea). Cell extracts were separated on 10% polyacrylamide gels and then transferred to nitrocellulose membranes using standard procedures. The membranes were developed using an ECL reagent (Amersham; Arlington Heights, IL). 2.5. ROS measurement Generation of intracellular ROS was examined by flow cytometry using hydroethidine (HE) for O 2 and 6-carboxy-20 ,70 -dichlorofluorescein diacetate (H2DCFDA) for H2O2 (Molecular Probes; Eugene, OR) [20]. The HE is oxidized to ethidium bromide, whereas H2DCFDA is cleaved by nonspecific cellular esterases and oxidized in the presence of H2O2 and peroxidases to yield fluorescent 20 ,70 dichlorofluorescein (DCF). Briefly, 5 104 cells were plated in 60-mm dishes, allowed to attach overnight, and exposed to different concentrations of butein for 1 h. Cells were stained with 2 lM HE or 5 lM H2DCFDA for 30 min at 37 °C. Cells were collected and the fluorescence was analyzed using a flow cytometer. In some experiments, cells were pretreated with 10 mM NAC or 5 mM Glu prior to butein exposure and analysis of ROS generation. 2.6. In vitro caspase-3 activity assay Activity of caspase-like protease was measured using a caspase activation kit according to the manufacturer’s protocol (R&D systems; Minneapolis, MN). This assay is based on spectrophotometric detection of the color reporter molecule p-nitroaniline (pNA), which is linked to the end of the caspase-specific substrate. The caspase cleaves the peptide and releases the chromophore pNA, which can be quantified spectrophotometrically at a wavelength of 405 nm. 2.7. Cdc2 kinase activity Cdc2 kinase activity was measured by MESACUP Cdc2 kinase assay kit (MBL; Woburn, MA) according to the manufacturer’s instructions.
2.3. Flow cytometric analysis
2.8. Superoxide dismutase (SOD) activity
Flow cytometry was used to analyze cell cycle distribution. Cells (1 106) were fixed in 70% ethanol overnight at 4 °C and washed in phosphate-buffered saline (PBS) with 0.1% BSA. Cells were then incubated with 1 U/ml of RNase A (DNase free) and 10 lg/ml of propidium iodide (PI; Sigma) 1 h at room temperature in the dark. FACSCalibur flow cytometer (Becton Dickenson; San Jose, CA) was used to
HepG2 and Hep3B cells (5 105) were plated, allowed to attach overnight, and exposed to butein for 1 h at 37 °C. The cells were collected, washed with PBS, and pelleted by centrifugation at 500g for 6 min. The cell pellet was suspended in ice-cold 50 mM potassium phosphate (pH 7.0) containing 1 mM EDTA, sonicated and centrifuged at 10,000g for 15 min at 4 °C. The supernatant fraction was
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used for determination of SOD activity using kits from Cayman Chemical (Ann Arbor, MI) according to the manufacturer’s instructions. 2.9. Statistical analysis All data were derived from at least three independent experiments. The blots were visualized with the ChemiSmart 2000 (Vilber Lourmat; Marine, Cedex, France). Images were captured using Chemi-Capt software (Vilber Lourmat) and imported into Adobe Photoshop (Adobe Systems; San Jose, CA). Statistical analyses were conducted using SigmaPlot software (Aspire Software International; Ashburn, VA), and values are presented as mean ± standard deviation (SD). Significant differences between the groups were determined using the unpaired Student’s t-test. A value of P < 0.05 was accepted to be statistically significant. 3. Results 3.1. Butein suppresses the viability and growth of hepatoma cells through G2/ M phase arrest To determine the effect of butein on cell viability and growth, human hepatoma cells (HepG2 and Hep3B) were treated with one of various concentrations of butein for 24 h. Cell viability and growth were determined
by MTT and trypan blue exclusion assays, respectively. Treatment with butein resulted in a dose-dependent inhibition of HepG2 and Hep3B cell viability and growth (Fig. 1A and B). To further characterize the inhibitory effect of butein on cell proliferation, we monitored cell cycle progression using flow cytometry. Exposure to butein resulted in an increase of G2/M phase cells, accompanied by a decrease in G1 phase cells (Fig. 1C). The effect observed at 30 lM butein was the greatest, with approximately 58% of cells being in the G2/M phase, compared to approximately 20% in the control condition. Consequently, our results demonstrate that treatment with butein inhibits the proliferation of hepatoma cancer cells by inducing G2/M transition. 