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Department of Metabolism and Nutrition, Instituto del Frío, Consejo Superior de Investigaciones Científicas (CSIC), José Antonio Novais 10, Ciudad Universitaria, 28040, Madrid, Spain
* To whom correspondence should be addressed. E-mail: s.ramos{at}if.csic.es.
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ABSTRACT |
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Introduction |
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TOPABSTRACTIntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
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Epidemiological and intervention studies in both humans and animals have shown that regular consumption of fruits, vegetables, and tea is associated with reduced risk of cancer (3,4). Fruits, vegetables, spices, and tea provide essential nutrients and many diet-derived phenolics, in particular flavonoids, which have been reported to exert potential anticarcinogenic activities (3–6). Quercetin is one of the most common flavonoids found in the diet (4) and is extensively metabolized during absorption in the small intestine and in the liver (7). Recent studies have shown that quercetin has antiproliferative effects (5,8) and can induce death by an apoptotic mechanism in leukemia (9), breast (10), lung (11), hepatoma (12), oral (13), and colon (8) cancer cell lines. However, this flavonoid exerts this apoptotic effect in a selective manner insofar as using the same concentrations of quercetin has induced apoptosis in cancer cultured cells but not in their normal counterparts (14). Some evidence indicates that quercetin can modulate a number of key elements in cellular signal transduction pathways linked to the apoptotic (caspases and Bcl-2 genes) and cell survival or cell proliferation (MAPKs and Akt) processes (8,15), but its molecular mechanism of action remains to be elucidated. Quercetin activates caspase-3 and caspase-9 and releases cytochrome c in HL-60 cells (16), blocks the cell cycle at G1 in human gastric (17), nasopharingeal (13), endometrial (18), and hepatic cancer cells (19), and produces DNA fragmentation in other human hepatocarcinoma cell lines (20), leukemia (21), and mouse thymocytes (22). Quercetin also downregulates anti-apoptotic proteins of the Bcl-2 family, Bcl-xL and Bcl-2 (23,24), and upregulates pro-apoptotic members, Bax and Bad (23,24). Additionally, apoptosis may also be induced by oxidative stress commonly associated with a previous increase of intracellular reactive oxygen species (ROS), which can act as signaling molecules to trigger apoptosis under certain situations (25,26). Alternatively, the modulation of signaling through the serine/threonine kinase, Akt/protein kinase B (PKB), phosphatidylinositol-3-kinase (PI-3-kinase), and members of the mitogen-activated protein kinase (MAPK) family, such as extracellular regulated kinase (ERK), may also be relevant in the molecular mechanism of action of this flavonoid. In this regard, quercetin modulates the enzymes involved in proliferation and signal transduction pathways, including members of the MAPK family, such as ERK and c-Jun N-terminal kinase (JNK) (27,28), and to inhibits PI-3-kinase (10,21,29) and Akt (11,27,28).
The present study investigates the mechanisms underlying the cytotoxic effect of quercetin on a human hepatoma cell line (HepG2), assessing its influence on the balance between-prodeath pathways, such as apoptotic cascade through caspases (caspases-3, -8, and -9), and some Bcl-2 family members (Bcl-xS and Bax) and prosurvival pathways, namely, Bcl-xL, Akt/PI-3-kinase, and ERK.
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Materials and Methods |
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/ß was from Upstate NY Biotechnology. Caspase-3 and caspase-8 substrates (M Ac-DEVD-AMC and Ac-IETD-AMC, respectively) were purchased from Pharmingen. Materials and chemicals for electrophoresis were from BioRad. Cell culture dishes and cell culture medium were from Falcon and Biowhittaker Europe (Innogenetics), respectively. Cell culture and quercetin treatments. Human hepatoma HepG2 cells were a gift from Dr. Paloma Martin-Sanz (Centro Nacional de Investigaciones Cardiovasculares, Madrid). They were grown in DMEM-F12 medium supplemented with 2.5% fetal bovine serum and the following antibiotics: gentamicin, penicillin, and streptomycin (50 mg/L). Cells were maintained at 37°C in a humidified atmosphere of 5% CO2.
