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β-catenin pathway in hepatocellular carcinoma cells
Phytomedicine 43 (2018) 37–45
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Reversal effect of quercetin on multidrug resistance via FZD7/β-catenin pathway in hepatocellular carcinoma cells
T
Zhaolin Chena,b, Cheng Huanga, Taotao Maa, Ling Jiangb, Liqin Tangb, Tianlu Shib, ⁎ ⁎ Shantang Zhangb, Lei Zhangb, Pengli Zhub, Jun Lia, , Aizong Shenb, a
Institute for Liver Diseases of Anhui Medical University (AMU), Anhui Province Key Laboratory of Major Autoimmune Diseases, Anhui Institute of Innovative Drugs, School of Pharmacy, Anhui Medical University, Hefei, Anhui 230032, China b Department of Pharmacy, Anhui Provincial Hospital, Anhui Medical University, Hefei, Anhui 230001, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Quercetin Hepatocellular carcinoma Multiple drug resistance ATP-binding cassette transporters, FZD7/βcatenin pathway
Background: Chemotherapy has been widely used to treat cancer, but the appearance of multidrug resistance (MDR) is the biggest obstacle to successful chemotherapy. One of the conventional mechanisms of MDR is overexpression of ATP-binding cassette (ABC) transporters such as P-glycoprotein (P-gp/ABCB1) and multidrug resistance-associated proteins (MRPs/ABCCs) that limits the prolonged and efficient use of chemotherapeutic drugs. To enhance the chemosensitivity of tumor cells, attentions have been focused on effective MDR modulators. Purpose: This study aimed to investigate the reversal effect of quercetin on MDR, and explored its mechanism of action in vitro. Study design/methods: The effect and mechanism of quercetin on MDR was examined by using MTT assay, flow cytometry, real-time PCR and western blot analysis in human hepatocellular carcinoma cells. Results: Our data found that the intracellular accumulation of rhodamine-123 (Rh123) and doxorubicin (ADR) were increased, the sensitivity of BEL/5-FU cells to chemotherapeutic drugs were increased, and the expressions of ABCB1, ABCC1 and ABCC2 were all down-regulated, which indicated that the functions and expressions of ABCB1, ABCC1 and ABCC2 efflux pump were inhibited by quercetin treatment. Moreover, the suppression of ABCB1, ABCC1 and ABCC2 by quercetin was dependent on the FZD7 through the Wnt/β-catenin pathway. Further research revealed that reduction of FZD7 by RNA interference (siFZD7) enhanced the sensitivity to chemotherapeutic drugs, increased the cellular accumulation of Rh123 and ADR, and induced inhibitory effects on the expression of FZD7, ABCB1, ABCC1, ABCC2 and β-catenin, similar to quercetin. In the meanwhile, overexpression of FZD7 showed the inversely effect on the expressions. Interesting, it was confirmed that quercetin could inhibit the expression levels of FZD7, ABCB1, ABCC1, ABCC2 and β-catenin in BEL-7402 cells; furthermore, treatment by quercetin combined with siFZD7 in BEL/5-FU cells, the expressions of these genes were effectively decreased in comparison to quercetin combined with siRNA negative control (sncRNA). Conclusion: Overall, these data suggested the effectiveness of using quercetin, at least in part, via inhibiting FZD7 to combat chemoresistance and showed that quercetin could be developed into an efficient natural sensitizer for resistant human hepatocellular carcinoma.
Introduction Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related mortality worldwide and also one of the most common
malignant liver tumors in China (Torre et al., 2015). The available front-line treatment for HCC is surgical resection and liver transplantation, but only a minority of the patients are eligible because HCCs typically present at advanced-stage with macrovascular invasion or
Abbreviations: 5-FU, 5-fluorouracil; ABC, ATP-binding cassette; ATP, adenosine triphosphate; ADR, doxorubicin; FZD7, Frizzled homolog protein 7; GV144-FZD7, lentivirus-FZD7 vector; HCC, hepatocellular carcinoma; IC50, 50% inhibition concentration; MDR, multiple drug resistance; MRP1/ABCC1, multidrug resistance-associated protein 1; MRP2/ABCC2, multidrug resistance-associated protein 2; MMC, Mitomycin C; MTT, thiazolyl blue tetrazolium blue; NC-GV144, empty GV144 vector; P-gp/ABCB1, P-glycoprotein; quercetin, Q; qRTPCR, quantitative real-time polymerase chain reaction; Rh123, rhodamine-123; SDS-PAGE, sulphate-polyacrylamide gel electrophoresis; siRNA, small interfering RNA; siFZD7, FZD7 interfering small RNA; sncRNA, siRNA negative control; VRP, verapamil ⁎ Corresponding authors. E-mail addresses: [email protected] (J. Li), [email protected] (A. Shen). https://doi.org/10.1016/j.phymed.2018.03.040 Received 22 September 2017; Received in revised form 26 January 2018; Accepted 18 March 2018 0944-7113/ © 2018 Elsevier GmbH. All rights reserved.
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Fig.1. The relative expressions FZD7, ABCB1, ABCC1 and ABCC2 in BEL-7402 and BEL/5-FU cells. (A) Expressions of FZD7, ABCB1, ABCC1 and ABCC2 were detected by qRT-PCR. (B) Expressions of FZD7, ABCB1, ABCC1 and ABCC2 were detected by western blot. The assays were performed at least three times with similar results. Data are shown as the mean ± SD (n = 3). *P < 0.05, ⁎⁎P < 0.01, versus control.
the KB/VCR oral cancer resistant cell lines (Yuan et al., 2015). Besides, quercetin might target the MAPK/ERK/JNK pathway and transcriptionally down-regulate ABCB1, suggesting that quercetin may reverse MDR by targeting the MAPK/ERK/JNK/ABCB1 pathway in multidrug resistant leukemia K562 cells (Chen et al., 2015). Furthermore, quercetin effectively inhibited tumor growth and enhanced the sensitivity to chemotherapy in a xenograft mouse model of HCC (Dai et al., 2016). Research carried out in the past few decades has shown that quercetin plays a vital role in the development of HCC. However, studies on the function and mechanisms of quercetin on MDR in HCC are still limited. Our previous study firstly revealed that the activation of FZD7/βcatenin pathway plays an important role in the MDR (Chen et al., 2013), but whether or not quercetin reverses MDR by suppressing FZD7/β-catenin pathway in HCC is not yet clear. Therefore, the present study aimed to investigate the effect and the underlying molecular mechanisms of quercetin on human hepatocellular carcinoma MDR BEL-7402/5-fluorouracil (BEL/5-FU) cells.
Table 1 The sensitivity of BEL-7402 and BEL/5-FU cells treated with chemotherapeutic drugs. IC50(μg/ml)
5-FU MMC ADR
BEL/5-FU
BEL-7402
Resistance index
69.648 ± 1.23 4.151 ± 0.30 8.882 ± 1.18
4.076 ± 0.27 2.065 ± 0.25 4.021 ± 0.13
17.08 2.01 2.21
metastases (Ling et al., 2017). Systemic chemotherapy is adopted as an effective nonsurgical therapeutic strategy for most HCCs. However, the emergence of multidrug resistance (MDR) to pharmacologically and structurally distinct class of clinical drugs severely blocks the successful management of HCC (Bruix et al., 2014; Wu et al., 2016). The well recognized mechanism underlying MDR is the significant overexpression of adenosine triphosphate (ATP)-binding cassette (ABC) super-family of transporters, such as P-glycoprotein (P-gp/ABCB1), multidrug resistance-associated protein 1 (MRP1/ABCC1) and multidrug resistance-associated protein 2 (MRP2/ABCC2), et al., which acts as an efflux pump on cell surface (Chen et al., 2016). Hence, the strategy consists in inhibiting the activity, or lowering the expression of the efflux transporter, which is highly warranted to improve the outcomes of HCC chemotherapy. Quercetin, a typical flavonol-type flavonoid compound found ubiquitously distributed in many plants and vegetables, which has been exhibited to possess antioxidative, anti-inflammatory, immunomodulatory, and vasodilating activities (Chen et al., 2010). In recent years, increasing evidence suggests that quercetin can be useful in cancer treatment by inducing cell apoptosis in vitro and in vivo (Hashemzaei et al., 2017; Lee et al., 2015; Nguyen et al., 2017). Quercetin could be suggested as a potential modulator of ABCB1 which inhibits the ABCB1 expressions in a concentration-dependent manner in
Material and methods Cell culture The parental sensitive human hepatocellular carcinoma cell line BEL-7402 and multidrug resistant cell line BEL/5-FU (all obtained from keygen biotech, Nanjing, China) were grown in RPMI-1640 medium (Gibco, KeyGen Biotech, Nanjing, China) containing 10% fetal calf serum (Sijiqing, Zhejiang Tianhang, China), 100 U/ml penicillin and 100 mg/ml streptomycin at 37 °C under a humidified atmosphere of 5% CO2. The BEL/5-FU cells were cultured in the media mentioned above that additionally contained 20 µg/ml 5-FU until at least two weeks before beginning experimentation to maintain its MDR phenotype.
