Journal of Nutrition Animal Diets/Enrichment Products...

Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Granado-Serrano, A. B.
Right arrow Articles by Ramos, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Granado-Serrano, A. B.
Right arrow Articles by Ramos, S.
© 2006 American Society for Nutrition J. Nutr. 136:2715-2721, November 2006

Biochemical, Molecular, and Genetic Mechanisms

Quercetin Induces Apoptosis via Caspase Activation, Regulation of Bcl-2, and Inhibition of PI-3-Kinase/Akt and ERK Pathways in a Human Hepatoma Cell Line (HepG2)1

Ana Belén Granado-Serrano, María Angeles Martín, Laura Bravo, Luis Goya and Sonia Ramos*

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.


   ABSTRACT
TOP
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 
Dietary polyphenols have been associated with the reduced risk of chronic diseases such as cancer, but the precise underlying mechanism of protection remains unclear. The aim of this study was to investigate the effect of quercetin on the activation of the apoptotic pathway in a human hepatoma cell line (HepG2). Treatment of cells for 18 h with quercetin induced cell death in a dose-dependent manner; however, a shorter treatment (4 h) had no effect on cell viability. Incubation of HepG2 cells with quercetin for 18 h induced apoptosis by the activation of caspase-3 and -9, but not caspase-8. Moreover, this flavonoid decreased the Bcl-xL:Bcl-xS ratio and increased translocation of Bax to the mitochondrial membrane. A sustained inhibition of the major survival signals, Akt and extracellular regulated kinase (ERK), also occurred in quercetin-treated cells. These data suggest that quercetin may induce apoptosis by direct activation of caspase cascade (mitochondrial pathway) and by inhibiting survival signaling in HepG2.


   Introduction
TOP
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 
Hepatocellular carcinoma (HCC)2 is a global health problem and the third-ranked cause of cancer-related deaths in the world (1). HCC is difficult to detect and in most cases is not noticed at an early enough stage, which, together with an HCC annual occurrence in more than a half-million people (2) and 600,000 deaths in 2002 (1), creates an important reason for searching chemotherapeutic/chemopreventive agents for HCC.

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.


   Materials and Methods
TOP
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 LITERATURE CITED
 
    Materials and chemicals. Quercetin, trypan blue, piruvate, NADH, gentamicin, penicillin G, and streptomycin were purchased from Sigma Chemical. The fluorescent probe 2',7'-dichlorofluorescin diacetate (DCFH-DA) was from Molecular Probes. Antiactive cleaved caspase-3, antiactive caspase-9, anti-Akt and antiphospho-Ser473-Akt, as well as anti-ERK1/2 and antiphospho-ERK1/2 recognizing phosphorylated Thr202/Thy204 of ERK1/2, and anti-ß-actin were obtained from Cell Signaling Technology (9661, 9507, 9271, 9272, 9101, 9102, and 4697, respectively). Anti-Bcl-x, anti-Bax, and anti-p110ß were purchased from Santa Cruz Biotechnology (sc-634, sc-526 and sc-7175, respectively. Anti-p85{alpha}/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.


   Results
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 LITERATURE CITED
 
    Cell viability and intracellular generation of ROS. To determine the accurate range of cytotoxic concentrations of quercetin in a human hepatoma cell line (HepG2) we used 2 different methods to assay cell viability, trypan blue exclusion and LDH leakage. Cells were exposed to 0–100 µmol/L of quercetin for 4 or 18 h. Quercetin induced cell death in a dose-dependent manner after 4 or 18 h of incubation, as determined by the trypan blue exclusion assay (Table 1). Incubation with quercetin for 18 h displayed a dramatic cell mortality (68% with the highest concentration, 100 µmol/L, P < 0.05) with an estimated 50% of cell death (IC50) value of 87 µmol/L (Table 1). In contrast, treatment for 4 h slightly but significantly reduced the cell viability compared with controls (up to 10% at the highest concentration, 100 µmol/L, P < 0.05).


