Generation of reactive oxygen species mediates butein-induced apoptosis in neuroblastoma cells

Flavonoids exhibit chemopreventive and chemotherapeutic effects. Butein, a bioactive flavonoid isolated from numerous native plants, has been shown to induce apoptosis in human cancer cells. In the current study, the molecular mechanisms of butein action on cell proliferation and apoptosis of neuroblastoma cells were evaluated. Treatment with butein decreased the viability of Neuro-2A neuroblastoma cells in a dose- and time-dependent manner. The dose-dependent nature of butein-induced apoptosis was characterized by an increase in the sub-G1 phase population. Treatment with butein significantly increased intracellular reactive oxygen species (ROS)levels and reduced the Bcl-2/Bax ratio, triggering the cleavage of pro-caspase 3 and poly-(ADP-ribose) polymerase (PARP). Pre-treatment with the antioxidant agent, N-acetyl cysteine (NAC), blocks butein-induced ROS generation and cell death. NAC also recovers butein-induced apoptosis-related protein alteration. In conclusion, butein-triggered neuroblastoma cells undergo apoptosis via generation of ROS, alteration of the Bcl-2/Bax ratio, and cleavage of pro-caspase 3 and PARP. Our results suggest that butein may serve as a potential therapeutic agent for the treatment of neuroblastoma.

Neuroblastoma, a highly prevalent solid tumor in children, accounts for 15% of all pediatric cancer-related deaths worldwide (16). Due to its easy metastasis and drug resistance, the mortality of neuroblastoma patients is very high. Therefore, determination of novel drugs that induce apoptosis in neuroblastoma cells without causing toxicity to normal cells is of utmost importance in the treatment of neuroblastoma. In the current study, we show for the first time that butein markedly triggers apoptosis in neuroblastoma cells through the generation of reactive oxygen species (ROS), alteration of Bcl-2 and Bax expression, and elevation of pro-caspase 3 activity. Pre-treatment with antioxidant agents reverse butein-induced effects. In general, butein may be a potential chemotherapeutic agent for the effective treatment of neuroblastoma.

Generation of reactive oxygen species mediates butein-induced apoptosis in neuroblastoma cells
purchased from the Sigma Chemical Company (St. Louis, MO, USA). Antibodies against pro-caspase 3 and PARP were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies against Bcl-2 and Bax were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antiβ-actin and HRP-conjugated secondary antibodies were obtained from Sigma Chemical Company.
MTT assay. Cells were seeded in 24-well plates at a density of 4x10 4 /ml and treated with the indicated concentrations of butein for 24 or 48 h. After removal of the supernatant, the cells were incubated with a medium containing 5.0 g/l MTT and incubated at 37˚C for an additional 3 h. The formazan was dissolved in 1 ml isopropanol and the absorbance was measured at 563 nm. The experiments were performed in duplicate and repeated at least 3 times.
Cell cycle distribution and apoptosis analysis. Cells exposed to different concentrations of butein for 24 h were collected and fixed with ice-cold ethanol overnight at -20˚C. Subsequently, cells were stained with 50 µg/ml propidium iodide (PI) in the dark for 15 min. The cell cycle distribution was analyzed on a BD Biosciences FACScan system using the CellQuest TM Pro software. The proportion of hypodiploid cells (sub-G1 phase) was also analyzed.
Reactive oxygen species (ROS) analysis. Cells were pretreated using 2 mM NAC for 1 h, followed by treatment with an indicated concentration of butein for an additional 24 h. The cells were then loaded with a 5 µM fluorescent probe 2' ,7'-dichlorodihydrofluorescein diacetate (H2DCFDA) at 37˚C for 1 h. The fluorescent intensity was analyzed using Molecular Devices Flexstation 3.
Western blot analysis. Cells were treated with the indicated concentration of butein for 24 h. Cells were rinsed with icecold phosphate buffered saline (PBS) and lysed in buffer containing 150 mM NaCl, 1% Triton X-100, 5 mM EDTA, 5 mM phenylmethylsulfonyl fluoride, 1% aprotinin, 1 g/ ml leupeptin, and 500 µM Na 3 VO 4 . The protein concentration was detected using Bradford protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA). Twenty micrograms of protein were separated by 10% polyacrylamide gel and transferred into a nitrocellulose membrane. The membrane was blocked by PBS containing 0.5% non-fat milk for 1 h at room temperature. After washing with PBS containing 0.1% Tween-20 (PBST), the membrane was probed with the indicated antibodies at 4˚C overnight, washed with PBST, and subsequently incubated with horseradish peroxidaseconjugated goat anti-mouse IgG antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA; 1:5,000 dilution) at room temperature for 1 h. The bands were detected by enhanced chemiluminescence kit (Perkin-Elmer Life Science, Boston, MA, USA). β-actin expression was used as the loading control.
Statistical analysis. Data reported are the mean ± SD deviation of 3 independent experiments and evaluated by Student t-tests. Significant differences were established at P<0.05.   were 36.70, 28.91, 18.54, 12.98 and 11.91% compared with the vehicle control when the cells were exposed to 6.25, 12.5, 25, 50 and 100 µM butein for 48 h. Collectively, treatment with butein resulted in a dose-and time-dependent inhibition of cell viability in Neuro-2A cells (Fig. 1).

