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. Author manuscript; available in PMC: 2020 Sep 24.
Published in final edited form as: Nutr Cancer. 2019 Jan 19;71(5):861–869. doi: 10.1080/01635581.2018.1559929

Regulation of the Intracellular ROS Level Is Critical for the Antiproliferative Effect of Quercetin in the Hepatocellular Carcinoma Cell Line HepG2

Ji-Sook Jeon a,*, Sora Kwon a,*, Kiwon Ban b, Young- Kwon Hong c, Curie Ahn d, Jung-Suk Sung e, Inho Choi a
PMCID: PMC7513677  NIHMSID: NIHMS1618224  PMID: 30661409

Abstract

Quercetin, an antioxidant flavonoid, has been known that it can induce the cell cycle arrest and apoptosis of hepatocellular carcinoma (HCC) cells by the stabilization or induction of p53. Here, we found that quercetin reduced the proliferation of HepG2 cells significantly, but not Huh7 cells. Interestingly, quercetin down-regulated the intracellular ROS level in HepG2 cells, but not Huh7 cells. Functional study using siRNA showed that the proliferation of HepG2 cells was still regulated by quercetin in the absence of p53. Furthermore, we confirmed the effect of quercetin on HepG2 cells by H2O2 supplementation. This study demonstrates that the antiproliferative effect of quercetin on HCC cells can be mediated by reducing intracellular ROS, which is independent of p53 expression.

Introduction

Hepatocellular carcinoma (HCC) is one of the most common types of cancer and the third leading cause of cancer-related deaths in the world (1). HCC is characterized by genetic changes affecting multiple signaling cascades, resulting in the uncontrolled growth of hepatocytes (2). Because of the heterogeneous etiology of HCC, only some HCC patients (about 30%) can be treated with appropriate treatment modalities such as liver resection, transplantation, or local ablation (3). To date, only one targeted chemotherapy agent sorafenib (Nexavar, Bayer), has been approved for treating HCC patients (4). However, not all HCC patients show a positive response to sorafenib (5). Therefore, it is vital to identify a molecular target specific for HCC and to develop suitable chemotherapy agents.

The activation of several signaling pathways has been implicated in the pathogenesis of HCC, such as reactive oxygen species (ROS), which can trigger the oxidative damage of biomolecules. Indeed, one of the main features of cancer cells is a persistent pro-oxidative state that leads to intrinsic oxidative stress (6,7). ROS participate in carcinogenesis from initiation to malignant conversion, by mutation of the proto-onco-genes and tumor suppressor genes and subsequent activation of the signal transduction pathways (8,9). Therefore, numerous studies have suggested that antioxidants that can quench intracellular ROS would be helpful for reducing cancer risk (10,11).

Quercetin (3, 3′, 4′, 5, 7-pentahydroxyflavone), a major polyphenol and flavonoid found in vegetables, fruits and wines, is known to prevent free radical-mediated cytotoxicity as an antioxidant. Because of phenolic hydroxyl groups, quercetin has been reported to possess a broad range of pharmacological properties, including strong antioxidant activity and hepatoprotective effects against oxidative stress (12,13). Recent studies have also reported that quercetin can upregulate intracellular ROS level, thus triggering cell death (14,15). Furthermore, quercetin can induce the cell cycle arrest and apoptosis of HCC cells by the stabilization or induction of p53 (2,16). p53, a tumor suppressor protein involved in cancer prevention, can regulate the cell cycle, apoptosis, and DNA repair (17). Accordingly, several compounds that upregulate p53 function have been reported to inhibit the proliferation of cancer cells by inducing cell cycle arrest or apoptosis (18). Despite the significant roles of ROS and p53 in cancer survival, there are few reports showing their relationship in the antiproliferative effect of quercetin on HCC.

In this study, the antiproliferative effect of quercetin was investigated using two different HCC cell lines, HepG2 and Huh7. Quercetin reduced the proliferation of HepG2 cells significantly, but not Huh7 cells. Interestingly, it was found that quercetin down-regulated the intracellular ROS level of HepG2 cells, but not that of Huh7 cells. In addition, the expression of p53 was up-regulated by quercetin. However, functional studies using small-interfering RNA (siRNA) showed that quercetin was still able to regulate the proliferation of HepG2 cells in the absence of p53. Moreover, the antiproliferative effect of quercetin on HepG2 cells was reduced by H2O2 supplementation. Overall, the present study demonstrates that quercetin can regulate the proliferation of HCC cells by reducing intracellular ROS, which is independent of p53 expression, and suggests that quercetin is useful for HCC treatment as an antioxidant.