View this table:
[in this window]
[in a new window]
  		TABLE 1 Effect of quercetin on viability, LDH leakage, and intracellular ROS in HepG2 cells1

 
Intracellular lactate dehydrogenase (LDH) released to the culture medium was also used as an indicator of cytotoxicity. After 4 h of treating HepG2 cells with quercetin, no cytotoxic effects occurred at any concentration (Table 1). In contrast, 18 h of treatment with 10–100 µmol/L quercetin increased LDH activity in the cell culture medium, indicating cell damage. It is remarkable that the highest leakage of LDH to the medium occurred at 50 µmol/L quercetin and was signficantly less at higher concentrations (75 and 100 µmol/L).

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.


View this table:
[in this window]
[in a new window]
  		TABLE 2 Effect of quercetin on caspase-3 activity.

 

Figure 1
View larger version (23K):
[in this window]
[in a new window]
  		Figure 1  Quercetin induces caspase-specific cleavage of caspase-3 and caspase-9 in HepG2 cells. Lysates (100 µg of total protein) were analyzed by Western blot with cleaved-caspase-3 and caspase-9 antibodies (A). Densitometric quantification of cleaved caspase-3 (B). Densitometric measurement of bands of caspase-9 (C) and cleaved caspase-9 (D). Values are expressed relative to the control and are means ± SEM, n = 6. Means without a common letter differ, P < 0.05.

 
Active caspase-9 appeared after 18 h incubation with 25µ mol/L quercetin (Fig. 1), being caspase-9 processing coincident with caspase-3 activation. Moreover, caspase-9 (zymogen) expression levels increased at 25–75 µmol/L quercetin concentrations.

    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).


Figure 2
View larger version (26K):
[in this window]
[in a new window]
  		Figure 2  Quercetin decreases the Bcl-xL/Bcl-xs ratio and induces Bax translocation to the mitochondria in HepG2. Total protein (100 µg) was analyzed for Bcl-x and mitochondrial or cytosolic fraction (25 µg) were analyzed for Bax (A). Densitometric quantification of the Bcl-xL:Bcl-xs ratio (B). Densitometric quantification of bands of mitochondrial (C) and cytosolic Bax (D). Values are expressed as a percentage relative to the control condition and are means ± SEM, n = 5 (A, B) or 6 (C, D). Means without a common letter differ, P < 0.05.

 
Bax has been shown to translocate from the cytoplasm to the outer mitochondrial membrane to form pores and mediate the apoptotic death (36). Mitochondrial and cytoplasmatic fractions were evaluated by Western blot, and we observed that after treatment with quercetin for 18 h, translocation of Bax to the mitochondria increased to its highest level at 50 µmol/L, decreased at 75 µmol/L, and reached control levels at 100 µmol/L (Figs. 2A and 2C); as expected, the opposite pattern occurred in the cytosolic fraction (Figs. 2A and 2D).

    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).


Figure 3
View larger version (27K):
[in this window]
[in a new window]
  		Figure 3  Differential effects of quercetin on the basal levels of p-Akt and Akt, and p85 and p110 subunits of PI-3-kinase. Total protein (100 µg) was analyzed by Western blot with antiphospho-Akt, anti-Akt, anti-p85, and anti-p110 antibodies (A). Densitometric quantification of the ratio p-Akt:Akt (B). Densitometric quantification of bands of PI-3-kinase subunit p85 (C) and p110 (D). Values are expressed as a percentage of the ratio p-AKT:AKT relative to the control condition and are means ± SEM, n = 6. Means without a common letter differ, P < 0.05.

 
The PI-3-kinase pathway is regulated by a variety of growth factors, and the activation of the PI-3-kinase/Akt signaling pathway is strongly implicated in the regulation and survival or protection of cells (37). Thus, we also examined the effect of quercetin on the levels of the p85 regulatory subunit and the p110 catalytic subunit of PI-3-kinase. The expression of both subunits was not altered by quercetin treatment (Fig. 3).