Table 2 Enhancement of quercetin on the cytotoxicity of chemotherapeutic drugs in BEL/5-FU cells. RF represents the reversal effect of modulator, the greater the RF magnitude, the more significant the effect. Reversal resistance of 5-FU
Quercetin (0 µM) Quercetin (40 µM) Quercetin (80 µM) Quercetin (160 µM) Verapamil (5 µM) MK571 (5 µM)
Reversal resistance of MMC
Reversal resistance of ADR
IC50(μg/ml) of 5-FU
RF
IC50(μg/ml) of MMC
RF
IC50(μg/ml) of ADR
RF
69.65 42.49 34.28 20.42 22.94 23.82
1.63 2.03 3.41 3.03 2.92
4.15 3.04 2.63 1.65 1.97 2.04
1.36 1.58 2.51 2.11 2.03
8.88 6.67 5.00 3.18 3.46 3.65
1.33 1.78 2.79 2.57 2.43
± ± ± ± ± ±
1.23 0.72 1.69 1.52 0.68 1.57
38
± ± ± ± ± ±
0.30 0.12 0.20 0.28 0.06 0.24
± ± ± ± ± ±
1.18 0.49 0.21 0.35 0.42 0.34
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Fig.2. The mRNA expressions of ABCB1, ABCC1 and ABCC2 in BEL/5-FU cells. Cells were treated with various concentrations of quercetin for 12 h, 24 h and 48 h. The mRNA levels of ABCB1 (A), ABCC1 (B) and ABCC2 (C), were analyzed by qRT-PCR. Data are shown as the mean ± SD (n = 3). *P < 0.05, ⁎⁎P < 0.01, versus control.
Fig.3. The intracellular accumulation of Rh123 and ADR by flow cytometry. The fluorescence intensity indicated the intracellular concentration of Rh123 and ADR. (A) Representative FACS histograms of Rh123 accumulation and intensity of Rh123 fluorescence in BEL-7402 and BEL/5-FU cells. (B) Representative FACS histograms of ADR accumulation and intensity of ADR fluorescence in BEL-7402 and BEL/5-FU cells. The assays were performed at least three times with similar results. Data are shown as the mean ± SD (n = 3). ⁎⁎P < 0.01, versus BEL-7402 cells. #P < 0.05, ##P < 0.01, versus BEL/5-FU control cells.
Fig. 1). To ensure reversion effect of quercetin in MDR, the BEL/5-FU and parent BEL-7402 cells were plated in 96-well plates. When the cells allowed to adhere overnight, different conventional chemotherapeutic drugs including 5-FU, MMC and ADR in various concentrations (0.2, 1, 5, 25, 125 µg/ml) with or without a fixed combination of quercetin were added to the wells. Positive control of 5 µM VRP or MK571 was used in parallel. After 48 h, cell viability was measured by an MTT [3(4,5-dimethylthiazol-2-yl)-2,5-diphenylte -trazolium bromide] (Sigma, USA) assay. The concentration at which each drug produced the 50% inhibition concentration (IC50) was estimated by the relative survival curve. Resistance index (RI) of BEL/5-FU cells to anticancer drugs was calculated by dividing IC50 for the MDR cells by that for parental sensitive cells. BEL/5-FU cells were transfected with the siFZD7, GV144FZD7 or corresponding control. At the end of transfection, the cells were plated in 96-well plates and incubated at 37 °C in a humidified 5% CO2 atmosphere for 16 h. Then anticancer drugs in various concentrations were added and conducted as above. Independent experiments were performed at least three times.
Cell transfection The BEL/5-FU cells in exponential phase of growth were plated in plates at 2 × 105 cells/ml and cultured for 16 h. The FZD7 interfering small RNA (siFZD7) and a negative scrambled siRNA (sncRNA) (Generay, Shanghai, China) were transfected into cells with Lipofectamine 2000 (Invitrogen, USA) based on our previous study (Chen et al., 2013). On the other hand, the cells were infected with the lentivirus-FZD7 vector (GV144-FZD7) and the empty GV144 vector (NC-GV144) (Generay, Shanghai, China) likewise according to the manufacturer's protocol. The effects were examined in triplicate at 24 h post-transfection by qRT-PCR or at 48 h post-transfection by western blot analysis.
Drug sensitivity assay The in vitro cytotoxicity of quercetin was measured by MTT assay. Concentrations of quercetin with more than 80% of cell survival (40, 80, 160 µM) were utilized in subsequent experiments (Supplementary 39
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(caption on next page)
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Fig.4. Involvement effect of FZD7/β-catenin on the MDR in the BEL/5-FU cells. (A) Representative photos of BEL/5-FU cells transfected with siFZD7 or GV144-FZD7. Light microscopy (left, 200×); fluorescent microscopy (right, 200×). (B) BEL/5-FU cells transfected with siFZD7, GV144-FZD7 or respective negative control. The FZD7, β-catenin, ABCB1, ABCC1 and ABCC2 mRNA expressions were examined by qRT-PCR. (C) The FZD7, nuclear β-catenin, cytoplasmic β-catenin, ABCB1, ABCC1 and ABCC2 protein expressions were examined by western blot analysis. (D) The FZD7, β-catenin, ABCB1, ABCC1 and ABCC2 mRNA and protein expressions were detected by qRT-PCR and western blot analysis after treated with 25 µM iCRT-3 in BEL/5-FU cells. (E) The sensitivities of cells to different doses of anticancer drugs (5-FU, MMC and ADR) after transfection with siFZD7, GV144-FZD7 or respective control. (F) Representative FACS histograms of Rh123 accumulation and intensity of Rh123 fluorescence in BEL-7402 and BEL/5-FU cells. (H) Representative FACS histograms of ADR accumulation and intensity of ADR fluorescence in BEL-7402 and BEL/5-FU cells. The assays were performed at least three times with similar results. Data are shown as the mean ± SD (n = 3). *P < 0.05, ⁎⁎P < 0.01, versus control. # P < 0.05, ##P < 0.01, versus corresponding-transfected control.
Accumulation of Rh123 by flow cytometry has been previously described (Shi et al., 2008). Briefly, the BEL/5-FU cells were cultured for 48 h in the absence or presence of quercetin, and then cells were collected and incubated with Rh123 at a final concentration of 5 µg/ml at 37 °C, 5% CO2 for 30 min. Positive control of 5 µM verapamil (VRP), a typical inhibitor of ABCB1, was used in parallel (Shen et al., 2015). On the other hand, BEL/5-FU cells were transfected with siFZD7 or sncRNA for 48 h, and then incubated with Rh123 as described above. After incubation, the cells were washed three times with ice-cold PBS and resuspended in 500 µl PBS. Thus, the mean fluorescence intensity of intracellular Rh123 was determined by a BD LSR flow cytometer (BD Biosciences) with excitation/emission wavelengths of 488 nm/525 nm.
sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred onto a PVDF membrane (Millipore Corp, Billerica, USA). Membranes were then blocked in TBST containing 5% skim milk and incubated with the following antibodies: anti-ABCB1 (Millipore, Merck KGaA, Germany), anti-ABCC1 (Abcam, Cambridge, MA), anti-ABCC2 (Abcam, Cambridge, MA), anti-FZD7 (Santa Cruz Biotechnology Inc., Santa Cruz, CA), anti-β-catenin (Cell Signaling Technology Inc., USA), anti-β-actin (Santa Cruz Biotechnology Inc., Santa Cruz, CA) as a total protein internal control, anti-Lamin A (Santa Cruz Biotechnology Inc., Santa Cruz, CA) as a nuclear protein internal control. Horseradish peroxidase conjugated anti-mouse, anti-goat, anti-rabbit antibodies were used as secondary antibodies correspondingly. The bands were visualized by chemiluminescence reagents (Model No.ChemiQ 4600, Bioshine).