Materials and Methods

Cell Culture, Transfection, and Reagents

HCC cell lines, HepG2, Huh7, PLC/PRF-5 and Hep3B, were purchased from the Korean Cell Line Bank (Seoul National University, Korea). Cells were cultured in Dulbecco’s modified Eagle medium (DMEM) with 10% fetal bovine serum (FBS), 5% penicillin–streptomycin, and 5% sodium pyruvate, and grown in an atmosphere containing 5% CO2 at 37 °C. Cells were incubated with or without quercetin (Sigma-Aldrich, MO) as indicated in each experiment. Resveratrol was also purchased from Sigma-Aldrich. Cell images were captured by microscopy (Leica, Germany). For functional experiments, HepG2 cells were transfected with siRNA against the p53 gene (sip53, forward; 5′-GACUCCAGUGGUAAUCUACTT-3′ and reverse; 5′-GUAGAUUACCACUGGAGUCTT-3′) using Lipofactor-2000 (Aptabio, Korea).

Cell Proliferation Assay

Cell proliferation was measured using a Premixed WST-1 Cell Proliferation Assay kit (TaKaRa, Japan), as described previously (19). Briefly, HepG2 cells (2 × 104 cells/well) or Huh7 cells3 (1 × 104 cells/well) were seeded in 24-well plates (SPL Inc., Korea) and cultured overnight. The media were then replaced with fresh conditional media (2% FBS) containing quercetin. After cells were grown for 48 h, WST-1 reagent was added to each well, and the cells were incubated for an additional 4 h. The absorbance was measured at a wavelength of 450 nm using a microplate reader (Molecular Devices, CA) and the cell proliferation count was estimated based on a standard curve that was prepared in parallel. H2O2 (10 μM) was added to HepG2 cells for ROS supplementation.

Measurement of the Intracellular ROS Level

The intracellular generation of ROS was analyzed with the probe dichlorofluorescein diacetate (H2DCFDA, Sigma-Aldrich, MO), a membrane-permeant fluorescent probe that is widely used to monitor intracellular ROS production. After treatment with quercetin, cells were incubated with 10 μM H2DCFDA at 37 °C for 30min in the dark. Subsequently, the cells were harvested, washed three times with PBS, and DCF fluorescence distribution was measured using Guava EasyCyte (Millipore, MA).

Protein Extraction and Western Blot Analysis

HepG2 cells (5 × 105 cells/dish) were seeded in 60 mm dishes and treated with the indicated concentrations of quercetin for 0 – 48 h. Cell lysates were prepared using RIPA buffer (50 mM Tris-HCl [pH7.4], 150 mM NaCl, 1% Nonidet P-40, 0.1% sodium dodecyl sulfate[SDS], and 0.5% sodium deoxycholate) containing protease and phosphatase inhibitors (Sigma-Aldrich, CA). The samples were quantified using the Bradford Protein Assay kit (Pierce Biotechnology, IL) according to the manufacturer’s instructions. Then, 20 μg of protein was separated by 10% SDS polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (PVDF; GE Healthcare, UK). Western blot analysis was performed with anti-p53 antibody (sc-6243; Santa Cruz Biotechnology, CA), anti-cyclin A antibody (sc-751; Santa Cruz Biotechnology, CA), anticyclin E antibody (sc-247; Santa Cruz Biotechnology, CA), anti-CHK1 (checkpoint kinase 1) antibody (sc-8408; Santa Cruz Biotechnology, CA) anti-HO-1 (heme oxygenase-1) antibody (sc-136960; Santa Cruz Biotechnology, CA), anti-SOD1 (superoxide dismutase 1) antibody (HPA001404; Sigma-Aldrich, CA), anti-β-actin antibody (sc-47778; Santa Cruz Biotechnology, CA), and the appropriate secondary antibodies. The enhanced chemiluminescence detection reagent (Amersham Pharmacia Biotech, NJ) was used for visualization.

Statistical analysis

The measurements were expressed as mean ± standard deviation (SD) for each experiment. All analyses were performed in triplicates and each experiment was repeated at least two times. Student t-test was used to determine whether the differences between the experimental and control groups were statistically significant. P values less than 0.05 were considered statistically significant (*P < 0.05; **P < 0.01; ***P < 0.001).