    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.


Figure 4
View larger version (37K):
[in this window]
[in a new window]
  		Figure 4  Differential effect of quercetin on the basal levels of p-ERK1/2 and ERK1/2. Total protein (100 µg) was analyzed for antiphospho ERK and anti-ERK (A). Densitometric quantification of the ratio p-ERK:ERK (B). Values are expressed as a percentage of the ratio p-ERK:ERK relative to control and are means ± SEM, n = 6. Means without a common letter differ, P < 0.05.

 

   Discussion
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
LITERATURE CITED
 
Epidemiological studies have demonstrated the relation between diet and cancer and the potential of dietary components, in particular, polyphenols, as antiproliferative agents (3–6). Targets for chemoprevention in carcinogenesis could be multiple (3,4), and induction of apoptosis is becoming an important preventive approach. In this context, a continuous daily intake of polyphenols like quercetin might result in steady-state plasmatic concentrations, even in levels that might provoke an apoptotic cell death (10–50 µmol/L), which has been observed after the intake of dietary supplements (39). Our study showed that quercetin modulates apoptosis induction in HepG2 cells; that is, it stimulates proteolytic activities of caspase-3 and -9, alters the expression of some of the Bcl-2 family of proteins, and dephosphorylates Akt and ERK1/2.

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.


   FOOTNOTES
 
1 This work was supported by grants AGL2004-302 from the Spanish Ministry of Science and Technology and 200570M050 from the Comunidad de Madrid; A. B. G-S. is a predoctoral fellow of the Spanish Ministry of Science and Education; S. R. has a Ramón y Cajal contract from the Spanish Ministry of Science and Technology. Back

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. Back

Manuscript received 10 May 2006. Initial review completed 7 July 2006. Revision accepted 28 August 2006.


   LITERATURE CITED
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 

1. Roberts L, Gores G. Hepatocellular carcinoma: molecular pathways and new therapeutic targets. Semin Liver Dis. 2005;25:212–25.[Medline]

2. Bosch F, Ribes J, Cleries R, Diaz M. Epidemiology of hepatocellular carcinoma. Clin Liver Dis. 2005;9:191–211.[Medline]

3. Surh Y-J. Cancer chemoprevention with dietary phytochemicals. Nat Rev Cancer. 2003;3:768–80.[Medline]

4. Yang C, Landau J, Huang M, Newmark H. Inhibition of carcinogenesis by dietary polyphenolic compounds. Annu Rev Nutr. 2001;21:381–97.[Medline]

5. Watson W, Cai J, Jones D. Diet and apoptosis. Annu Rev Nutr. 2000;20:485–505.[Medline]

6. Middleton E, Jr., Kandaswami C, Theoharides TC. The effects of plant flavonoids on mammalian cells: implications for inflammation, heart disease, and cancer. Pharmacol Rev. 2000;52:673–751.[Abstract/Free Full Text]

7. Spencer J, Chowrimootoo G, Choudhury R, Debnam E, Srai S, Rice-Evans C. The small intestine can both absorb and glucuronidate luminal flavonoids. FEBS Lett. 1999;458:224–30.[Medline]

8. van Erk M, Roepman P, van der Lende T, Stierum R, Aarts J, van Bladeren P, van Ommen B. Integrated assessment by multiple gene expression analysis of quercetin bioactivity on anticancer-related mechanisms in colon cancer cells in vitro. Eur J Nutr. 2005;44:143–56.[Medline]

9. Shen S, Chen Y, Hsu F, Lee W. Differential apoptosis-inducing effect of quercetin and its glycosides in human promyeloleukemic HL-60 cells by alternative activation of the caspase 3 cascade. J Cell Biochem. 2003;89:1044–55.[Medline]