Intracellular accumulation of doxorubicin (ADR)
Statistical analysis
Studies were carried out in BEL-7402/5-FU cells as described by Shi et al. (2008). Simply, BEL-7402/5-FU cells were treated as the intracellular Rh123 accumulation assay described above. ADR was added to the cells to a final concentration of 8 µM at 37 °C, 5% CO2 for 2 h. Positive control of 5 µM MK571, an inhibitor of ABCC1/2, was used in parallel (Roundhill and Burchill, 2012; Shen et al., 2015). The cells then were harvested and washed three times with ice-cold PBS. The mean fluorescence intensity of intracellular ADR was detected using flow cytometry with excitation/emission wavelengths of 480 nm/575 nm.
Data are presented as mean ± SD. Comparisons between the groups were performed by using Student's t test when only two groups were compared and one-way analysis of variance (ANOVA) when more than two groups were compared. Values of P < 0.05 indicate statistical significance.
Intracellular accumulation of rhodamine-123 (Rh123)
Results Aberrant expressions of FZD7, ABCB1, ABCC1 and ABCC2 in BEL-7402 and BEL/5-FU cells
Real-time reverse-transcription-PCR analysis At the beginning of this project, we evaluated the FZD7, ABCB1, ABCC1 and ABCC2 expressions in BEL-7402 and BEL/5-FU cell lines using qRT-PCR and western blot, respectively. The relative expression levels of FZD7, ABCB1, ABCC1 and ABCC2 were strongly ascended in the 5-FU-resistant BEL/5-FU cells compared to the parental BEL-7402 cells (P < 0.05, Fig. 1A and B).
The cells were incubated with quercetin for 12 h, 24 h and 48 h. Moreover, after transfected with siFZD7, GV144-FZD7 and respective negative control, the cells were then incubated for 48 h in the absence or presence of quercetin. In the meanwhile, cells were cultured for 16 h and subsequently treated with an antagonist of β-catenin (iCRT-3, 25 µM, Merck KGaA, Germany) for 48 h (Narayanan et al., 2012). Total RNA was extracted from the cultured cells using the TRIzol reagents (Invitrogen, USA), and the first strand cDNA synthesis was performed using a ThermoScript RT-PCR synthesis kit (Fermentas, USA) according to the manufacturer's protocol. Quantitative real-time PCR analyses for mRNA of ABCB1, ABCC1, ABCC2, FZD7, β-catenin and β-actin were performed with a SYBR Green (QIAGEN, Germany) using quantitative real-time PCR detection system (Themal 5100). All amplifications were normalized by β-actin. Data were analyzed using the 2−ΔΔct method and expressed as fold change relative to the respective control. Each sample was performed from three different experiments. The sequences of the synthetic primers are shown in Supplementary Table 1.
Reversal effect of quercetin on multidrug resistance in MDR cells To demonstrate the functions of quercetin on MDR, the resistance of MDR cells and the parental cells to different concentrations of several clinically relevant chemotherapeutic drugs were evaluated using MTT assay. We observed a 17.08-fold increase in resistance to 5-FU, a 2.01fold increase in resistance to MMC and a 2.21-fold increase in resistance to ADR for BEL/5-FU cells compared with BEL-7402 cells (Table 1). Therefore, the BEL/5-FU cells considered to be a multidrug resistance human cancer cell line, which was suitable for the study of MDR modulation. The ability of quercetin to reverse the resistance of BEL/5FU cells to anticancer drugs was shown in Table 2. The reversal effect of the modulator was evaluated by reversal fold (RF), which was scored as follows: RF > 1 indicates enhanced drug sensitivity in the presence of modulator, RF = 1 indicates no effect and RF < 1 indicates decreased drug sensitivity; the higher the RF magnitude, the more significant the effect (Shi et al., 2008). Quercetin enhanced the chemosensitivity of BEL/5-FU cells to 5-FU by 1.63-, 2.03-, 3.41-fold at the concentrations of 40, 80, 160 µM. The cross-resistance to other anticancer drugs (MMC, ADR) was also compared, and the sensitivity of BEL/5-FU cells was improved by 1.36–2.51-fold and 1.33–2.79-fold, respectively.
Western blot analysis After treatment as we previously described, cells were harvested and suspended in RIPA lysis buffer (Beyotime, China) and quantified with a Bicin Choninic Acid (BCA) protein assay kit (Boster, Wuhan, China). Nuclear and cytoplasmic extracts were prepared using a nuclear and cytoplasmic protein extraction kit (Bestbio, China) according to the manufacturer's recommendation. After boiled in sample buffer, total proteins were separated by denaturing 8% or 10% sodium dodecyl 41
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Fig.5. Quercetin reverses the drug-resistance via activating the FZD7/β-catenin pathway in hepatocellular carcinoma cells. (A) BEL/5-FU cells were treated with different concentrations of quercetin. The FZD7, β-catenin, ABCB1, ABCC1 and ABCC2 mRNA expressions were examined by qRT-PCR. (B) The FZD7, nuclear βcatenin, cytoplasmic β-catenin, ABCB1, ABCC1 and ABCC2 protein expressions were examined by western blot analysis. (C) BEL-7402 cells were treated with 160 µM quercetin; BEL/5-FU cells were treated with 160 µM quercetin with or without the transfection of siFZD7 or a negative control. The FZD7, β-catenin, ABCB1, ABCC1 and ABCC2 mRNA expressions were examined by qRT-PCR. (D) The FZD7, nuclear β-catenin, cytoplasmic β-catenin, ABCB1, ABCC1 and ABCC2 protein expressions were examined by western blot analysis. The assays were performed at least three times with similar results. Data are shown as the mean ± SD (n = 3). *P < 0.05, ⁎⁎ P < 0.01, versus control. #P < 0.05, versus Q + sncRNA.
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(P < 0.05, Fig. 4E). Besides, the IC50 values of 5-FU, MMC and ADR in BEL/5-FU cells transfected with GV144-FZD7 or NC-GV144 were increased by 1.87-, 1.76-, 1.54-fold (P < 0.05, Fig. 4E). More in more, Fig. 4F and H suggested that the accumulation of Rh123 and ADR in siFZD7 transfected BEL/5-FU cells were all significantly increased by 2.63-fold and 2.25-fold over sncRNA transfected BEL/5-FU cells. Collectively, our data confirmed the hypothesis that upregulation of FZD7/ β-catenin could decrease the MDR in BEL/5-FU cells.
Additionally, the positive control VRP and MK571 also showed the reversal ability. Our observations indicate that quercetin could increase the 5-FU, MMC and ADR sensitivity on drug-resistant BEL/5-FU cells. In order to uncover the effect of quercetin on the MDR, the mRNA expressions of ABCB1, ABCC1 and ABCC2 were detected by qRT-PCR after treatment with quercetin at a different time point in the BEL/5-FU cells. Our results showed that ABCB1, ABCC1 and ABCC2 mRNA expression were clearly decreased by quercetin in the dose- and timedependent manner. Notably, compared with the control cells, the relative mRNA expression of ABCB1 was lower (32.5%, 58.0% and 68.7% decrease) by incubation with quercetin at 48 h (P < 0.05, Fig. 2A). Besides, the ABCC1 and ABCC2 mRNA expression levels were declined entirely by almost 30.4%∼67.4% in the drug treatment group than that in control at 48 h (P < 0.05, Fig. 2B and C). Taken together, these data strongly suggest that quercetin has an effect on the multidrug resistance in MDR cells.