Cells were seeded and routinely grown in DMEM-F12 medium and 2.5% fetal bovine serum, but they were changed to serum-free medium 24 h before the assay. Cells were treated with different concentrations of quercetin (10, 25, 50, 75, and 100 µmol/L) during 4 or 18 h.
Viability cell assay. Viability was calculated by counting the cells in a Neubauer chamber. An aliquot of the total cell suspension (1.5 x 105 cells) was mixed with an equal volume of trypan blue and incubated for 5 min at room temperature.
Cytotoxicity assay. Lactate dehydrogenase leakage assay (LDH) was carried out as previously described (30,31). In brief, culture medium was collected separately and the cells were scraped. Cell suspension (1.5 x 106 cells) was sonicated to ensure breaking down the cell membrane to release the total amount of LDH. A mixture of 5 mmol/L pyruvate, 0.35 mmol/L NADH, and 84 mmol/L Tris was added to the sample and read at 340 nm in a microplate ELISA reader (Bio-Rad). LDH leakage was estimated as the ratio between the LDH activity in the culture medium and that of the whole cell content.
Determination of ROS. Cellular oxidative stress was quantified by the dichlorofluorescin (DCFH) assay using a microplate reader (30,32). After being oxidized by intracellular oxidants, DCFH becomes dichlorofluorescein (DCF) and emits fluorescence. By quantifying fluorescence at an excitation wavelength of 485 nm and emission wavelength of 530 nm, a fair estimation of the overall oxygen species generated under the different conditions was obtained.
Purification of mitochondrial and cytosolic extracts. Cells were incubated in a hypotonic buffer [1 mmol/L EDTA, 10 mmol/L HEPES, 50 mmol/L sucrose (pH 7.6)] at 37°C and homogenized. Then, after the addition of a hypertonic solution [1 mmol/L EDTA, 10 mmol/L HEPES, and 450 mmol/L sucrose (pH 7.6)], a supernatant was obtained corresponding to the cytosolic fraction and a pellet with the mitochondria. Mitochondria were resuspended in an isotonic buffer [1 mmol/L EDTA, 10 mmol/L HEPES, and 250 mmol/L sucrose (pH 7.6)].
Western blot analysis. Total cell extracts were obtained as previously described (33) except for the analysis of the levels of Akt, phospho-Akt, ERK1/2, phospho-ERK1/2, and PI-3-kinase subunits (p85 and p110), which were prepared according to Fabregat et al. (34).
Equal amounts of proteins (25–100 µg) were separated by SDS-polyacrylamide gel electrophoresis, and transferred to polyvinylidene difluoride filters (PVDF, Protein Sequencing Membrane, Bio-Rad) that were probed with the corresponding primary antibody. Western blots were developed with the ECL system (GE Healthcare). Normalization was ensured by ß-actin and bands were quantified by laser scanning densitometry (Molecular Dynamics).
Fluorometric analysis of caspase-3 and -8 activities. Caspase-3 and -8 activities were measured as previously described (33). Briefly, cells were lysed in a buffer containing 5 mmol/L Tris (pH 8), 20 mmol/L EDTA, and 0.5% Triton-X100. For caspase-3 activity, reaction mixture contained 20 mmol/L HEPES (pH 7), 10% glycerol, 2 mmol/L dithiothreitol, 30 µg protein per condition, and 20 µmol/L Ac-DEVD-AMC (N-acetyl-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin) as substrate. Reaction mixture for caspase-8 activity contained 20 mmol/L piperazine. N,N'-bis(2-ethane sulfonic acid) (pH 7.2), 100 mmol/L NaCl, 10 mmol/L dithiothreitol, 1 mmol/L EDTA, 0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, 10% sucrose, 70 µg protein/condition, and 22.6 µmol/L Ac-IETD-AMC (N-acetyl-Ile-Glu-Thr-Asp-7-amino-trifluoromethylcoumarin) as substrate. Enzymatic activity was measured at excitation wavelength of 380 nm and emission wavelength of 440 nm.