10. Singhal R, Yeh YA, Praja N, Olah E, Sledge GJ, Weber G. Quercetin down-regulates signal transduction in human breast carcinoma cells. Biochem Biophys Res Commun. 1995;208:425–31.[Medline]

11. Nguyen T, Tran E, Nguyen T, Do P, Huynh T, Huynh H. The role of activated MEK-ERK pathway in quercetin-induced growth inhibition and apoptosis in A549 lung cancer cells. Carcinogenesis. 2004;25:647–59.[Abstract/Free Full Text]

12. Chi YS, Jong HG, Son KH, Chang HW, Kang SS, Kim HP. Effects of naturally occurring prenylated flavonoids on enzymes metabolising arachidonic acid: cyclooxigenases and lypoxygenases. Biochem Pharmacol. 2001;62:1185–91.[Medline]

13. Ong C, Tran E, Nguyen T, Ong C, Lee S, Lee J, Ng C, Leong C, Huynh H. Quercetin-induced growth inhibition and cell death in nasopharyngeal carcinoma cells are associated with increase in Bad and hypophosphorylated retinoblastoma expressions. Oncol Rep. 2004;11:727–33.[Medline]

14. Chowdhury S, Kishino K, Satoh R, Hashimoto K, Kikuchi H, Nishikawa H, Shirataka Y, Sakagami H. Tumor-specificity and apoptosis-inducing activity of stilbenes and flavonoids. Anticancer Res. 2005;25:2055–63.[Medline]

15. Mertens-Talcott S, Talcott S, Percival S. Low concentrations of quercetin and ellagic acid synergistically influence proliferation, cytotoxicity and apoptosis in MOLT-4 human leukemia cells. J Nutr. 2003;133:2669–74.[Abstract/Free Full Text]

16. Wang I, Lin-Shiau S, Lin J. Induction of apoptosis by apigenin and related flavonoids through cytochrome c release and activation of caspase-9 and caspase-3 in leukaemia HL-60 cells. Eur J Cancer. 1999;35:1517–25.[Medline]

17. Yoshida M, Sakai T, Hosokawa N, Marui N, Matsumoto K, Fujioka A, Nishino H, Aoike A. The effect of quercetin on cell cycle progression and growth of human gastric cancer cells. FEBS Lett. 1990;260:10–3.[Medline]

18. Kaneuchi M, Sasaki M, Tanaka Y, Sakuragi N, Fujimoto S, Dahiya R. Quercetin regulates growth of Ishikawa cells through the suppression of EGF and cyclin D1. Int J Oncol. 2003;22:159–64.[Medline]

19. Ramos S, Alia M, Bravo L, Goya L. Comparative effects of food-derived polyphenols on the viability and apoptosis of a human hepatoma cell line (HepG2). J Agric Food Chem. 2005;53:1271–80.[Medline]

20. Shi M, Wang F, Wu Z. Synergetic anticancer effect of combined quercetin and recombinant adenoviral vector expressing human wild-type p53, GM-CSF and B7–1 genes on hepatocellular carcinoma cells in vitro. World J Gastroenterol. 2003;9:73–8.[Medline]

21. Csokay B, Prajda N, Weber G, Olah E. Molecular mechanisms in the antiproliferative action of quercetin. Life Sci. 1997;60:2157–63.[Medline]

22. Lee J, Kim J, Park J, Chung G, Jang Y. The antioxidant, rather than prooxidant, activities of quercetin on normal cells: quercetin protects mouse thymocytes from glucose oxidase-mediated apoptosis. Exp Cell Res. 2003;291:386–97.[Medline]

23. Chang J, Hsu Y, Kuo P, Kuo Y, Chiang L, Lin C. Increase of Bax/ Bcl-XL ratio and arrest of cell cycle by luteolin in immortalized human hepatoma cell line. Life Sci. 2005;76:1883–93.[Medline]