Results

Effect of quercetin on the proliferation and ROS level of HepG2 cells

The effect of quercetin on cellular proliferation was first investigated using two different HCC cell lines, HepG2 and Huh7. In comparison with control cells (treated with the vehicle [DMSO] alone), the proliferation of HepG2 cells was down-regulated by quercetin (Fig. 1(A) and (B)). Cell proliferation was observed until 2 days after quercetin treatment by microscopy (Fig. 1(A)) and WST-1 assay (Fig. 1(B)). The proliferation and morphology of HepG2 cells were affected by quercetin after 48h. However, Huh7 cells were not significantly affected by quercetin up to 80 μM (Fig. 1(E) and (F)). These results indicated that quercetin can regulate the proliferation of HepG2 cells but not Huh7 cells. The antiproliferative effect of quercetin was also confirmed in other HCC cell lines, PLC/PRF-5 and Hep3B (Supplementary Information Figure S1). The variation in the ROS level in HepG2 and Huh7 cells after quercetin treatment was measured using H2DCFDA as a probe for detecting intracellular ROS. The number of fluorescence-positive cells in the M2 region was reduced to half after treatment with quercetin, indicating that quercetin reduced the intracellular ROS level in HepG2 cells. On the other hand, the cell number in the fluorescence-negative region was increased up to two-fold (Fig. 1(C) and (D)). However, the ROS level in Huh7 cells was not affected by quercetin (Fig. 1(G) and (H)). The relationship between the regulation of intracellular ROS level and the antiproliferative effect was also investigated using another antioxidant, resveratrol (Supplementary Information Figure S2). These results demonstrated that quercetin can regulate the ROS level in HepG2 cells, but not Huh7 cells.

Figure 1.

Figure 1.

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Quercetin down-regulated the proliferation of HepG2 cells. (A) HepG2 cells were cultured in conditional media (2% FBS) with or without quercetin (80 μM). Cell morphology was visualized by microscopic analysis. Scale bars represent 500 μm. (B) The proliferation of HepG2 cells treated with quercetin was measured after 48 h. **P < 0.01. (C) The intracellular ROS level in HepG2 cells treated with or without quercetin was measured using H2DCFDA. (D) Flow cytometry analysis indicated that quercetin increased the number of fluorescence-negative cells (M1) but decreased the number of fluorescence-positive cells (M2). **P < 0.01. (E) Huh7 cells were treated with or without quercetin, and cell shape was analyzed by microscopy. (F) The proliferation of Huh7 cells treated with quercetin was measured after 48 h. (G) The intracellular ROS level in Huh7 cells was not affected by quercetin treatment. (H) Flow cytometry analysis indicated that quercetin did not affect the intracellular ROS level of Huh7 cells.

Effect of quercetin on the expression of proteins regulating the proliferation and protection of cells

The expression of proteins responsible for proliferation and protection in HepG2 cells was investigated after quercetin treatment. Quercetin increased the expression of p53 and HO-1 but decreased that of cyclin A and CHK1 (Fig. 2). The expression of p53 and HO-1 was upregulated by quercetin after 12 and 24 h, respectively. However, the expression of cyclin E and SOD1 was not affected by quercetin (Fig. 2(A)). The relative analysis comparing each protein with β-actin showed the statistical significance (Fig. 2(B)). The variation in protein expression was also verified in HepG2 cells treated with various concentrations of quercetin (Fig. 2(C)). The expression of p53 and HO-1 was increased but that of CHK1 was decreased in response to the increase in quercetin up to 100 μM. The relative analysis also showed the significant change of protein expression (Fig. 2(D)). These results indicated that quercetin can regulate the proliferation and antioxidant activity of HepG2 cells by increasing the expression of p53 and HO-1.

Figure 2.

Figure 2.

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Quercetin increased the expression of p53 and HO-1 but decreased the expression of cyclin A and CHK1. (A) HepG2 cells were treated with the vehicle (DMSO) or quercetin and harvested from 0 to 48 h. The protein expression in HepG2 cells was analyzed by western blot assay. (B) The relative analysis of (A) was performed by comparing with each protein with β-actin. *P < 0.05, **P < 0.01, ***P < 0.001. (C) HepG2 cells were treated with various concentrations of quercetin from 20 to 100 μM. After 48 h, the cells were harvested and analyzed by western blot assay. (D) The relative analysis data of (C) by comparing with each protein with β-actin. *P < 0.05, **P < 0.01, ***P < 0.001.