3.2. Butein controls the expression level of proteins that regulate G2/M transition The generation of ROS has an important role in the effects of various anticancer agents on cell cycle transition [6]. We thus investigated the possibility that butein induces G2/M phase arrest by allowing for the accumulation of ROS. Intracellular ROS generation in control and butein-treated Hep3B cells was assessed by flow cytometry after staining with HE and H2DCFDA. Butein treatment induced a dose-dependent increase in mean HE and DCF fluorescence, when compared with control cells (Fig. 2A). We then determined the effect of butein treatment on SOD enzyme activity. Treatment with butein for 1 h resulted in a modest, but statistically significant, increase of SOD activity, as compared with controls (Fig. 2B). To elucidate the mechanism of G2/M phase arrest in butein-treated cells, we investigated effects associated with the expression of pivotal protein for G2/M transition. This was done after treating cells with one of various concentrations of butein for 24 h. Butein treatment downregulated both cdc25C and Cdc2 in HepG2 and Hep3B cells
Fig. 1. Butein dose-dependently triggers G2/M phase arrest in hepatoma cells. Cells were seeded at 5 104 cells/ml and treated with the indicated concentrations of butein for 24 h. Cell viability (A) and number (B) were determined by MTT assay and hemocytometer counts of trypan blue-excluding cells, respectively. (C) In a parallel experiment, cells were harvested and stained with PI for 1 h; 10,000 events were analyzed for each sample. DNA content is on the x-axis; the number of cells counted is on the y-axis. Each point represents mean ± SD of three independent experiments. Statistically significant differences were determined using Student’s t-test (, P < 0.05 vs. vehicle control).
D.-O. Moon et al. / Cancer Letters 288 (2010) 204–213 (Fig. 2C). In contrast, butein did not influence the expression levels of cyclin B1. Further, butein treatment decreased Cdc2 kinase activity (Fig. 2D). Cdc2 is inactive when phosphorylated at the Thr-14 and Tyr-15 site. However, activated cdc25C dephosphorylates Cdc2 at these residues, leading to activation of the cyclin B1/Cdc2 complex [21]. We thus hypothesized that treatment with butein might result in an accumulation of Thr14/Tyr-15-phosphorylated Cdc2. We examined this possibility by Western blot analysis, using an antibody specific for p-Cdc2 (Thr-14/Tyr15). Compared with untreated control cells, treatment with butein was associated with a dose-dependent increase in the Thr-14/Tyr-15-phosphorylation of Cdc2 (Fig. 2C). After Chk1 and Chk2 phosphorylate cdc25C on Ser-216, thereby cdc25C is undergone to degradation. Next, we examined whether treatment with butein lead to an accumulation of phosphorylated Chk1 (Ser345) or Chk2 (Thr-68). Western blot analysis revealed the phosphorylated forms of Chk1 and Chk2 to be more abundant after 24 h of treatment with
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butein, as compared to a control condition (Fig. 2C). ATM is an upstream kinase implicated in the phosphorylation/activation of Chk1 and Chk2, and is known to be activated in response to DNA damage/genomic stress in eukaryotic cells [22]. Western blot analysis using an antibody specific for p-ATM (Ser-1981) revealed that treatment with butein increased the phosphorylation of ATM (Fig. 2C). These findings indicate that butein induces G2/M phase arrest, and that this coincides with the activation of ATM, Chk1, and Chk2, and with the inactivation of Cdc2. 3.3. Antioxidants NAC and Glu prevent butein-induced G2/M phase cell cycle arrest NAC and Glu scavenge ROS in cells by interacting with OH and H2O2 [3,4], and thus affect ROS-mediated signaling pathways. Because of this, we investigated whether these antioxidants affect butein-induced G2/M phase arrest. Pretreatment with NAC or Glu significantly blocked the
Fig. 2. Butein affects the expression of proteins involved in regulating the G2/M transition associated with the generation of ROS. Cells were treated with one of various concentrations of butein for 1 h, or for one of various times up to 24 h (C and D). (A) Hep3B cells were treated with the indicated concentration of butein, and then with either 2 lM HE or 5 lM H2DCFDA for 30 min at 37 °C and subsequent FACS analysis was used to assess the intracellular accumulation of ROS. (B) SOD activity in the lysates from HepG2 and Hep3B cells treated for 1 h with the indicated concentrations of butein. (C) The cells were lysed for protein extraction. Samples (50 lg) were subjected to 10% SDS–PAGE and Western blotting for the detection of specific proteins, as indicated [cyclin B1, Cdc2, cdc25C, p-Cdc2 (Thr-14/Tyr-15), p-ATM (Ser-1981), p-Chk1 (Ser-345), and p-Chk2 (Thr-68)]. b-Actin was used as a loading control. (D) Cdc2 kinase activity was determined as per the manufacturer’s protocol. Each point represents the mean ± SD of three independent experiments. Statistically significant differences were determined using Student’s t-test (, P < 0.05 vs. vehicle control).