Effects of butein on the viability and apoptosis of neuro
To further examine the inhibitory effects of butein on cell viability, cell cycle progression was conducted by flow cytometry analysis. Increased sub-G1 phase percentages were found in proportion to the butein treatments (0.58, 0.71, 1.45, 3.63, 23.60 and 15.89% in the presence of 0, 6.25, 12.5, 25, 50, and 100 µM butein). Accordingly, treatment with butein diminished cell viability mainly by triggering apoptosis (Fig. 2).

Effects of butein on apoptosis-related protein expression.
Bcl-2 family proteins are involved in apoptosis (17,18). Decreased Bcl-2/Bax ratios may trigger the release of cytochrome C from the mitochondria and activation of procaspase 3 (18). To elucidate the role of Bcl-2 family proteins in butein-induced apoptosis, the expressions of Bcl-2, Bax, procaspase 3, and PARP were measured in cell lysates derived from butein-treated cells. In the presence of 25, 50, and 100 µM butein, Bcl-2 expression decreased by 15, 25 and 30%, respectively, compared with the vehicle control. Bax expression increased by 1.52, 1.47, 1.65, 1.74 and 2.14-fold compared with the control (Fig. 3). Significantly reduced pro-forms and increased cleavage forms of caspase 3 and PARP were also observed in the butein-treated groups (Fig. 3).

Effect of antioxidant agents on butein-induced changes.
Recent studies demonstrated that generation of ROS may function as a mediator in flavonoid-induced apoptosis in cancer cells (19,20). To determine whether or not butein induces oxidative stress, ROS generation by butein was determined using the DCFDA staining method. As expected, treatment with 25, 50 and 100 µM butein significantly induced ROS production. ROS generation was diminished by pretreatment with the antioxidant agent NAC (Fig. 4A).
To determine whether or not elevated ROS levels mediate the cytotoxic effects of butein, the effects of antioxidant agents on cell viability was examined. Cells were pretreated with 2 mM NAC and then treated with 25, 50 and 100 µM butein for an additional 24 h. As shown in Fig. 4B, butein-induced cell death was significantly prevented by NAC. In the presence of NAC, cell viability recovered to about 61.56, 57.53 and 43.50% in the presence of 25, 50, and 100 µM butein compared to the vehicle-treated group, respectively.
To determine whether or not NAC reverses apoptosisrelated protein expression, cell lysates derived from combined treatments with NAC and butein were subjected to Western blot analysis. NAC significantly recovered the expression of Bcl-2 and attenuated the expression of Bax expression (Fig. 5). Cleavage form of caspase 3 and PARP was also prevented by NAC. Taken together, our results show that ROS plays a critical role in butein-induced apoptosis.

