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Department of Dermatology (J.-H.Y.), Chung Shan Medical University Hospital, Taichung, Taiwan; Department of Internal Medicine (T.-C.H.), China Medical University Hospital, Taichung, Taiwan; Departments of Parasitology (H.-M.K.) and Pharmacy (P.-D.L.C.), China Medical University, Taichung, Taiwan; Department of Veterinary Medicine (C.-C.C.), National Chung-Hsin University, Taichung, Taiwan; Department of Biochemistry (Y.H.-W.), National Yang-Ming University, Taipei, Taiwan; and School of Biological Science and Technology (J.-G.C.), China Medical University, Taichung, Taiwan, Republic of China
(Received April 30, 2005; Accepted November 8, 2005)
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Abstract |
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). Small-cell lung carcinoma (SCLC), a form of lung cancer characterized by a neuroendocrine phenotype, represents approximately 20% of primary lung cancers and exhibits the most malignant phenotype of lung cancer (Chua et al., 2004). Most SCLC patients are not suitable for surgery, and the standard treatment for SCLC is chemotherapy or radiotherapy (Chua et al., 2004); however, the treatment outcome of SCLC or other types of lung cancer is far from satisfactory.
Flavonoids are naturally occurring polyphenolic compounds and have profound pharmacological properties, and a daily intake of flavonoids is associated with a lower risk of cancer (Middleton et al., 2000). Quercetin (3,3',4',5,7-pentahydroxyflavone) is one of the major dietary flavonoids particularly abundant in fruits and vegetables (Hertog et al., 1993). Analysis of plasma of volunteers fed with a quercetin-supplemented diet shows that quercetin is mainly circulating as quercetin glucuronides (Fig. 1) (Manach et al., 2004). Most of the biological studies for assessing the properties of quercetin are performed on quercetin (aglycone), and there is limited information available regarding the biological effects of quercetin glucuronides.
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), although the mechanisms are still obscure. Quercetin treatment caused cell cycle arrest either at the G1/S or G2/M transition, depending on cell type (Choi et al., 2001; Nguyen et al., 2004; Ong et al., 2004). Moreover, quercetin-mediated apoptosis may be related to many factors such as stress proteins, disruption of microtubules, nuclear factor 
B, Cox-2, p53, survivin, c-Jun NH2-terminal kinase, mitogen-activated protein kinase kinase-extracellular signal-regulated kinase, Bcl-2 family proteins, heat shock proteins, DNA topoisomerase II, release of cytochrome c, and activation of caspases (Yoshida et al., 1990; Orzechowski et al., 2000; Choi et al., 2001; Jakubowicz-Gil et al., 2002; Kaneuchi et al., 2003; Cheong et al., 2004; Kuo et al., 2004; Nguyen et al., 2004; Ong et al., 2004). Quercetin has been reported to inhibit the growth of various human cancers, including leukemia, breast, esophagus, colon, prostate, nasopharyngeal, endometrial, and lung cancers (Yoshida et al., 1990; Orzechowski et al., 2000; Choi et al., 2001; Jakubowicz-Gil et al., 2002; Kaneuchi et al., 2003; Cheong et al., 2004; Kuo et al., 2004; Nguyen et al., 2004; Ong et al., 2004). It is imperative to investigate the biological effects of quercetin glucuronides that are more close to the in vivo situation. Although quercetin-induced apoptosis has been documented in human lung cancer cells (Kuo et al., 2004; Nguyen et al., 2004), there are no reports addressing the molecular mechanisms of quercetin glucuronide-induced apoptosis in human lung cancer cells. Here, we report the findings that quercetin glucuronides inhibited cell proliferation through cell cycle arrest and induction of apoptosis in human lung cancer NCI-H209 cells.
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Materials and Methods |
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Preparation of Quercetin Glucuronides. Quercetin glucuronides were prepared from the serum of rabbits that were administrated with quercetin, and the concentration of quercetin glucuronides was determined by high-performance liquid chromatography after hydrolysis with ß-glucuronidase as described previously (Wittig et al., 2001; Kuo et al., 2002).
Human Lung Cancer Cell Line NCI-H209. NCI-H209 is a SCLC cell line procured from The Wellcome Trust Sanger Institute (Cambridge, UK), and we purchased it from the Food Industry Research and Development Institute (Hsinchu, Taiwan). The cells were immediately transferred into 75-cm3 tissue culture flasks and grown at 37°C under a humidified 5% CO2 and 95% air at one atmosphere in RPMI 1640 medium supplemented with 10% FBS, 1% penicillin-streptomycin (10 ng/ml penicillin and 10 ng/ml streptomycin), and 1% glutamine.