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Fig. 3. The butein-induced generation of ROS is correlated with G2/M phase arrest. (A) Hep3B cells were pretreated with either 10 mM NAC or 5 mM Glu for 1 h before being exposed to 30 lM butein for 1 h following which either 2 lM HE or 5 lM H2DCFDA was administered for 30 min at 37 °C. FACS analysis was used to assess the intracellular accumulation of ROS. (B) In a parallel experiment, cell cycle distribution was analyzed by flow cytometry after 24 h. Cells were harvested and 10,000 events analyzed for each sample. Fluorescence intensity is plotted on the x-axis, and the relative number of cells plotted on the y-axis. (C) Equal amounts of cell lysates (50 lg) were resolved by SDS–PAGE, transferred to nitrocellulose, and probed with specific antibodies against pChk2 (Thr-68), cdc25C, Cdc2, and p-Cdc2. b-Actin was used as a loading control. (D) Cdc2 kinase activity was determined as per the manufacturer’s protocol. Each point represents the mean ± SD of three independent experiments. Statistically significant differences were determined using Student’s t-test (, P < 0.05 vs. vehicle control).
D.-O. Moon et al. / Cancer Letters 288 (2010) 204–213 butein-mediated generation of ROS, including O 2 and H2O2, (Fig. 3A), and completely abrogated butein-induced G2/M phase arrest (Fig. 3B). We used Western blot analysis to further examine the role of ROS in butein-induced G2/M phase arrest. Pretreatment with NAC or Glu suppressed the phosphorylation of Chk2 and Cdc2 proteins induced by butein (Fig. 3C). The phosphorylation status of these proteins was not affected by NAC or Glu administered in isolation of butein as compared with a control condition. Indeed, pretreatment with these antioxidants produced a significant restoration of cdc25C and Cdc2 expression levels (Fig. 3C), and Cdc2 kinase activity (Fig. 3D). Therefore, these data indicate that cdc25C and Cdc2 have important roles in regulating butein-mediated G2/M phase arrest.
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3.4. The role of JNK activation in butein-induced cell cycle arrest It is well known that ROS-mediated DNA damage induces JNK activation, and subsequently induces cell death [12,23]. Accordingly, we assessed the status of JNK at 24 h after treatment with butein. Treatment with butein resulted in the activation of JNK (Fig. 4A). On the other hand, the total amount of JNK (unphosphorylated form) was not altered by treatment with butein. We performed Western blot analysis to investigate whether butein-mediated JNK activation depends on the intracellular level of ROS. Pretreatment with NAC significantly blocked butein-induced JNK activation (Fig. 4B). We next investigated the possible roles of these alterations of JNK activity in butein-induced G2/M phase arrest. As shown
Fig. 4. Butein induces G2/M phase arrest through JNK activation. (A) HepG2 and Hep3B cells were treated with the indicated concentration of butein for 24 h. Equal amounts of cell lysates (50 lg) were resolved on SDS–polyacrylamide gels, transferred to nitrocellulose membranes, and probed with antibodies against anti-p-JNK and anti-JNK. (B) Hep3B cells were pretreated with the indicated concentration of NAC for 1 h before exposure to 30 lM butein for 24 h. Western blot analysis was performed to detect p-JNK and JNK. (C) HepG2 and Hep3B cells were stimulated with 30 lM butein for 24 h after pretreatment with SP600125 (20 or 40 lM) for 1 h. The cells were stained with PI and analyzed by flow cytometry after 24 h. (D) Portions of the G2/M phase are presented. (E) Equal amounts of cell lysates (50 lg) were resolved by SDS–PAGE, transferred to nitrocellulose, and probed with specific antibodies (anti-p-cjun, anti-c-jun, anti-p-Chk2, and anti-cdc25C). b-Actin was used as a loading control. SP, SP600125.