Statistics. Statistical analysis was as follows: data were analyzed using 2-way ANOVA with concentration and time as the 2 factors tested. Significant time x concentration interactions were found for all variables, and subgroups were analyzed further by testing the effect of concentration within each group using 1-way ANOVA. Data were tested prior to analysis for homogeneity of variances using Levene's test. In experiments when only one factor was studied (i.e., concentration), data were evaluated using 1-way ANOVA followed by the Bonferroni test when variances were homogeneous or by the Tamhane test when variances were not homogeneous (LDH and ROS, 18-h treatment). Differences with P < 0.05 were considered significant. The SPSS version 12.0 program was used.
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Results |
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We assayed ROS production to test whether different concentrations of quercetin had an effect on the generation of oxygen radicals in HepG2 in culture. Quercetin at 10–75 µmol/L enhanced ROS generation after 4 h treatment but did not have an effect at 100 µmol/L (Table 1). Interestingly, at 18 h, intracellular ROS levels were significantly decreased in the presence of 10–100 µmol/L quercetin at 18 h in a dose-dependent fashion. Leakage of probe was not observed in cells throughout the assay, as determined in previous tests during method set-up in our laboratory (35). Thus, any potential contribution of extracellularly oxidized DCF to the final fluorescence can be ruled out.
Caspase processing in the induced apoptosis. We tested the effect of quercetin on the cascade of caspases that are crucial initiators or effectors in the cell death pathways. Enzymatic activity of caspase-8 was unchanged after 4 or 18 h of incubation at all quercetin concentrations (data not shown). A significant activation of caspase-3 occurred at 25 µmol/L quercetin after 18 h of incubation. Activity was greater at 50 µmol/L quercetin but decreased at 75 and 100 µmol/L (Table 2). Proteolytic caspase-3 activity was not affected by any quercetin concentration after 4 h treatment (Table 2) and increased only slightly (P = 0.05) due to 50 µmol/L after 8 h (data not shown). The activation of caspase-3 after 18 h was confirmed by Western blot analysis (Fig. 1). Parallel to the cytotoxic effect and the enhanced caspase-3 activity, treatment of HepG2 cells with quercetin for 18 h increased the levels of active caspase-3; in fact, the cleaved caspase-3 level increased (P < 0.05) after 18 h of treatment with 25 µmol/L quercetin (Fig. 1A and 1B), and increased further at higher concentrations, especially at 50 µmol/L. Consistent with our previous results (19), the short-term treatment (4 h) did not have a prominent cytotoxic effect nor did it greatly induce apoptosis (data not shown). Therefore, the remaining assays were conducted using the 18-h quercetin treatment.
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Anti-apoptotic and pro-apoptotic Bcl-2 family members. The balance of the expression of anti- and pro-apoptotic members of the Bcl-2 gene family is one of the major mechanisms that regulates apoptosis in mammalian cells (36). Therefore, to determine whether quercetin-induced apoptosis in HepG2 was also associated with the modulation of members of this protein family, we examined the expressions of Bcl-x and Bax.
Bcl-xL has been proposed to be a caspase substrate, and the product of Bcl-xL cleavage, Bcl-xS, has a pro-apoptotic function. This proteolytic fragment (Bcl-xS) was detected when the cells were incubated for 18 h and the levels were greater at all quercetin concentrations compared with controls (Fig. 2A). The Bcl-xL:Bcl-xS ratio was decreased by quercetin, which reached a minimum value at 50 µmol/L (Figs. 2A and 2B).
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Akt phosphorylation and PI-3-kinase protein levels. Phosphorylation activation of Akt is associated with protection of cells from apoptosis (37). To analyze whether inhibition of Akt phosphorylation is related to quercetin-induced apoptosis, we measured total and phosphorylated levels of this protein. Concentrations of quercetin above 50 µmol/L inhibited Akt by decreasing the level of phosphorylated active Akt and, in contrast, lower concentrations of quercetin (10 and 25 µmol/L) resulted in the activation of Akt (Figs. 3A and 3B).