Cell Viability Determination by Using Trypan Blue Exclusion and Flow Cytometry. NCI-H209 cells were plated in 12-well plates at a density of 5 x 105 cells/well and grown for 24 h. They were then added with different concentrations of quercetin glucuronides to reach final concentrations of 0, 0.5, 1, 2.5, 5, and 10 µM, while only adding DMSO (solvent) for the control and grown at 37°C, 5% CO2 and 95% air for a different period of time. For determination of cell viability, the trypan blue exclusion protocol was used. Briefly, approximately 10 µl of cell suspensions in PBS were mixed with 40 µl of trypan blue, and the numbers of stained (dead cells) and unstained cells (live cells) were counted using a hemocytometer (Pettit et al., 1996) or using flow cytometric assay as described previously (Lee et al., 2003).
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).
Analysis of DNA Content by Flow Cytometry in NCI-H209 Cells. Approximately 5 x 105 cells/well of NCI-H209 cells in six-well plate were incubated with concentrations (0, 0.5, 1, 2.5, 5, and 10 µM) of quercetin glucuronides for different time periods, and then the cells were harvested by centrifugation. The cells were fixed gently (drop by drop) by putting 70% ethanol (in PBS) in ice overnight and were then resuspended in PBS containing 40 µg/ml propidium iodide and 0.1 mg/ml RNase (Sigma-Aldrich) and 0.1% Triton X-100 in a dark room. After 30 min at 37°C, the cells were analyzed on a flow cytometer (BD Biosciences, San Jose, CA) equipped with an argon laser at 488 nm wavelength. The cell cycle was then analyzed (Ormerod, 1990).
Detection of Reactive Oxygen Species (ROS) in NCI-H209 Cells by Flow Cytometry. The level of ROS of the NCI-H209 cells was determined by flow cytometry (Becton Dickinson FACS Calibur; BD Biosciences), using 2,7-dichlorodihydrofluorescein diacetate (Sigma-Aldrich). NCI-H209 cells were treated with or without various concentrations (0.5, 1, 2.5, 5, and 10 µM) of quercetin glucuronides for 24 h, and the levels of ROS were determined. The cells were harvested and washed twice, resuspended in 500 µl of 2,7-dichlorodihydrofluorescein diacetate (10 µM) and incubated at 37°C for 30 min and analyzed by flow cytometry (Kalbacova et al., 2003).
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).
Determination of Caspase-3 Activity in NCI-H209 Cells. NCI-H209 cells were plated in 12-well plates at a density of 5 x 105 cells/well and grown for 24 h. Various concentrations of quercetin glucuronides were added to the medium, and the cells were grown at 37°C in a humidified 5% CO2 for 12 h. Approximately 5 x 106 cells were lysed in the lysis buffer (1% Triton X-100, 0.32 M sucrose, 5 mM EDTA, 10 mM Tris-HCl, pH 8.0, 2 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, and 1 mg/ml leupeptin) for 30 min at 4°C followed by centrifugation at 10,000g for 30 min. For caspase-3 activity determination, 50-µl reaction mixtures with fluorogenic peptide substrates were specifically used for caspase-3. The peptide substrate (200 µM) was incubated at 37°C with cytosolic extracts (15 µg of total protein) in the reaction buffer (100 mM HEPES, 10% sucrose, 10 mM dithiothreitol, and 0.1% 3-[3-chloamidopropyl] dimethylammonio-1-propane-sulfonate). Fluorescence was determined after 2 h (excitation wavelength, 400 nm; emission wavelength, 505 nm) with a fluorescence plate reader (Fluoroskan Ascent; Thermo Electron Corporation, Waltham, MA) (Lu et al., 2004).
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Poly(ADP-Ribose) Monoclonal Antibody Assay for Apoptosis of NCI-H209 Cells. NCI-H209 cells were plated in 12-well plates at a density of 2 x 105 cells/well and grown for 24 h. Various concentrations of quercetin glucuronides were added to the medium, and the cells were grown at 37°C in a humidified 5% CO2 for 24 h for poly(ADP-ribose) monoclonal antibody assay (Alexis, San Diego, CA) as described in previous studies (Ormerod, 1990; Ferreira et al., 2002).