Quercetin modulates multidrug resistance by inhibition of FZD7/β-catenin pathway
Rh123, known as a typical ABCB1 substrate, was conducted to investigate the ABCB1 inhibition effect of quercetin in BEL/5-FU cells. Apparently, the activity of the ABCB1 drug pump was evaluated by the degree of Rh123 intracellular accumulation, which can in turn be determined by the measurement of intracellular fluorescence using flow cytometry (Clark et al., 1996; Shi et al., 2008). As described in Fig. 3A, compared with sensitive BEL-7402 cells, lesser accumulation of Rh123 was distinctly observed in resistant BEL/5-FU cells (P < 0.01). After treatment with quercetin (40, 80 and 160 µM), Rh123 accumulation in BEL/5-FU cells was strongly significant increased 1.68-, 2.59-, 3.07-fold compared to control group cell levels (P < 0.05). Furthermore, the effect of 5 µM VRP, positive control, was similar to 160 µM quercetin (P < 0.01). On the other hand, the inhibition of MRP2 function by the quercetin was assessed in BEL/5-FU cells using ADR uptake assay. The fluorescence characteristics of ADR were utilized to determine ADR intracellular concentration by flow cytometry. The results exhibited that an apparent increase in ADR accumulation was noted following treatment with either MK571 (positive control) or quercetin (40, 80, 160 µM) (P < 0.05, Fig. 3B).
It was found that quercetin treatment significantly decreased the ABCB1, ABCC1 and ABCC2 mRNA expressions and activities in BEL/5FU cells. We further investigate whether FZD7/β-catenin pathway played a role in quercetin-induced suppression of ABCB1, ABCC1 and ABCC2. The results were shown in Fig. 5A and B; quercetin treatment yielded a significant and dose-dependent manner decrease in FZD7, nuclear and cytoplasmic β-catenin, ABCB1, ABCC1 and ABCC2 expressions with the control group (P < 0.05). It needs to be emphasized that quercetin at the concentrations of 40, 80 and 160 µM overtly suppress the FZD7 protein expression by almost 33.6%, 46.5% and 58.1%, respectively (P < 0.05, Fig. 5B). Simultaneously, as described in Fig. 5C and D, the results were consistent with the previous findings after treatment with 160 µM quercetin in BEL/5-FU cells. More interesting, when BEL/5-FU cells transfected with siFZD7 and then cotreated with 160 µM quercetin, we found that compared to its corresponding control-transfected cells (Q+sncRNA), the protein expressions of FZD7, nuclear β-catenin, cytoplasmic β-catenin, ABCB1, ABCC1 and ABCC2 were apparently decreased by 38.0%, 31.7%, 30.8%, 36.1%, 23.0% and 27.5% (Fig. 5D), similar to the mRNA levels (Fig. 5C). Consistently, the expressions of FZD7, nuclear and cytoplasmic β-catenin, ABCB1, ABCC1 and ABCC2 were significantly downregulated in BEL-7402 cells treatment with 160 µM quercetin as compared with the control, it was decreased by over 30% (P < 0.05, Fig. 5C and D). Remarkably, we found evidence that overcoming multidrug resistance effect by quercetin, at least in part, was associated with the suppression of FZD7/β-catenin pathway.
FZD7/β-catenin is involved in multidrug resistance
Discussion
To evaluate the effect of the FZD7/β-catenin pathway on MDR, the FZD7 siRNA (siFZD7), lentivirus-FZD7 vector (GV144-FZD7) or the corresponding negative control was transfected into the BEL/FU cells (Fig. 4A). As shown in Fig. 4B and C, knockdown of FZD7 by transferring siFZD7 markedly decreased the mRNA and protein expressions of FZD7 by over 72%, and led to decrease the expressions of the βcatenin mRNA level (P < 0.05) and the nuclear and cytoplasmic β-catenin protein levels (P < 0.05) sharply. Moreover, the mRNA and protein expression levels of ABCB1, ABCC1 and ABCC2 were much downregulated by approximately 54.8%∼67.1% (P < 0.05), compared controls. Consistently, overexpression of FZD7 by GV144-FZD7 transfection dramatically induced the FZD7 expression (2.06-fold, P < 0.05), increased the expressions of the β-catenin mRNA level (2.07-fold, P < 0.05) and the nuclear and cytoplasmic β-catenin protein levels (1.99-, 1.86-fold, P < 0.05), thus up-regulated the mRNA and protein expressions of ABCB1, ABCC1 and ABCC2 by roughly 2-fold. As expected, compared with control group, the ABCB1, ABCC1 and ABCC2 expressions were much lower after adding the inhibitor of β-catenin (iCRT-3) (P < 0.05, Fig. 4D). To further assess the functions of FZD7 on chemoresistance, we determined the sensitivity of BEL/5-FU cells to chemotherapeutic drugs after transferring by MTT assay. Briefly, it revealed that BEL/5-FU cells transfected with siFZD7 enhanced the sensitivity to 5-FU, MMC and ADR greatly by comparison to those transfected with sncRNA, as indicated by distinctly decreased IC50 values by 68.7%, 42.8% and 45.3%
MDR is accepted as a common critical limitation for the treatment of liver cancer in chemotherapy. ABC transporters serve as primary regulators in the distribution and elimination of chemotherapeutic agents, as well as intrinsic and acquired drug resistance of liver cancers. Overexpression of these transporters in HCC is one of the most important mechanisms of MDR, which expedites a broad range of anticancer drugs efflux out of tumor cells (Wang et al., 2017). Thus, targeting ABC transporters provides a promising strategy to suppress or eliminate MDR in cancer treatment. To date, enormous efforts have been devoted to the development of ABC transporter inhibitors. Three generation of ABC transporter inhibitors has been developed to circumvent anticancer MDR. Unfortunately, there has been a limited success due to the inherited side effects or unexpected pharmacokinetic interactions, which restricted the clinical application of these ABC transporter inhibitors (Bugde et al., 2017). Contemporarily, an increasing number of Chinese herbal medicines such as flavonoids, coumarins, terpenoids, and alkaloids were indicated to be an efficient way in MDR reversal, also according to their low cost and lower toxicity (Chen et al., 2016). In recent years, quercetin, a flavonoid compound, was reported to interact with ABC transporters in the development of MDR in HCC. Nevertheless, the function and molecular mechanisms of regulation of ABC transporters including ABCB1, ABCC1 and ABCC2 in the MDR have not been well elucidated. Some studies demonstrate that quercetin is a MDR modulator and thus a potential chemosensitizer in a plenty variety of cancers. It is
Quercetin inhibits the Rh123 and ADR efflux from BEL/5-FU cells
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and cytoplasmic β-catenin, ABCB1, ABCC1 and ABCC2 were declined compared to the control group (Q+sncRNA). Contemporary, quercetin could inhibit the FZD7, nuclear and cytoplasmic β-catenin, ABCB1, ABCC1 and ABCC2 expressions in the FZD7-lower expressed BEL-7402 cells. Therefore, we have presented that the ability of quercetin to reverse multidrug resistance may result from reduced ABCB1 and ABCC1/ 2 expressions via suppressing FZD7/β-catenin signaling pathway. Collectively, our study provides the first in vitro evidence that quercetin could enhance the efficacy of conventional chemotherapeutic agents in ABCB1, ABCC1 and ABCC2-overexpressing multidrug resistant BEL/5-FU cells, at least in part, by blocking the FZD7/β-catenin signaling pathway. Thus, quercetin may be used as an ABCB1 or ABCC1/2-mediated MDR reversal agent and is promising in the reversal of MDR for the clinic.