Antiproliferative effect of quercetin via ROS scavenging

After identifying the effects of quercetin on ROS and p53, knock-down experiments were performed to investigate the significance of p53 in ROS reduction. The knock-down effect of sip53 was confirmed by western bot assay (Fig. 3(A)). Quercetin up-regulated the expression of p53 in control siRNA (siCTR)-transfected cells but not in sip53-transfected cells. Then, the antiproliferative effect of quercetin was measured. Interestingly, quercetin down-regulated the proliferation of HepG2 cells transfected with sip53 as well as siCTR (Fig. 3(B)), which indicated that quercetin can regulate the proliferation of cells in the absence of p53 expression. In addition, intracellular ROS was regulated by quercetin in HepG2 cells transfected with sip53 as well as siCTR (Fig. 3(C)). The number of fluorescence-positive cells (M2 region) and fluorescence-negative cells (M1 region) was decreased and increased, respectively, by quercetin for HepG2 cells transfected with sip53 as well as siCTR (Fig. 3(D)), indicating that quercetin can regulate intracellular ROS in the absence of p53 expression. These results revealed that the downregulation of intracellular ROS is critical for the antiproliferative effect of quercetin on HepG2 cells.

Figure 3.

Figure 3.

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Down-regulation of intracellular ROS level is important for the antiproliferative effect of quercetin on HepG2 cells. (A) The knockdown effect of siRNA against p53 mRNA (sip53) was verified by western blot analysis. Cells were transfected with sip53 or control siRNA (siCTR). After 24 h, the cells were treated with the vehicle (DMSO; D) or quercetin (Q; 80 μM) for another 12 h. (B) HepG2 cells were transfected with sip53 or siCTR. After 24 h, the cells were treated with the vehicle (DMSO; D) or quercetin (Q; 80 μM) for another 48 h. (C) The representative fluorescence profile revealed that quercetin reduced the intracellular ROS level in HepG2 cells transfected with sip53 as well as siCTR. (D) Flow cytometry analysis indicated that quercetin reduced the intracellular ROS level in sip53-transfected HepG2 cells (higher number of fluorescence-negative cells [M1] and lower number of fluorescence-positive cells [M2]) and siCTR-transfected HepG2 cells. *P < 0.05, **P < 0.01.

ROS level as a critical factor in cancer cell proliferation

The results showing the significance of ROS in the proliferation of HepG2 cells led us to investigate the antiproliferative effect of quercetin after replenishing ROS levels. First, a suitable concentration of H2O2 that does not affect cellular proliferation but can compensate for ROS was determined (10 μM of H2O2). Then, the antiproliferative effect of quercetin was measured with or without H2O2 supplementation. Interestingly, the reduced proliferation of HepG2 cells by quercetin was recovered after treatment with H2O2 (Fig. 4(A)). The expression of proteins in HepG2 cells showed that the quercetin-induced up-regulation of p53 was decreased by H2O2 treatment. However, the expression of HO-1 was synergistically increased by quercetin and H2O2. Interestingly, CHK1 expression was up-regulated by H2O2 but down-regulated by quercetin (Fig. 4(B)). These results indicated that the antiproliferative effect of quercetin was mediated by ROS-scavenging activity, which was reduced by H2O2 supplementation.

Figure 4.

Figure 4.

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ROS level is critical for the proliferation of HepG2 cells. (A) The antiproliferative effect of quercetin on HepG2 cells was investigated with or without H2O2 supplementation (10 μM). Cell proliferation was measured by WST-1 assay after 48 h incubation. The proliferation of HepG2 cells was inhibited by quercetin and recovered by H2O2 treatment. **P < 0.01. (B) Western blot analysis revealed that p53 expression was upregulated by quercetin but down-regulated by H2O2 treatment, and HO-1 expression was synergistically increased by quercetin and H2O2. CHK1 expression was induced by H2O2 but inhibited by quercetin.

Discussion

Recent studies have shown that quercetin exhibits anticancer activity in some types of tumor cells including HCC cells (20–22). The evaluation of the anticancer effect of quercetin is mainly focused on the activation or induction of p53, a tumor suppressor protein (16,23,24) or the upregulation of intracellular ROS level (15,25). However, quercetin is well known as an antioxidant that can quench lipid peroxides or enhance the production of endogenous antioxidants such as glutathione (11,26). Despite the significant roles of ROS and p53 in cancer survival, there are few reports showing the relationship between the p53 molecule and intracellular ROS level in the anticancer effect of quercetin on HCC cells.