Western Blotting for Examination of Effect of Quercetin Glucuronides on the Expression of Apoptosis-Related Proteins. Total proteins were collected from human lung cancer NCI-H209 cells treated with or without various concentrations of quercetin glucuronides for 48 h. Western blotting was used to examine the expression levels of the apoptosis-related proteins including p21CIP1/WAF1, Cdc25C-ser-216-p (phosphorylation of Cdc25C on serine-216), Bax, Bad, Bcl-2, Bcl-xl, cytochrome c, caspase-9, caspase-3, and poly(ADP-ribose) polymerase (PARP) by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) as previously described (Ferreira et al., 2002).
Statistical Analysis. Student's t test was used to analyze the statistical significance of the differences between the quercetin glucuronide-treated and control groups.
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Results |
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Quercetin Glucuronides Induced Cell Cycle Arrest and Apoptosis in NCI-H209 Cells. The data indicated that during the 48-h time period, quercetin glucuronides increased the percentage of S and G2/M phases, and the percentage of G0/G1 phase was decreased. Nontreated control cells showed a typical pattern of DNA content that reflected G0/G1, S, and G2/M phases of the cell cycle. The quercetin glucuronide-treated cells showed a typical pattern of DNA content that reflected G0/G1, S, and G2/M phases of the cell cycle together with a subG0/G1 phase (corresponding to apoptotic cells) as shown in Fig. 3, A and B. Quercetin glucuronides induced a distinct subG1 peak, which represents the population of apoptotic cells. The percentage of apoptosis in various concentrations of quercetin glucuronides are shown in Fig. 3C.
Effect of Quercetin Glucuronides on the Expression of Wee1, Cdc25C-ser-216-p, CDK1, and Cyclin B in NCI-H209 cells. The results of SDS-PAGE are presented in Fig. 4. The results demonstrated that quercetin glucuronides increased WEE1 and Cdc25C-ser-216-p levels and inhibited cyclin B level, whereas they did not affect the CDK1 level. These results demonstrated that quercetin glucuronides induced cell cycle arrest at G2/M transition.
Quercetin Glucuronides Induced DNA Fragmentation in NCI-H209 Cells. DNA fragmentation was performed in DNA gel electrophoresis. The results indicate that high doses of quercetin glucuronides (10 µM) induced apoptosis because of the occurrence of DNA laddering (data not shown). This result was in agreement with the results from flow cytometric assays.
Effects of Quercetin Glucuronides on ROS in NCI-H209 Cells. The change in ROS levels of NCI-H209 cells in response to different concentrations of quercetin glucuronides were studied by staining cells with 2,7-dichlorodihydrofluorescein diacetate and then analyzed by flow cytometry. The results showed that ROS levels did not change significantly between the control and the treated cells (data not shown).
Effects of Quercetin Glucuronides on Mitochondrial Membrane Potential. The changes of mitochondrial membrane potential levels of NCI-H209 cells in response to the effect of different concentrations of quercetin glucuronides were studied by staining cells with DiOL6, then analyzed by flow cytometry. The representative profile is showed in Fig. 5, and the data are shown in Table 1, which revealed that mitochondrial membrane potential was decreased in NCI-H209 cells after treatment with quercetin glucuronides. This effect was dose-dependent.
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Inhibition of Quercetin Glucuronide-Induced Caspase Activation and Apoptosis by z-VAD-fmk. These experiments were performed to examine whether caspase-3 activation is involved in the apoptosis triggered by quercetin glucuronides. The results indicate that quercetin glucuronides increased caspase-3 activity, and these effects are dose- and time-dependent (Fig. 6, A and B). NCI-H209 cells had been pretreated with the cell-permeable broad-spectrum caspase inhibitor z-VAD-fmk 3 h prior to the treatment with quercetin glucuronides. The caspase inhibitor decreased caspase-3 activity (Fig. 6C). After treatment with quercetin glucuronides and z-VAD-fmk in NCI-H209 cells, inhibition of quercetin glucuronide-mediated caspase-3 activation was accompanied by the marked attenuation of quercetin glucuronide-induced apoptotic cell death (Fig. 6, C and D). The results revealed that activation of caspase-3 contributes to quercetin glucuronide-induced apoptosis in NCI-H209 cells.
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Effect of Quercetin Glucuronides on the Production of p21CIP1/WAF1, Bak, Bax, Cytochrome c, Bcl-2, and Caspase-3 from NCI-H209 Cells. Total proteins were prepared and followed by Western blotting, and the results from SDS-PAGE are presented in Fig. 8. The results also showed that quercetin glucuronides increased p21CIP1/WAF1, Bak, Bax, cytochrome c, and caspase-3 levels but decreased Bcl-2 levels. These findings suggest that quercetin glucuronides induced apoptosis through these molecular events.