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Extracellular regulated kinase phosphorylation. The ERK signaling pathway is activated in response to certain situations of cellular stress, and it is implicated in cellular death or survival signaling (38). Therefore, we investigated whether quercetin-induced apoptosis was related to ERKs. Quercetin evoked a dose-dependent inhibitory effect in the 2 bands corresponding to ERK1 and ERK2 at all concentrations that induced apoptosis (Fig. 4). Similar to Akt regulation, treatment of HepG2 cells for 18 h with quercetin enhanced the phosphorylation of ERK1/2 at 10 µmol/L and dephosphorylation at concentrations above 50 µmol/L.
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Discussion |
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Quercetin concentrations that significantly decrease HepG2 cell viability have similar effects on other cancer cell cultures, such as leukemia (9), human colon (8), prostatic (24,40), breast (10), lung (11) cancer cells, and murine hepatoma (12). Our result with quercetin further demonstrates the inhibitory effect of this flavonoid on tumor cell viability. Increased leakage of LDH from the cell was unexpectedly observed upon exposure to common reducing agents such as glutathione or ascorbate (41) and also after long quercetin treatments (30,42); however, decreased activity of LDH by oxidation of essential enzyme residues in the presence of metals, light, or various oxidants (i.e., hydrogen peroxide) has also been demonstrated (43). In this context, we observed that LDH leakage could be affected by quercetin, and yet we observed no inhibition of this enzyme activity by this flavonoid (data not shown).
ROS are highly reactive metabolites generated during normal cell metabolism; however, elevated intracellular ROS could be sufficient to trigger apoptosis (25,26). Moreover, apoptotic cell death is reported to be preceded by the following sequential facts: ROS production, loss of mitochondrial transmembrane potential, release of cytochrome c, and activation of caspase-3 in fetal rat hepatocytes (33). Our previous (19) and present results (4-h treatment) attribute the enhanced production of ROS to the initial oxidative stress prior to the apoptotic process, although quercetin might not require enhanced ROS production for the apoptosis induction, as previously described for TGF-ß1 in fetal rat hepatocytes (44) or IL-3 in murine myeloid cells (45). However, inhibition of ROS after 18 h of flavonoid treatment suggests that quercetin at high concentrations or long exposures might inhibit the mitochondrial respiratory chain (46). This feature, supported by present and previous results (19), could be related either to apoptosis or necrosis, and therefore, a small percentage of necrosis cannot be ruled out.
Caspase-8 was not activated in response to the quercetin treatment, indicating that the extrinsic pathway was not involved in quercetin-induced caspase-3 activation and in apoptosis. Several studies indicate the activation of the mitochondrial pathway, and therefore caspase-9, by quercetin in leukemia (16) and colon cancer cells (8); similarly, caspase-9 was also cleaved by other polyphenols such luteolin in a rat hepatoma cell line (47). In our study, caspase-9 processing was coincident with caspase-3 activation, which indicates that the highest activity of caspases implicated in the mitochondrial apoptosis pathway was found 18 h after treatment with 50 µmol/L quercetin.
As our results showed, expression of Bcl-xL, Bcl-xS, and Bax could be differently regulated by quercetin, suggesting that the balance in the expression of these proteins might be involved in the control of the apoptotic process. Quercetin decreased the Bcl-xL:Bcl-xS ratio; thus, regulation of Bcl-xL protein levels seems to be, at least in part, caspase-dependent in HepG2 cells, which agrees with previous results in fetal hepatocytes treated with TGF-ß1 (33). Herrera et al. (33) proposed that Bcl-xL is a caspase substrate. Quercetin also increased Bax translocation from the cytosol to the mitochondrial membrane, an event that promotes apoptotic death (44) and followed a similar pattern to caspase-3 and -9 activation and Bcl-xL/Bcl-xS decrease. Accordingly, increased levels of total Bax occurred in other studies of fetal hepatocytes (44) as well as in studies of human lung (11), prostate (24), and liver (23) cancer cells. Increased Bax translocation was also observed in HepG2 cells after luteolin treatment (48).