Discussion
Extensive reports have revealed the inverse relationship between the consumption of vegetables and fruits and several chronic diseases, such as allergic inflammation, hypertension, and cancer (21,22). Flavonoids, one of the major bioactive  compounds in vegetables and fruits, exhibit anti-atherosclerosis (23), anti-inflammation (4), and antitumor effects (4). Butein, an ingredient isolated from native plants, inhibits the proliferation of several human cancers, such as melanoma (10), leukemia (12), and osteosarcoma (9). In this presentation, the molecular mechanisms of butein on the apoptosis of neuroblastoma cells were delineated for the first time. Butein inhibited the cell viability of neuroblastoma cells in a doseand time-dependent manner. Treatment with butein induced apoptosis, as evidenced by an increased percentage of the sub-G1 population. In addition, butein decreased the Bcl-2/ Bax ratio, and enhanced the cleavage of caspase 3 and PARP. Pretreatment with antioxidant agents such as NAC clearly attenuated butein-induced effects.
The balance of expression of pro-apoptotic and antiapoptotic members of the Bcl-2 family proteins plays a critical role in apoptosis (24). An increased pro-apoptotic/ anti-apoptotic protein ratio altered the membrane potential of the mitochondria, released apoptogenic factors to the cytoplasma, activated the caspase cascade, and eventually caused apoptosis (18,24). Crude acetone extracts of R. verniciflua Stokes, which is rich in fustin and fisetin, and butein-triggered osteosarcoma cells undergo apoptosis via the activation of Bax and the down-regulation of Bcl-2 expression (9). Similarly, butein increased Bax expression and decreased Bcl-2 expression in HL60 leukemia cells (12). In accordance with previous reports, our findings also showed that butein upregulated Bax expression and attenuated Bcl-2 expression in neuroblastoma cells. Moreover, activation of caspase 3 and cleavage of PARP, 2 hallmarks of apoptosis, were also observed in butein-treated Neuro-2A cells. Collectively, our data suggest that butein-induced apoptosis may mediate by increase ROS level and change the pro-apoptotic/antiapoptotic protein ratio.
Alternation of intracellular ROS production may modulate several physiological functions (25). Elevated ROS can bind to lipids, proteins, or DNA to produce oxidative stress and eventually cause cell death (25). Flavonoid-induced apoptosis in cancer cells via elevation of intracellular ROS levels has been shown in several reports (19,20), although previous reports have also shown that butein induces ROS generation, modulates JNK, ATM, and Chk activity, and causes G2/M arrest in hepatoma cells (26). The consequence of butein-induced ROS production in cell death remains unclear. In the current presentation, butein enhanced ROS levels up to 1.5-fold and caused cell death. Blockage of ROS generation by an antioxidant agent, NAC, significantly increased cell viability in the presence of butein. Cotreatment with NAC recovered the anti-apoptotic protein Bcl-2 and decreased pro-apoptotic Bax levels, which, in turn, attenuated pro-caspase 3 activity. ROS appears to play a pivotal role in butein-induced apoptosis in neuroblastoma cells.
In summary, to the best of our knowledge, this is the first report to show that butein inhibits cell viability in neuroblastoma cells. Butein triggers apoptosis, as evidenced by an increase in the sub-G1 population. Butein also elevated ROS levels, altered Bcl-2/Bax ratios, induced pro-caspase 3 and PARP cleavage, and consequently caused cells to undergo apoptosis. Pre-treatment with NAC obviously abrogated the butein-induced effects. Collectively, our findings suggest that butein may serve as a chemotherapeutic agent for the future treatment of neuroblastoma. Figure 5. NAC reverses butein-induced apoptotic protein expression. Neuro-2A cells were pretreated with 2 mM NAC, followed by co-treated with the indicated concentrations of butein for additional 24 h. Cell lysates were harvested and subjected to Western blot analysis using antibodies against Bcl-2, Bax, pro-caspase 3, and PARP. The data represent one of the 3 independent experiments. β-actin was used as a loading control. The intensities of Bcl-2 and Bax were quantified and compared to the vehicle treated group.