reported that all the hydrogen and hydrophobic interactions of quercetin with the amino acid residues present in the drug-binding pocket of P-gp/ABCB1 (Mohana et al., 2016). The same situation exhibited that quercetin interacted with MRP1/ABCC1 at the drug-binding site and mediated inhibition of ABCC1 function. It was also found that quercetin could inhibit the ATPase activity of purified reconstituted ABCC1 and ABCC4, suggesting quercetin is likely to be a substrate of both ABCC1 and ABCC4 (Wu et al., 2005). A study by van et al. showed that quercetin repressed ABCC1-mediated transport of dinitrophenyl glucuronide (DNP-SG) in ABCC1-transfected MDCKII cells to an extent similar to the typical ABCC inhibitor, MK571; whereas, similar inhibition was not obtained for ABCC2-mediated DNP-SG transport in ABCC2-transfected MDCKII. Moreover, quercetin affected ABCC1 but not ABCC2 mediated transport activity in a similar way as observed in the MCF7 cells (van Zanden et al., 2004). Based on the above, we confirmed to determine the potential role of quercetin and ABC transporter-mediated MDR in HCC. In our present study, the expression of ABCB1, ABCC1 and ABCC2 were all significantly high in BEL/5-FU cell line. Treatment with quercetin evidently increased the sensitivity of BEL/5-FU cells to chemotherapeutic drugs. Contemporary, we assessed the impact of quercetin exposure on the efflux function of ABCB1 by Rh123 transport assay (Qiu et al., 2017) and ABCC1/2 by ADR transport assay (Zhang et al., 2014) in the BEL/5-FU cells. Our results found that quercetin treatment distinctly increased the intracellular accumulation of substrate drugs Rh123 and ADR. Also, the mRNA and protein expressions of ABCB1, ABCC1 and ABCC2 were down-regulated after quercetin treatment. Consistent with these findings, these results demonstrate that quercetin is a potential MDR modulator in cancer chemotherapy. Considerable researches showed that Wnt/β-catenin signaling pathway acts essential roles in cell proliferation, differentiation, invasion and metastasis, and activation of the Wnt/β-catenin pathway results in many types of human cancers (Ahmad et al., 2017; Chen et al., 2017; Yang et al., 2017). Meanwhile, the Wnt/β-catenin signaling pathway was significantly linked to the expression of ABC transporters (Huang et al., 2017; Luo et al., 2016; Zhou et al., 2017). As far as we know, the canonical Wnt/β-catenin signaling cascade is commonly triggered through the binding of Wnt ligands to a seven-pass transmembrane Frizzled (FZD) receptor proteins and the lipoprotein receptor-related protein 5/6 (LRP5/6) co-receptor, and this interaction leads to the stabilization of cytosolic β-catenin which translocates into the nucleus, thus controlling key developmental gene expression. The FZD family contains ten members in humans (Li et al., 2017; McDonald and Silver, 2009). FZD7 located on human chromosome 2q33 is one of the most abundant Frizzled family proteins. FZD7 were markedly upregulated in various cancers including breast cancer, hepatocellular carcinoma and colon cancer, thereby activating Wnt signaling, and contributed to cancer malignancy by driving proliferation and invasion in cancer cells (Chakrabarti et al., 2014; Ueno et al., 2009; Wu et al., 2017; Yang et al., 2011). Lately, FZD7 has been reported to promote drug resistance of cancer cells through activation of Wnt signaling (Liu et al., 2017). Our previous study proved that FZD7 silencing promotes the sensitivity of cancer cells to 5-FU which related to downregulation of P-gp expression by blocking Wnt signaling. However, whether quercetin enhances the chemosensitivity of chemotherapeutic drugs by the correlation between ABC transporters expression and FZD7/β-catenin signaling pathway remains unknown. In this research, we further confirmed that knockdown of FZD7 with small interfering RNA significantly enhanced sensitivity to chemotherapeutic drugs, increased the cellular accumulation of ABCB1 substrate Rh123 and ABCC1/2 substrate ADR, and decreased the expression levels of the nuclear and cytoplasmic β-catenin, ABCB1, ABCC1 and ABCC2. Additionally, overexpression of FZD7 showed the inverse effect as expected, emphasizing the importance of FZD7 dysfunction in the development of MDR in HCC cells. What's more, in treatment with quercetin combined with siFZD7, the expression levels of FZD7, nuclear
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Phytomedicine journal homepage: www.elsevier.com/locate/phymed
Reversal effect of quercetin on multidrug resistance via FZD7/β-catenin pathway in hepatocellular carcinoma cells
T
Zhaolin Chena,b, Cheng Huanga, Taotao Maa, Ling Jiangb, Liqin Tangb, Tianlu Shib, ⁎ ⁎ Shantang Zhangb, Lei Zhangb, Pengli Zhub, Jun Lia, , Aizong Shenb, a
Institute for Liver Diseases of Anhui Medical University (AMU), Anhui Province Key Laboratory of Major Autoimmune Diseases, Anhui Institute of Innovative Drugs, School of Pharmacy, Anhui Medical University, Hefei, Anhui 230032, China b Department of Pharmacy, Anhui Provincial Hospital, Anhui Medical University, Hefei, Anhui 230001, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Quercetin Hepatocellular carcinoma Multiple drug resistance ATP-binding cassette transporters, FZD7/βcatenin pathway
Background: Chemotherapy has been widely used to treat cancer, but the appearance of multidrug resistance (MDR) is the biggest obstacle to successful chemotherapy. One of the conventional mechanisms of MDR is overexpression of ATP-binding cassette (ABC) transporters such as P-glycoprotein (P-gp/ABCB1) and multidrug resistance-associated proteins (MRPs/ABCCs) that limits the prolonged and efficient use of chemotherapeutic drugs. To enhance the chemosensitivity of tumor cells, attentions have been focused on effective MDR modulators. Purpose: This study aimed to investigate the reversal effect of quercetin on MDR, and explored its mechanism of action in vitro. Study design/methods: The effect and mechanism of quercetin on MDR was examined by using MTT assay, flow cytometry, real-time PCR and western blot analysis in human hepatocellular carcinoma cells. Results: Our data found that the intracellular accumulation of rhodamine-123 (Rh123) and doxorubicin (ADR) were increased, the sensitivity of BEL/5-FU cells to chemotherapeutic drugs were increased, and the expressions of ABCB1, ABCC1 and ABCC2 were all down-regulated, which indicated that the functions and expressions of ABCB1, ABCC1 and ABCC2 efflux pump were inhibited by quercetin treatment. Moreover, the suppression of ABCB1, ABCC1 and ABCC2 by quercetin was dependent on the FZD7 through the Wnt/β-catenin pathway. Further research revealed that reduction of FZD7 by RNA interference (siFZD7) enhanced the sensitivity to chemotherapeutic drugs, increased the cellular accumulation of Rh123 and ADR, and induced inhibitory effects on the expression of FZD7, ABCB1, ABCC1, ABCC2 and β-catenin, similar to quercetin. In the meanwhile, overexpression of FZD7 showed the inversely effect on the expressions. Interesting, it was confirmed that quercetin could inhibit the expression levels of FZD7, ABCB1, ABCC1, ABCC2 and β-catenin in BEL-7402 cells; furthermore, treatment by quercetin combined with siFZD7 in BEL/5-FU cells, the expressions of these genes were effectively decreased in comparison to quercetin combined with siRNA negative control (sncRNA). Conclusion: Overall, these data suggested the effectiveness of using quercetin, at least in part, via inhibiting FZD7 to combat chemoresistance and showed that quercetin could be developed into an efficient natural sensitizer for resistant human hepatocellular carcinoma.
Introduction Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related mortality worldwide and also one of the most common
malignant liver tumors in China (Torre et al., 2015). The available front-line treatment for HCC is surgical resection and liver transplantation, but only a minority of the patients are eligible because HCCs typically present at advanced-stage with macrovascular invasion or
Abbreviations: 5-FU, 5-fluorouracil; ABC, ATP-binding cassette; ATP, adenosine triphosphate; ADR, doxorubicin; FZD7, Frizzled homolog protein 7; GV144-FZD7, lentivirus-FZD7 vector; HCC, hepatocellular carcinoma; IC50, 50% inhibition concentration; MDR, multiple drug resistance; MRP1/ABCC1, multidrug resistance-associated protein 1; MRP2/ABCC2, multidrug resistance-associated protein 2; MMC, Mitomycin C; MTT, thiazolyl blue tetrazolium blue; NC-GV144, empty GV144 vector; P-gp/ABCB1, P-glycoprotein; quercetin, Q; qRTPCR, quantitative real-time polymerase chain reaction; Rh123, rhodamine-123; SDS-PAGE, sulphate-polyacrylamide gel electrophoresis; siRNA, small interfering RNA; siFZD7, FZD7 interfering small RNA; sncRNA, siRNA negative control; VRP, verapamil ⁎ Corresponding authors. E-mail addresses: [email protected] (J. Li), [email protected] (A. Shen). https://doi.org/10.1016/j.phymed.2018.03.040 Received 22 September 2017; Received in revised form 26 January 2018; Accepted 18 March 2018 0944-7113/ © 2018 Elsevier GmbH. All rights reserved.