24. Vijayababu M, Kanagaraj P, Arunkumar A, Ilangovan R, Aruldhas M, Arunakaran J. Quercetin-induced growth inhibition and cell death in prostatic carcinoma cells (PC-3) are associated with increase in p21 and hypophosphorylated retinoblastoma proteins expression. J Cancer Res Clin Oncol. 2005;131:765–71.[Medline]

25. Curtin J, Donovan M, Cotter T. Regulation and measurement of oxidative stress in apoptosis. J Immunol Methods. 2002;265:49–72.[Medline]

26. Chandra J, Samali A, Orrenius S. Triggering and modulation of apoptosis by oxidative stress. Free Radic Biol Med. 2000;29:323–33.[Medline]

27. Spencer J, Rice-Evans C, Williams R. Modulation of pro-survival Akt/protein kinase B and ERK1/2 signaling cascades by quercetin and its in vivo metabolites underlie their action on neuronal viability. J Biol Chem. 2003;278:34783–93.[Abstract/Free Full Text]

28. Yoshizumi M, Tsuchiya K, Kirima K, Kyaw M, Suzaki Y. Tamaki. T. Quercetin inhibits Shc- and phosphatidylinositol 3-kinase-mediated c-Jun N-terminal kinase activation by angiotensin II in cultured rat aortic smooth muscle cells. Mol Pharmacol. 2001;60:656–65.[Abstract/Free Full Text]

29. Agullo G, Gamet-Payrastre L, Manenti S, Viala C, Remesy C, Chap H, Payrastre B. Relationship between flavonoid structure and inhibition of phosphatidylinositol 3-kinase: a comparison with tyrosine kinase and protein kinase C inhibition. Biochem Pharmacol. 1997;53:1649–57.[Medline]

30. Alia M, Mateos R, Ramos S, Lecumberri E, Bravo L, Goya L. Influence of quercetin and rutin on growth and antioxidant defense system of a human hepatoma cell line (HepG2). Eur J Nutr. 2006;45:19–28.[Medline]

31. Welder A, Acosta D. Enzyme leakage as an indicator of cytotoxicity in culture cells. In: Tyson CA, Franzier JM, editors, In vitro toxicity indicators: methods in toxicology. New York: Academic Press 1994:46–9.

32. Wang H, Joseph J. Quantifying cellular oxidative stress by dichlorofluorescein assay using microplate reader. Free Radic Biol Med. 1999;27:612–16.[Medline]

33. Herrera B, Fernandez M, Alvarez A, Roncero C, Benito M, Gil J, Fabregat I. Activation of caspases occurs downstream from radical oxygen species production, Bcl-xL down-regulation, and early cytochrome C release in apoptosis induced by transforming growth factor beta in rat fetal hepatocytes. Hepatology. 2001;34:548–56.[Medline]

34. Fabregat I, Herrera B, Fernandez M, Alvarez AM, Roncero C, Ventura JJ, Valverde AM, Benito M. Epidermal growth factor impairs the cytochrome C/caspase-3 apoptotic pathway induced by transforming growth factor beta in rat fetal hepatocytes via a phosphoinositide 3-kinase-dependent pathway. Hepatology. 2000;32:528–35.[Medline]

35. Alia M, Ramos S, Mateos R, Bravo L, Goya L. Response of the antioxidant defense system to tert-butyl hydroperoxide and hydrogen peroxide in a human hepatoma cell line (HepG2). J Biochem Mol Toxicol. 2005;19:119–28.[Medline]

36. Adams J, Cory S. Life-or-death decisions by the Bcl-2 protein family. Trends Biochem Sci. 2001;26:61–6.[Medline]

37. Nicholson K, Anderson N. The protein kinase B/Akt signalling pathway in human malignancy. Cell Signal. 2002;14:381–95.[Medline]

38. Ballif B, Blenis J. Molecular mechanisms mediating mammalian mitogen-activated protein kinase (MAPK) kinase (MEK)-MAPK cell survival signals. Cell Growth Differ. 2001;12:397–408.[Free Full Text]