Akt promotes cell survival by inhibiting apoptosis, and its phosphorylation has been considered a critical factor in the aggressiveness of HCC (49). Quercetin induced inactivation of Akt by decreasing the level of phosphorylated Akt in a concentration-dependent manner, contributing to the promotion of apoptosis. Although the precise anti-apoptotic effects of Akt are still unclear, Akt directly phosphorylates and inactives procaspase-9 and blocks caspase-9-mediated apoptosis (50). This could explain the observed effects of low concentrations of quercetin (10 and 25µ mol/L) that induce a sharp increase in Akt phosphorylation levels (Fig. 3B) and accumulation of procaspase-9 (Fig. 1C) but did not result in increased levels of active caspase-3 and -9 and, consequently, showed reduced cytotoxic effects (Table 1). Alternatively, inhibition of Akt promotes phosphorylation of the proapoptotic Bad, a fact that favors progress of the apoptotic process (1,49).
In contrast to the marked effects of quercetin on Akt phosphorylation, quercetin did not affect PI-3-kinase protein levels. This observation has been previously demonstrated in studies of human breast (10), lung (11), prostate (40) cancer cells, and of rat aortic smooth muscle cells (28). Quercetin and other flavonoids are reported to be PI-3-kinase inhibitors, reducing enzymatic PI-3-kinase activity without changing either p85 or p110 subunit levels (10,29,40), which agrees with the lack of effect of quercetin on PI-3-kinase levels observed in our study. Therefore, a reduction of PI-3-kinase activity by quercetin might be taking place, which might explain the observed decrease of Akt phosphorylation, its downstream target. PI-3-kinase activity will be investigated further in future studies.
Survival-signaling cascade in many cells involves PI-3-kinase, Akt, and also cross-communication between PI-3-kinase and ERKs (38). Because a sustained activation of ERK1/2 is necessary for cell survival and cell proliferation (38), the inhibition of ERK1/2 by quercetin contributes to the increased occurrence of cell death. However, quercetin exposure at low concentrations (10 µmol/L) results in an increased phosphorylation of ERK1/2, similar to what was observed for Akt, which suggests an activation of cell-survival pathways. Therefore, survival of HepG2 cells seems to depend on both ERK and PI-3-kinase/Akt pathways as previously reported in this cell line (51). A similar prosurvival effect of low quercetin concentrations (10 µmol/L) with increased phosphorylation of Akt and ERK was observed in neuronal cultures by Spencer et al. (27).
In summary, our studies showed that inhibition of Akt and ERK phosphorylation, induced by high quercetin concentrations, was coupled with a significant increase of caspase-3 and -9 levels and activities, higher expression of proapoptotic Bcl-2 family members (Bcl-xS and Bax), and lower levels of anti-apoptotic Bcl-xL that contributed directly to the apoptotic process. Interestingly, the highest quercetin concentrations (75 and 100 µmol/L) reduced the expression of proapoptotic (caspase-3, -9, and Bax) and prosurvival signals (Akt and ERK) compared with lower concentrations of quercetin (50 µmol/L). Thus, the caspases implicated in the mitochondrial apoptosis pathway that showed the highest activity after 18 h of treatment with 50 µmol/L quercetin provoked the greatest activation of their Bcl-2 family substrates, whereas the apoptotic effect might have been enhanced by the inhibition of Akt and ERK. Higher concentrations further decreased cell viability due to the activation of executor apoptotic signal and the more pronounced inhibition of prosurvival pathways (Akt). Although special attention must be given to flavonoid concentrations, quercetin may be a potential chemopreventive or therapeutic agent in HCC, and further efforts to investigate these possibilities are needed.
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FOOTNOTES |
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2 Abbreviations used: ERK, extracellular regulated kinase; HCC, hepatocellular carcinoma; LDH, lactate dehydrogenase; MAPK, mitogen-activated protein kinase; PI-3-kinase, phosphatidylinositol-3-kinase; PKB, protein kinase B; ROS, reactive oxygen species.
Manuscript received 10 May 2006. Initial review completed 7 July 2006. Revision accepted 28 August 2006.
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LITERATURE CITED |
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