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by flow cytometry analysis of HepG2 cells (Fig. 4C and D), pretreatment with SP600125 (an inhibitor of JNK) resulted in a greater decrease in the number of G2/M cells (32 ± 3%) than treatment with butein alone (60 ± 3%). We found similar results for Hep3B cells. In order to confirm that this effect was specific for the JNK pathway, the phosphorylation status of c-jun (a downstream of JNK) was assessed by Western blot analysis after 24 h. Butein-induced c-jun phosphorylation was significantly attenuated by treatment with JNK inhibitors (Fig. 4E). We also investigated whether SP600125 modulates protein expression associated with butein-induced G2/M phase arrest. Treatment with SP600125 significantly blocked the phosphorylation of the Chk2 and Cdc2 proteins induced by butein (Fig. 4E). Indeed, pretreatment with SP600125 restored the levels to which cdc25C was expressed (Fig. 4E). These data suggest that the phosphorylation of JNK regulates butein-induced G2/M phase arrest. 3.5. Butein induces apoptotic cell death by triggering mitochondrial apoptosis pathway In order to investigate whether treatment with butein affects apoptosis, HepG2 and Hep3B cells were stimulated with various concentrations of butein (0–40 lM) for 48 h. Treatment with butein significantly increased the proportion of annexin-V in both hepatoma cell lines (Fig. 5A). The Bcl-2 protein family contains both antiapoptotic and proapoptotic members. These proteins play pivotal regulatory roles in deciding cell fate, through interactions that integrate a wide array of diverse upstream survival and distress signals [24]. In order to examine the role of the Bcl-2 family in butein-induced apoptosis, we used Western blot analysis to assess the effect of butein on the levels of anti-apoptotic Bcl-2 and pro-apoptotic Bax. Treatment with butein induced a dose-
dependent increase in Bax, and a dose-dependent decrease in Bcl-2 (Fig. 5B). Recent studies have demonstrated that caspases are important regulators of apoptosis [25]. Therefore, we investigated the involvement of caspase-3 in butein-induced apoptosis. Treatment with butein resulted in the activation of caspase-3 in HepG2 and Hep3B cells (Fig. 5C). There data suggest that treatment with butein induces apoptosis via increases in the Bax/Bcl-2 ratio and caspase-3 activity. 3.6. ROS are involved in butein-induced apoptosis We investigated whether the ROS generation is directly associated with butein-induced apoptosis, by assessing these outcomes in hepatoma cells pretreated for 1 h with either 10 lM NAC or 5 lM Glu followed by treatment with 30 lM butein. Pretreatment with NAC or Glu prevented the butein-induced increases in sub-G1 phase DNA content (Fig. 6A) and annexin-V (Fig. 6B). Additionally, these antioxidants decreased the butein-mediated upregulation of Bax and cleavage of PARP, and abrogated the butein-induced downregulation of Bcl-2 (Fig. 6C). Furthermore, the butein-induced activation of caspase-3 was also attenuated by pretreatment with NAC or Glu (data not shown). These results suggest that the generation of ROS plays an upstream role in butein-mediated mitochondrial apoptotic pathways.
4. Discussion It is well known that O 2 and H2O2 are the two major ROS in aerobic organisms. The functions of ROS include stimulating mitotic cell division and inducing cellular
Fig. 5. Butein induces apoptosis. HepG2 and Hep3B cells were seeded at 5 104 cells/ml and treated with the indicated concentrations of butein for up to 48 h. (A) Cells were treated with one of various concentrations of butein for 48 h, harvested, and 10,000 events analyzed for apoptotic annexin-V+ population. (B) Cells treated with the indicated concentrations of butein for 48 h were lysed for protein extraction. Samples were subjected to 10% SDS– PAGE and Western blotting for the detection of specific proteins, as indicated (anti-Bax and anti-Bcl-2). b-Actin was used as a loading control. (C) Caspase-3 activity was determined using a caspase-3 assay kit. Each point represents the mean ± SD of three independent experiments. Statistically significant differences were determined using Student’s t-test (, P < 0.05 vs. vehicle control).