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Fig.1. The relative expressions FZD7, ABCB1, ABCC1 and ABCC2 in BEL-7402 and BEL/5-FU cells. (A) Expressions of FZD7, ABCB1, ABCC1 and ABCC2 were detected by qRT-PCR. (B) Expressions of FZD7, ABCB1, ABCC1 and ABCC2 were detected by western blot. The assays were performed at least three times with similar results. Data are shown as the mean ± SD (n = 3). *P < 0.05, ⁎⁎P < 0.01, versus control.
the KB/VCR oral cancer resistant cell lines (Yuan et al., 2015). Besides, quercetin might target the MAPK/ERK/JNK pathway and transcriptionally down-regulate ABCB1, suggesting that quercetin may reverse MDR by targeting the MAPK/ERK/JNK/ABCB1 pathway in multidrug resistant leukemia K562 cells (Chen et al., 2015). Furthermore, quercetin effectively inhibited tumor growth and enhanced the sensitivity to chemotherapy in a xenograft mouse model of HCC (Dai et al., 2016). Research carried out in the past few decades has shown that quercetin plays a vital role in the development of HCC. However, studies on the function and mechanisms of quercetin on MDR in HCC are still limited. Our previous study firstly revealed that the activation of FZD7/βcatenin pathway plays an important role in the MDR (Chen et al., 2013), but whether or not quercetin reverses MDR by suppressing FZD7/β-catenin pathway in HCC is not yet clear. Therefore, the present study aimed to investigate the effect and the underlying molecular mechanisms of quercetin on human hepatocellular carcinoma MDR BEL-7402/5-fluorouracil (BEL/5-FU) cells.
Table 1 The sensitivity of BEL-7402 and BEL/5-FU cells treated with chemotherapeutic drugs. IC50(μg/ml)
5-FU MMC ADR
BEL/5-FU
BEL-7402
Resistance index
69.648 ± 1.23 4.151 ± 0.30 8.882 ± 1.18
4.076 ± 0.27 2.065 ± 0.25 4.021 ± 0.13
17.08 2.01 2.21
metastases (Ling et al., 2017). Systemic chemotherapy is adopted as an effective nonsurgical therapeutic strategy for most HCCs. However, the emergence of multidrug resistance (MDR) to pharmacologically and structurally distinct class of clinical drugs severely blocks the successful management of HCC (Bruix et al., 2014; Wu et al., 2016). The well recognized mechanism underlying MDR is the significant overexpression of adenosine triphosphate (ATP)-binding cassette (ABC) super-family of transporters, such as P-glycoprotein (P-gp/ABCB1), multidrug resistance-associated protein 1 (MRP1/ABCC1) and multidrug resistance-associated protein 2 (MRP2/ABCC2), et al., which acts as an efflux pump on cell surface (Chen et al., 2016). Hence, the strategy consists in inhibiting the activity, or lowering the expression of the efflux transporter, which is highly warranted to improve the outcomes of HCC chemotherapy. Quercetin, a typical flavonol-type flavonoid compound found ubiquitously distributed in many plants and vegetables, which has been exhibited to possess antioxidative, anti-inflammatory, immunomodulatory, and vasodilating activities (Chen et al., 2010). In recent years, increasing evidence suggests that quercetin can be useful in cancer treatment by inducing cell apoptosis in vitro and in vivo (Hashemzaei et al., 2017; Lee et al., 2015; Nguyen et al., 2017). Quercetin could be suggested as a potential modulator of ABCB1 which inhibits the ABCB1 expressions in a concentration-dependent manner in
Material and methods Cell culture The parental sensitive human hepatocellular carcinoma cell line BEL-7402 and multidrug resistant cell line BEL/5-FU (all obtained from keygen biotech, Nanjing, China) were grown in RPMI-1640 medium (Gibco, KeyGen Biotech, Nanjing, China) containing 10% fetal calf serum (Sijiqing, Zhejiang Tianhang, China), 100 U/ml penicillin and 100 mg/ml streptomycin at 37 °C under a humidified atmosphere of 5% CO2. The BEL/5-FU cells were cultured in the media mentioned above that additionally contained 20 µg/ml 5-FU until at least two weeks before beginning experimentation to maintain its MDR phenotype.
Table 2 Enhancement of quercetin on the cytotoxicity of chemotherapeutic drugs in BEL/5-FU cells. RF represents the reversal effect of modulator, the greater the RF magnitude, the more significant the effect. Reversal resistance of 5-FU
Quercetin (0 µM) Quercetin (40 µM) Quercetin (80 µM) Quercetin (160 µM) Verapamil (5 µM) MK571 (5 µM)
Reversal resistance of MMC
Reversal resistance of ADR
IC50(μg/ml) of 5-FU
RF
IC50(μg/ml) of MMC
RF
IC50(μg/ml) of ADR
RF
69.65 42.49 34.28 20.42 22.94 23.82
1.63 2.03 3.41 3.03 2.92
4.15 3.04 2.63 1.65 1.97 2.04
1.36 1.58 2.51 2.11 2.03
8.88 6.67 5.00 3.18 3.46 3.65
1.33 1.78 2.79 2.57 2.43
± ± ± ± ± ±
1.23 0.72 1.69 1.52 0.68 1.57
38
± ± ± ± ± ±
0.30 0.12 0.20 0.28 0.06 0.24
± ± ± ± ± ±
1.18 0.49 0.21 0.35 0.42 0.34
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Fig.2. The mRNA expressions of ABCB1, ABCC1 and ABCC2 in BEL/5-FU cells. Cells were treated with various concentrations of quercetin for 12 h, 24 h and 48 h. The mRNA levels of ABCB1 (A), ABCC1 (B) and ABCC2 (C), were analyzed by qRT-PCR. Data are shown as the mean ± SD (n = 3). *P < 0.05, ⁎⁎P < 0.01, versus control.
Fig.3. The intracellular accumulation of Rh123 and ADR by flow cytometry. The fluorescence intensity indicated the intracellular concentration of Rh123 and ADR. (A) Representative FACS histograms of Rh123 accumulation and intensity of Rh123 fluorescence in BEL-7402 and BEL/5-FU cells. (B) Representative FACS histograms of ADR accumulation and intensity of ADR fluorescence in BEL-7402 and BEL/5-FU cells. The assays were performed at least three times with similar results. Data are shown as the mean ± SD (n = 3). ⁎⁎P < 0.01, versus BEL-7402 cells. #P < 0.05, ##P < 0.01, versus BEL/5-FU control cells.
Fig. 1). To ensure reversion effect of quercetin in MDR, the BEL/5-FU and parent BEL-7402 cells were plated in 96-well plates. When the cells allowed to adhere overnight, different conventional chemotherapeutic drugs including 5-FU, MMC and ADR in various concentrations (0.2, 1, 5, 25, 125 µg/ml) with or without a fixed combination of quercetin were added to the wells. Positive control of 5 µM VRP or MK571 was used in parallel. After 48 h, cell viability was measured by an MTT [3(4,5-dimethylthiazol-2-yl)-2,5-diphenylte -trazolium bromide] (Sigma, USA) assay. The concentration at which each drug produced the 50% inhibition concentration (IC50) was estimated by the relative survival curve. Resistance index (RI) of BEL/5-FU cells to anticancer drugs was calculated by dividing IC50 for the MDR cells by that for parental sensitive cells. BEL/5-FU cells were transfected with the siFZD7, GV144FZD7 or corresponding control. At the end of transfection, the cells were plated in 96-well plates and incubated at 37 °C in a humidified 5% CO2 atmosphere for 16 h. Then anticancer drugs in various concentrations were added and conducted as above. Independent experiments were performed at least three times.
Cell transfection The BEL/5-FU cells in exponential phase of growth were plated in plates at 2 × 105 cells/ml and cultured for 16 h. The FZD7 interfering small RNA (siFZD7) and a negative scrambled siRNA (sncRNA) (Generay, Shanghai, China) were transfected into cells with Lipofectamine 2000 (Invitrogen, USA) based on our previous study (Chen et al., 2013). On the other hand, the cells were infected with the lentivirus-FZD7 vector (GV144-FZD7) and the empty GV144 vector (NC-GV144) (Generay, Shanghai, China) likewise according to the manufacturer's protocol. The effects were examined in triplicate at 24 h post-transfection by qRT-PCR or at 48 h post-transfection by western blot analysis.