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Discussion |
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; Norbury and Zhivotovsky, 2004). In general, the initiation of cellular apoptosis is through two distinct pathways: one is the extrinsic pathway involved the death receptor signaling, and the other is the intrinsic pathway involved the mitochondrial cascades (Green and Amarante-Mendes, 1998; Kroemer and Reed, 2000; Henry-Mowatt et al., 2004). Activation of either one of the pathways by cleavage of the procaspase-8 or -9 will in turn result in downstream activation of caspase-3, caspase-activated DNase, and finally lead to DNA fragmentation (Ferreira et al., 2002; Norbury and Zhivotovsky, 2004). Activation of caspase-3 is often considered as the point-of-no-return in the apoptotic signaling cascade (Green and Amarante-Mendes, 1998).
In this study, we demonstrated that the possible roles of quercetin glucuronides on the human lung cancer NCI-H209 cells were 1) to decrease the percentage of viable cells in a dose- and time-dependent effect, 2) to arrest the cell cycle at the G2/M transition via cyclin B, WEE1, Cdc25C-ser-216-p, and p21 checkpoints, 3) to induce apoptosis via promoting the caspases-9 and -3 activities, and 4) to block apoptosis by cotreatment with the caspase inhibitor z-VAD-fmk. Our results provide evidence that quercetin glucuronide-induced apoptosis results from intrinsic pathway involved the mitochondrial cascades in NCI-H209 cells.
Although DNA damage due to reactive oxygen species (ROS) is involved in apoptosis, levels of hydrogen peroxide were not affected and the percentage of apoptosis was not influenced by cotreatment with catalase (the scavenger of H2O2) in NCI-H209 cells. This indicates that quercetin glucuronides may have antioxidant activity, and its induction of apoptosis is not dependent on the presence of H2O2, which is in agreement with the observation by Park et al. (2003).
It has been suggested that the intranuclear mechanisms after DNA damage are highly conserved in initiating apoptosis, cell cycle arrest, or DNA repair (Ferreira et al., 2002) The G2/M phase checkpoint plays a key role in providing time for DNA repair, and it provides an alternative pathway to apoptosis to remove irreparably damaged cells (Kroemer and Reed, 2000; Castedo et al., 2002). We demonstrated that quercetin glucuronides induced G2/M arrest (at 12 h) before the onset of apoptosis (at 24 h or later) from cell cycle analysis (Fig. 2, A and B). The Cdk1/cyclin B complex has been well known as the regulators governing the G2 to M progression or inducing apoptosis (Castedo et al., 2002). Wee1, Cdc25C, and p21CIP1/WAF1 can be G2/M checkpoints in human cells (Pines and Hunter, 1991; McGowan and Russel, 1993; Jackson et al., 2000). Wee1 delays mitosis, whereas Cdc25C advances mitosis (Russel and Nuse, 1986, 1987; Lew and Kornbluth, 1996); moreover, G2 DNA damage checkpoint simultaneously signals via both up-regulation of Wee1p (phosphorylation of Wee1) and down-regulation of Cdc25p (phosphorylation of Cdc25), thus providing a double-lock mechanism to ensure cell cycle arrest and genomic stability (Raleigh et al., 2000). The p21CIP1/WAF1 (an inhibitor of Cdk) binds to and inactivates most Cdk-cyclin complexes and induces cell cycle arrest either at G0/G1 or G2/M transition (Dulic et al., 1998). Cdc25C is a dual-specific protein phosphatase that controls the entry into mitosis by dephosphorylating the protein kinase Cdc2; however, Cdc25C-ser-216-p creates a binding site for 14-3-3 protein and inhibits the function of Cdc25C phosphatase (Peng et al., 1997; Sanchez et al., 1997). G2 arrest is initiated via phosphorylation of Cdc25C on serine-216 by Chk1 or Chk2, and the maintenance of G2 arrest is highly p53-dependent and involves its transcriptional targets p21 (Cdk inhibitor) and protein 14-3-3 (the adaptor) (Ohi and Gould, 1999; Takizawa and Morgan, 2000; Taylor and Stark, 2001). Charrier-Savournin et al. (2004) has recently demonstrated that p21 inhibits cyclin B1-Cdk1 activation by binding to and sequestering them in the nucleus, implying that p21 could efficiently block initiation of early mitotic events. Our results showed that quercetin glucuronide-induced G2/M arrest is closely related to the increase of Wee1 and Cdc25C-ser-216-p levels and to the decrease of cyclin B level (Fig. 3, C and D). Therefore, signals elicited by DNA damage in S and G2 phases prevent mitotic entry by inhibiting both activation (via inactivation of Cdc25) and import of cyclin B1/Cdk1 in quercetin glucuronide-treated NCI-H209 cells.