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senescence. ROS are also important regulators of apoptosis. ROS production has been shown to be part of the signaling process, whereby many bioactive agents activate a cascade that functions to eliminate cancer cells [26,27]. Accordingly, excessive generation of ROS has been shown to cause cell damage and initiate various effects [28]. Recent reports have documented that many forms of apoptosis, as well as cell death in ischemia and neurodegenerative disease are associated with ROS production [29,30]. In the present study, we found that butein induces ROS-dependent cell cycle arrest at the G2/M phase, followed by a late apoptosis, in HepG2 and Hep3B cells. Butein arrested cell cycle progression by upregulating the phosphorylation of Cdc2, which is required for G2/M transition. It is well known that cdc25C is a specific tyrosine phosphatase that can directly
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induce Cdc2 activation [21], and the phosphorylation and degradation of cdc25C are important regulatory steps by which cells can delay or block mitotic entry under normal conditions. It is therefore not surprising that we also found a decreased accumulation of cdc25C as being associated with butein-induced G2/M arrest. Chk1 and Chk2 are known to be activated in response to DNA damage/genomic stress via phosphorylations at Ser-345/Ser-317 and Thr-68, respectively [31,32]. Upon activation, Chk1 and Chk2 can inactivate cdc25C via phosphorylation at Ser216, blocking downstream activation of Cdc2 and mitosis [33]. Our results show the abundance of the phosphorylated forms of Chk1 and Chk2 as dose-dependently increasing in cells treated with butein for 24 h. Chk1 and Chk2 are important intermediaries of DNA damage check-
Fig. 6. Antioxidants prevent butein-induced apoptosis. HepG2 and Hep3B cells were pretreated with either 10 mM NAC or 5 mM Glu for 1 h before exposure to 30 lM butein for 48 h. FACS analysis for DNA contents (A) and annexin-V (B). (C) In a parallel experiment, equal amounts of cell lysates (50 lg) were resolved by SDS–PAGE, transferred to nitrocellulose, and probed with specific antibodies against Bcl-2, Bax, and PARP. b-Actin was used as a loading control.
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point pathways, and ATM is an upstream kinase implicated in the phosphorylation/activation of Chk1 and Chk2 [32]. Western blot analysis using an antibody specific for pATM (Ser-1981) revealed an increase of p-ATM in buteintreated cells. The results of the present study indicate that an increased degradation of cdc25C in butein-treated cells is associated with ATM-dependent activation of Chk1 and Chk2. Furthermore, Western blot analysis also revealed that the Bax/Bcl-2 ratio was affected by treatment with butein. Moreover, although the activity of caspase-3 was elevated in the presence of butein, cell death was not stopped by a caspase-3 specific inhibitor, Ac-DEVD-CHO, nor by the pan-caspase inhibitor z-VAD-fmk (data not shown). These findings suggest that butein-induced apoptosis involves ROS generation, but not involve a caspase-dependent pathway. However, further study is necessary to clarify if there is a role for caspases in butein-induced apoptosis, given that caspases are the main regulators of apoptosis in various types of cancer cells. JNKs have been implicated in the apoptotic response of cells exposed to UV irradiation, heat shock, chemotherapy, and proinflammatory cytokines [34,35]. A number of anticancer drugs have been reported to kill cancer cells via the JNK apoptotic pathway, so it is to be expected that a blockade of JNK activation could inhibit anticancer activity. In the present study, we have shown that treatment with butein resulted in the in vitro accumulation of p-JNK in HepG2 and Hep3B cancer cells. JNK activation was found to correlate well with the butein-mediated upregulation of JNK activity, as measured by the JNK substrate p-c-Jun. Furthermore, by administering the JNK specific inhibitor SP600125, we found that preventing the butein-induced activation of JNK blocks butein-mediated G2/M arrest, and that this occurs through a downregulation of the ATM-Chk1/2 signaling pathway induced by butein. However, the inhibition of JNK did not prevent butein-induced apoptosis (data not shown), suggesting that JNK does not cooperate with a mitochondrial apoptotic pathway in generating butein-induced apoptosis. In conclusion, our data indicate that human hepatoma cancer cells are highly sensitive to butein, which inhibits their growth and induces apoptosis. Underlying butein-induced cell cycle arrest is a generation of ROS and a subsequent activation of JNK. Conflict of interest None declared. Acknowledgment This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2009-0076654). References [1] A.K. Nowak, P.K.H. Chow, M. Findlay, Systemic therapy for advanced hepatocellular carcinoma: a review, Eur. J. Cancer 40 (2004) 1474– 1484.
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