Drug sensitivity assay The in vitro cytotoxicity of quercetin was measured by MTT assay. Concentrations of quercetin with more than 80% of cell survival (40, 80, 160 µM) were utilized in subsequent experiments (Supplementary 39
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Fig.4. Involvement effect of FZD7/β-catenin on the MDR in the BEL/5-FU cells. (A) Representative photos of BEL/5-FU cells transfected with siFZD7 or GV144-FZD7. Light microscopy (left, 200×); fluorescent microscopy (right, 200×). (B) BEL/5-FU cells transfected with siFZD7, GV144-FZD7 or respective negative control. The FZD7, β-catenin, ABCB1, ABCC1 and ABCC2 mRNA expressions were examined by qRT-PCR. (C) The FZD7, nuclear β-catenin, cytoplasmic β-catenin, ABCB1, ABCC1 and ABCC2 protein expressions were examined by western blot analysis. (D) The FZD7, β-catenin, ABCB1, ABCC1 and ABCC2 mRNA and protein expressions were detected by qRT-PCR and western blot analysis after treated with 25 µM iCRT-3 in BEL/5-FU cells. (E) The sensitivities of cells to different doses of anticancer drugs (5-FU, MMC and ADR) after transfection with siFZD7, GV144-FZD7 or respective control. (F) Representative FACS histograms of Rh123 accumulation and intensity of Rh123 fluorescence in BEL-7402 and BEL/5-FU cells. (H) Representative FACS histograms of ADR accumulation and intensity of ADR fluorescence in BEL-7402 and BEL/5-FU cells. The assays were performed at least three times with similar results. Data are shown as the mean ± SD (n = 3). *P < 0.05, ⁎⁎P < 0.01, versus control. # P < 0.05, ##P < 0.01, versus corresponding-transfected control.
Accumulation of Rh123 by flow cytometry has been previously described (Shi et al., 2008). Briefly, the BEL/5-FU cells were cultured for 48 h in the absence or presence of quercetin, and then cells were collected and incubated with Rh123 at a final concentration of 5 µg/ml at 37 °C, 5% CO2 for 30 min. Positive control of 5 µM verapamil (VRP), a typical inhibitor of ABCB1, was used in parallel (Shen et al., 2015). On the other hand, BEL/5-FU cells were transfected with siFZD7 or sncRNA for 48 h, and then incubated with Rh123 as described above. After incubation, the cells were washed three times with ice-cold PBS and resuspended in 500 µl PBS. Thus, the mean fluorescence intensity of intracellular Rh123 was determined by a BD LSR flow cytometer (BD Biosciences) with excitation/emission wavelengths of 488 nm/525 nm.
sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred onto a PVDF membrane (Millipore Corp, Billerica, USA). Membranes were then blocked in TBST containing 5% skim milk and incubated with the following antibodies: anti-ABCB1 (Millipore, Merck KGaA, Germany), anti-ABCC1 (Abcam, Cambridge, MA), anti-ABCC2 (Abcam, Cambridge, MA), anti-FZD7 (Santa Cruz Biotechnology Inc., Santa Cruz, CA), anti-β-catenin (Cell Signaling Technology Inc., USA), anti-β-actin (Santa Cruz Biotechnology Inc., Santa Cruz, CA) as a total protein internal control, anti-Lamin A (Santa Cruz Biotechnology Inc., Santa Cruz, CA) as a nuclear protein internal control. Horseradish peroxidase conjugated anti-mouse, anti-goat, anti-rabbit antibodies were used as secondary antibodies correspondingly. The bands were visualized by chemiluminescence reagents (Model No.ChemiQ 4600, Bioshine).
Intracellular accumulation of doxorubicin (ADR)
Statistical analysis
Studies were carried out in BEL-7402/5-FU cells as described by Shi et al. (2008). Simply, BEL-7402/5-FU cells were treated as the intracellular Rh123 accumulation assay described above. ADR was added to the cells to a final concentration of 8 µM at 37 °C, 5% CO2 for 2 h. Positive control of 5 µM MK571, an inhibitor of ABCC1/2, was used in parallel (Roundhill and Burchill, 2012; Shen et al., 2015). The cells then were harvested and washed three times with ice-cold PBS. The mean fluorescence intensity of intracellular ADR was detected using flow cytometry with excitation/emission wavelengths of 480 nm/575 nm.
Data are presented as mean ± SD. Comparisons between the groups were performed by using Student's t test when only two groups were compared and one-way analysis of variance (ANOVA) when more than two groups were compared. Values of P < 0.05 indicate statistical significance.
Intracellular accumulation of rhodamine-123 (Rh123)
Results Aberrant expressions of FZD7, ABCB1, ABCC1 and ABCC2 in BEL-7402 and BEL/5-FU cells
Real-time reverse-transcription-PCR analysis At the beginning of this project, we evaluated the FZD7, ABCB1, ABCC1 and ABCC2 expressions in BEL-7402 and BEL/5-FU cell lines using qRT-PCR and western blot, respectively. The relative expression levels of FZD7, ABCB1, ABCC1 and ABCC2 were strongly ascended in the 5-FU-resistant BEL/5-FU cells compared to the parental BEL-7402 cells (P < 0.05, Fig. 1A and B).
The cells were incubated with quercetin for 12 h, 24 h and 48 h. Moreover, after transfected with siFZD7, GV144-FZD7 and respective negative control, the cells were then incubated for 48 h in the absence or presence of quercetin. In the meanwhile, cells were cultured for 16 h and subsequently treated with an antagonist of β-catenin (iCRT-3, 25 µM, Merck KGaA, Germany) for 48 h (Narayanan et al., 2012). Total RNA was extracted from the cultured cells using the TRIzol reagents (Invitrogen, USA), and the first strand cDNA synthesis was performed using a ThermoScript RT-PCR synthesis kit (Fermentas, USA) according to the manufacturer's protocol. Quantitative real-time PCR analyses for mRNA of ABCB1, ABCC1, ABCC2, FZD7, β-catenin and β-actin were performed with a SYBR Green (QIAGEN, Germany) using quantitative real-time PCR detection system (Themal 5100). All amplifications were normalized by β-actin. Data were analyzed using the 2−ΔΔct method and expressed as fold change relative to the respective control. Each sample was performed from three different experiments. The sequences of the synthetic primers are shown in Supplementary Table 1.
Reversal effect of quercetin on multidrug resistance in MDR cells To demonstrate the functions of quercetin on MDR, the resistance of MDR cells and the parental cells to different concentrations of several clinically relevant chemotherapeutic drugs were evaluated using MTT assay. We observed a 17.08-fold increase in resistance to 5-FU, a 2.01fold increase in resistance to MMC and a 2.21-fold increase in resistance to ADR for BEL/5-FU cells compared with BEL-7402 cells (Table 1). Therefore, the BEL/5-FU cells considered to be a multidrug resistance human cancer cell line, which was suitable for the study of MDR modulation. The ability of quercetin to reverse the resistance of BEL/5FU cells to anticancer drugs was shown in Table 2. The reversal effect of the modulator was evaluated by reversal fold (RF), which was scored as follows: RF > 1 indicates enhanced drug sensitivity in the presence of modulator, RF = 1 indicates no effect and RF < 1 indicates decreased drug sensitivity; the higher the RF magnitude, the more significant the effect (Shi et al., 2008). Quercetin enhanced the chemosensitivity of BEL/5-FU cells to 5-FU by 1.63-, 2.03-, 3.41-fold at the concentrations of 40, 80, 160 µM. The cross-resistance to other anticancer drugs (MMC, ADR) was also compared, and the sensitivity of BEL/5-FU cells was improved by 1.36–2.51-fold and 1.33–2.79-fold, respectively.