In this study, the effect of quercetin on two different HCC cell lines, HepG2 and Huh7, was investigated and quercetin was found to inhibit the proliferation of HepG2 cells (Fig. 1(A) and (B)). However, the antiproliferative effect of quercetin (up to 80 μM) on Huh7 cells was not statistically significant (Fig. 1(E) and (F)). The anticyclin-dependent kinaseproliferative effect of quercetin was also investigated in other HCC cell lines, PLC/PRF-5 and Hep3B (Supplementary Information Figure S1), which showed that quercetin affected the proliferation of HCC cells differently. It’s necessary to find out which factors contribute differently to the quercetin sensitivity of these HCC cell lines in the future study. Measurement of intracellular ROS level showed that quercetin significantly reduced the ROS level in HepG2 cells but not Huh7 cells (Fig. 1(D) and (H)), and the intracellular ROS level in Huh7 cells was much higher than that in HepG2 cells (Fig. 1(C) and (G)). These results indicated that the different effects of quercetin on the proliferation of the two HCC cell lines (HepG2 and Huh7) may be attributed to the differently reduced intracellular ROS levels. This effect was also confirmed using another antioxidant, resveratrol (Supplementary Information Figure S1). The variation in protein expression in HepG2 cells showed that quercetin induced the expression of p53 as well as HO-1 (Fig. 2). These results are in agreement with previous studies demonstrating the anticancer effect of quercetin (2,25,27,28). On the other hand, the expression of cyclin A and CHK1 was down-regulated by quercetin. Cyclin A, a member of the cyclin protein family, plays an important role in regulating cell cycle progression by interacting with cyclin-dependent kinases (CDKs) (29,30). In this study, the up-regulation of p53 and the down-regulation of cyclin A by quercetin may be attributed to the regulation of cyclin expression by p53 indirectly through the up-regulation of p21, a CDK inhibitor (31,32). CHK1, a kinase that triggers a pleiotropic cellular response, participates in the regulation of cell cycle arrest, DNA repair, or cell death (33). Recently, CHK1 has been found to be overexpressed in a variety of cancer cells including HCC cells (34,35). Furthermore, there are several studies showing that CHK1 may contribute to therapy resistance (23,36). Therefore, knowledge of the effect of quercetin on CHK1 expression may be helpful for enhancing chemotherapy or radiotherapy.

To investigate the role of p53 in the antiproliferative effect of quercetin, knock-down experiments using siRNAs were conducted. After confirming the knock-down effect of siRNA against p53 (sip53; Fig. 3(A)), we measured cell proliferation with or without quercetin treatment. Interestingly, the antiproliferative effect of quercetin on HepG2 cells was comparable between the sip53-transfected group and the siCTR-transfected group (Fig. 3(B)). Moreover, quercetin reduced the intracellular ROS level in HepG2 cells transfected with sip53 (Fig. 3(C) and (D)). These results indicated that the ROS-scavenging activity of quercetin but not p53 is critical for antiproliferative activity. Previous studies have reported that intracellular ROS is increased in almost all cancer cells compared with their normal counterparts (6,37,38), suggesting that the ROS requirement is higher in cancer cells than in normal cells. Indeed, the down-regulation of intracellular ROS level in cancer cells can induce the inhibition of cell proliferation, resulting in apoptosis (39,40).

Finally, we investigated the effect of quercetin on cell proliferation after replenishing ROS levels. We found that 10 μM of H2O2 was a suitable concentration that can compensate for ROS without affecting cellular proliferation. Interestingly, the antiproliferative effect of quercetin was reduced by H2O2 supplementation (Fig. 4(A)). Western blot analysis showed that quercetin-induced increase in p53 expression was down-regulated after H2O2 treatment and HO-1 expression was synergistically up-regulated by quercetin and H2O2 (Fig. 4(B)). CHK1 expression was induced by H2O2, but inhibited by quercetin, indicating that the down-regulation of CHK1 expression by quercetin may be beneficial for enhancing chemotherapy regardless of ROS levels. The key points of this study were summarized as a graphical abstract (Fig. 5).

Figure 5.

Figure 5.

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Graphical representation summarizing this study.

Quercetin was known as a safe reagent for the treatment of human cancer (41). Despite the huge amount of previous reports, further investigation is still needed to find out the intracellular effects of quercetin and to increase its clinical application as an anticancer reagent. It is also necessary to try studies about the efficacy or chemical modification of quercetin for the treatment of cancer. In this study, we found that quercetin regulated the proliferation of HepG2 cells by down-regulating intracellular ROS level as well as up-regulating p53. Functional studies using siRNA against p53 showed that intracellular ROS level is one of the critical factors determining the anticancer effect of quercetin. Overall, this study demonstrates that the regulation of intracellular ROS level is one of factors affecting HCC cell proliferation and suggests that antioxidants such as quercetin may be useful for HCC treatment in combination with drugs used in anticancer therapy.

Supplementary Material

Supplementary Material 1
Supplementary Material 2

Acknowledgments

Funding

This study was supported by the Ministry of Science, ICT and Future Planning in Republic of Korea (2016M3A9B6903411).

Footnotes

Disclosure Statement

The authors have no conflict of interest to declare.

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Supplementary Material 2
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