39. Wätjen W, Michels G, Steffan B, Niering P, Chovolou Y, Kampkötter A, Thi QHT, Proksch P, Kahl R. Low concentrations of flavonoids are protective in rat H4IIE cells whereas high concentrations cause DNA damage and apoptosis. J Nutr. 2005;135:525–31.[Abstract/Free Full Text]

40. Huynh H, Nguyen T, Chan E, Tran E. Inhibition of ErbB-2 and ErbB-3 expression by quercetin prevents transforming growth factor alpha (TGF-alpha)- and epidermal growth factor (EGF)-induced human PC-3 prostate cancer cell proliferation. Int J Oncol. 2003;23:821–9.[Medline]

41. Cardoso SM, Pereira C, Oliveira R. Mitochondrial function is differentially affected upon oxidative stress. Free Radic Biol Med. 1999;26:3–13.[Medline]

42. Haghiac M, Walle T. Quercetin induces necrosis and apoptosis in SCC-9 oral cancer cells. Nutr Cancer. 2005;53:220–31.[Medline]

43. Andersson MM, Breccia JD, Hatti-Kaul R. Stabilizing effect of chemical additives against oxidation of lactate dehydrogenase. Biotechnol Appl Biochem. 2000;32:145–53.

44. Herrera B, Alvarez A, Sanchez A, Fernandez M, Roncero C, Benito M, Fabregat I. Reactive oxygen species (ROS) mediates the mitochondrial-dependent apoptosis induced by transforming growth factor (beta) in fetal hepatocytes. FASEB J. 2001;15:741–51.[Abstract/Free Full Text]

45. Packham G, Ashmun R, Cleveland J. Cytokines suppress apoptosis independent of increases in reactive oxygen levels. J Immunol. 1996;156:2792–800.[Abstract]

46. Dorta DJ, Pigoso AA, Mingatto FE, Rodrigues T, Prado IM, Helena AF, Uyemura A, Santos AC, Curti C. The interaction of flavonoids with mitochondria: effects on energetic processes. Chem Biol Interact. 2005;152:67–78.[Medline]

47. Michels G, Watjen W, Niering P, Steffan B, Thi Q, Chovolou Y, Kampkotter A, Bast A, Proksch P, Kahl R. Pro-apoptotic effects of the flavonoid luteolin in rat H4IIE cells. Toxicology. 2005;206:337–48.[Medline]

48. Lee H-J, Wang C-J, Kuo H-C, Chou F-P, Jean L-F, Tseng T-H. Induction apoptosis of luteolin in human hepatoma HepG2 cells involving mitochondria translocation of Bax/Bak and activation of JNK. Toxicol Appl Pharmacol. 2005;203:124–31.[Medline]

49. Nakanishi K, Sakamoto M, Yamasaki S, Todo S, Hirohashi S. Akt phosphorylation is a risk factor for early disease recurrence and poor prognosis in hepatocellular carcinoma. Cancer. 2005;103:307–12.[Medline]

50. Cardone M, Roy N, Stennicke H, Salvesen G, Franke T, Stanbridge E, Frisch S, Reed JC. Regulation of cell death protease caspase-9 by phosphorylation. Science. 1998;282:1318–21.[Abstract/Free Full Text]

51. Mitsui H, Takuwa N, Maruyama T, Maekawa H, Hirayama M, Sawatari T, Hashimoto N, Takuwa Y, Kimura S. The MEK1-ERK map kinase pathway and the PI 3-kinase-Akt pathway independently mediate anti-apoptotic signals in HepG2 liver cancer cells. Int J Cancer. 2001;92:55–62.[Medline]





This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Granado-Serrano, A. B.
Right arrow Articles by Ramos, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Granado-Serrano, A. B.
Right arrow Articles by Ramos, S.

Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]