Recently, quercetin-induced growth inhibition and apoptosis has been demonstrated in human lung cancer cells (Kuo et al., 2004; Nguyen et al., 2004). Nguyen et al. (2004) found that alterations of Bcl-2 family proteins, inactivation of Akt-1, and activation of mitogen-activated protein kinase kinase-extracellular signal-regulated kinase all play a role during quercetin-induced apoptosis in A549 lung epidermoid cancer cells. Kuo et al. (2004) found that survivin reduces the cell growth inhibition and apoptosis, and p53 elevates the p21 level, which may delay cell death in the quercetin-treated A549 (p53-contained) and H1299 (p53-null) human nonsmall cell lung cancer cell lines. On the other hand, we demonstrated loss of mitochondria membrane potential and release of mitochondrial cytochrome c to cytosol in quercetin glucuronide-treated cells. We also showed the up-regulation of Bax and Bak and down-regulation of Bcl-2 and increased levels of caspase-9 and caspase-3, accompanied by cleavage of PARP in NCI-H209 cells. These data support that quercetin glucuronides induce apoptosis in human small-cell lung carcinoma NCI-H209 cells and the molecular mechanisms of apoptosis involved in the mitochondria-dependent pathway. Furthermore, z-VAD-fmk, a caspase inhibitor, inhibited the caspase-3 activity and totally blocked the apoptotic effect, indicating that quercetin glucuronide-induced apoptosis is caspase-3-dependent.
The release of cytochrome c from mitochondria to cytosol, related to loss of mitochondrial membrane potential, is the central gate in turning on/off apoptosis and is closely regulated by the Bcl-2 family proteins. The proapoptotic members of the Bcl-2 family proteins include Bax, Bak, Bad, Mtd, Diva, and BH3-only proteins, and the antiapoptotic members include Bcl-2, Bcl-XL, Bcl-w, Mcl-1, Bfl-1, and Boo/Diva, and a specific class of inhibitors of apoptosis proteins (IAPs) including c-IAP1, c-IAP2, XIAP, survivin, and others (Norbury and Zhivotovsky, 2004). Bax and Bak, normally located in the cytosol, are activated and then translocated to the mitochondria and permeabilize the outer mitochondrial membrane, facilitating release of cytochrome c from mitochondria to cytosol in response to apoptotic stimuli. These events subsequently trigger the formation of apoptosome, consisting of procaspase-9, Apaf-1, and cytochrome c in the presence of dATP, and turns on the executioner caspases-9 and -3 (Li et al., 1997; Green and Reed, 1998). Bax and Bak also act on the endoplasmic reticulum (ER), and their role on ER may be the release of Ca2+ ions and the activation of caspase-12, which subsequently activates the caspase-3 cascade (Li et al., 1997; Wei et al., 2001; Scorrano et al., 2003; Zong et al., 2003). Therefore, it is of great interest to further clarify the cross-talk between mitochondria and ER by quercetin glucuronides in cancer cells.
In conclusion, we demonstrated that quercetin glucuronides inhibited cell proliferation through cell cycle arrest at G2/M phase and induction of mitochondria-mediated apoptosis in human lung cancer cells (NCI-H209). Delineation of the biological effects of specific major quercetin metabolites on chemotherapeutic potential or chemoprevention of human cancers warrants further investigation.
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Footnotes |
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
ABBREVIATIONS: SCLC, small-cell lung carcinoma; DMSO, dimethyl sulfoxide; FBS, fetal bovine serum; PBS, phosphate-buffered saline; z-VAD-fmk, benzyloxycarbonyl-Val-Ala-Asp fluoromethylketone; PARP, poly(ADP-ribose) polymerase; PAGE, polyacrylamide gel electrophoresis; ROS, reactive oxygen species; CDK, cyclin-dependent kinase; IAP, inhibitor of apoptosis protein; ER, endoplasmic reticulum.
Address correspondence to: Dr. Jing-Gung Chung, Department of Microbiology, China Medical University, No 91, Hsueh-Shih Road, Taichung 404, Taiwan, Republic of China. E-mail: jgchung{at}mail.cmu.edu.tw
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