Western blot analysis After treatment as we previously described, cells were harvested and suspended in RIPA lysis buffer (Beyotime, China) and quantified with a Bicin Choninic Acid (BCA) protein assay kit (Boster, Wuhan, China). Nuclear and cytoplasmic extracts were prepared using a nuclear and cytoplasmic protein extraction kit (Bestbio, China) according to the manufacturer's recommendation. After boiled in sample buffer, total proteins were separated by denaturing 8% or 10% sodium dodecyl 41
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Fig.5. Quercetin reverses the drug-resistance via activating the FZD7/β-catenin pathway in hepatocellular carcinoma cells. (A) BEL/5-FU cells were treated with different concentrations of quercetin. The FZD7, β-catenin, ABCB1, ABCC1 and ABCC2 mRNA expressions were examined by qRT-PCR. (B) The FZD7, nuclear βcatenin, cytoplasmic β-catenin, ABCB1, ABCC1 and ABCC2 protein expressions were examined by western blot analysis. (C) BEL-7402 cells were treated with 160 µM quercetin; BEL/5-FU cells were treated with 160 µM quercetin with or without the transfection of siFZD7 or a negative control. The FZD7, β-catenin, ABCB1, ABCC1 and ABCC2 mRNA expressions were examined by qRT-PCR. (D) The FZD7, nuclear β-catenin, cytoplasmic β-catenin, ABCB1, ABCC1 and ABCC2 protein expressions were examined by western blot analysis. The assays were performed at least three times with similar results. Data are shown as the mean ± SD (n = 3). *P < 0.05, ⁎⁎ P < 0.01, versus control. #P < 0.05, versus Q + sncRNA.
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(P < 0.05, Fig. 4E). Besides, the IC50 values of 5-FU, MMC and ADR in BEL/5-FU cells transfected with GV144-FZD7 or NC-GV144 were increased by 1.87-, 1.76-, 1.54-fold (P < 0.05, Fig. 4E). More in more, Fig. 4F and H suggested that the accumulation of Rh123 and ADR in siFZD7 transfected BEL/5-FU cells were all significantly increased by 2.63-fold and 2.25-fold over sncRNA transfected BEL/5-FU cells. Collectively, our data confirmed the hypothesis that upregulation of FZD7/ β-catenin could decrease the MDR in BEL/5-FU cells.
Additionally, the positive control VRP and MK571 also showed the reversal ability. Our observations indicate that quercetin could increase the 5-FU, MMC and ADR sensitivity on drug-resistant BEL/5-FU cells. In order to uncover the effect of quercetin on the MDR, the mRNA expressions of ABCB1, ABCC1 and ABCC2 were detected by qRT-PCR after treatment with quercetin at a different time point in the BEL/5-FU cells. Our results showed that ABCB1, ABCC1 and ABCC2 mRNA expression were clearly decreased by quercetin in the dose- and timedependent manner. Notably, compared with the control cells, the relative mRNA expression of ABCB1 was lower (32.5%, 58.0% and 68.7% decrease) by incubation with quercetin at 48 h (P < 0.05, Fig. 2A). Besides, the ABCC1 and ABCC2 mRNA expression levels were declined entirely by almost 30.4%∼67.4% in the drug treatment group than that in control at 48 h (P < 0.05, Fig. 2B and C). Taken together, these data strongly suggest that quercetin has an effect on the multidrug resistance in MDR cells.
Quercetin modulates multidrug resistance by inhibition of FZD7/β-catenin pathway
Rh123, known as a typical ABCB1 substrate, was conducted to investigate the ABCB1 inhibition effect of quercetin in BEL/5-FU cells. Apparently, the activity of the ABCB1 drug pump was evaluated by the degree of Rh123 intracellular accumulation, which can in turn be determined by the measurement of intracellular fluorescence using flow cytometry (Clark et al., 1996; Shi et al., 2008). As described in Fig. 3A, compared with sensitive BEL-7402 cells, lesser accumulation of Rh123 was distinctly observed in resistant BEL/5-FU cells (P < 0.01). After treatment with quercetin (40, 80 and 160 µM), Rh123 accumulation in BEL/5-FU cells was strongly significant increased 1.68-, 2.59-, 3.07-fold compared to control group cell levels (P < 0.05). Furthermore, the effect of 5 µM VRP, positive control, was similar to 160 µM quercetin (P < 0.01). On the other hand, the inhibition of MRP2 function by the quercetin was assessed in BEL/5-FU cells using ADR uptake assay. The fluorescence characteristics of ADR were utilized to determine ADR intracellular concentration by flow cytometry. The results exhibited that an apparent increase in ADR accumulation was noted following treatment with either MK571 (positive control) or quercetin (40, 80, 160 µM) (P < 0.05, Fig. 3B).
It was found that quercetin treatment significantly decreased the ABCB1, ABCC1 and ABCC2 mRNA expressions and activities in BEL/5FU cells. We further investigate whether FZD7/β-catenin pathway played a role in quercetin-induced suppression of ABCB1, ABCC1 and ABCC2. The results were shown in Fig. 5A and B; quercetin treatment yielded a significant and dose-dependent manner decrease in FZD7, nuclear and cytoplasmic β-catenin, ABCB1, ABCC1 and ABCC2 expressions with the control group (P < 0.05). It needs to be emphasized that quercetin at the concentrations of 40, 80 and 160 µM overtly suppress the FZD7 protein expression by almost 33.6%, 46.5% and 58.1%, respectively (P < 0.05, Fig. 5B). Simultaneously, as described in Fig. 5C and D, the results were consistent with the previous findings after treatment with 160 µM quercetin in BEL/5-FU cells. More interesting, when BEL/5-FU cells transfected with siFZD7 and then cotreated with 160 µM quercetin, we found that compared to its corresponding control-transfected cells (Q+sncRNA), the protein expressions of FZD7, nuclear β-catenin, cytoplasmic β-catenin, ABCB1, ABCC1 and ABCC2 were apparently decreased by 38.0%, 31.7%, 30.8%, 36.1%, 23.0% and 27.5% (Fig. 5D), similar to the mRNA levels (Fig. 5C). Consistently, the expressions of FZD7, nuclear and cytoplasmic β-catenin, ABCB1, ABCC1 and ABCC2 were significantly downregulated in BEL-7402 cells treatment with 160 µM quercetin as compared with the control, it was decreased by over 30% (P < 0.05, Fig. 5C and D). Remarkably, we found evidence that overcoming multidrug resistance effect by quercetin, at least in part, was associated with the suppression of FZD7/β-catenin pathway.
FZD7/β-catenin is involved in multidrug resistance
Discussion
To evaluate the effect of the FZD7/β-catenin pathway on MDR, the FZD7 siRNA (siFZD7), lentivirus-FZD7 vector (GV144-FZD7) or the corresponding negative control was transfected into the BEL/FU cells (Fig. 4A). As shown in Fig. 4B and C, knockdown of FZD7 by transferring siFZD7 markedly decreased the mRNA and protein expressions of FZD7 by over 72%, and led to decrease the expressions of the βcatenin mRNA level (P < 0.05) and the nuclear and cytoplasmic β-catenin protein levels (P < 0.05) sharply. Moreover, the mRNA and protein expression levels of ABCB1, ABCC1 and ABCC2 were much downregulated by approximately 54.8%∼67.1% (P < 0.05), compared controls. Consistently, overexpression of FZD7 by GV144-FZD7 transfection dramatically induced the FZD7 expression (2.06-fold, P < 0.05), increased the expressions of the β-catenin mRNA level (2.07-fold, P < 0.05) and the nuclear and cytoplasmic β-catenin protein levels (1.99-, 1.86-fold, P < 0.05), thus up-regulated the mRNA and protein expressions of ABCB1, ABCC1 and ABCC2 by roughly 2-fold. As expected, compared with control group, the ABCB1, ABCC1 and ABCC2 expressions were much lower after adding the inhibitor of β-catenin (iCRT-3) (P < 0.05, Fig. 4D). To further assess the functions of FZD7 on chemoresistance, we determined the sensitivity of BEL/5-FU cells to chemotherapeutic drugs after transferring by MTT assay. Briefly, it revealed that BEL/5-FU cells transfected with siFZD7 enhanced the sensitivity to 5-FU, MMC and ADR greatly by comparison to those transfected with sncRNA, as indicated by distinctly decreased IC50 values by 68.7%, 42.8% and 45.3%
MDR is accepted as a common critical limitation for the treatment of liver cancer in chemotherapy. ABC transporters serve as primary regulators in the distribution and elimination of chemotherapeutic agents, as well as intrinsic and acquired drug resistance of liver cancers. Overexpression of these transporters in HCC is one of the most important mechanisms of MDR, which expedites a broad range of anticancer drugs efflux out of tumor cells (Wang et al., 2017). Thus, targeting ABC transporters provides a promising strategy to suppress or eliminate MDR in cancer treatment. To date, enormous efforts have been devoted to the development of ABC transporter inhibitors. Three generation of ABC transporter inhibitors has been developed to circumvent anticancer MDR. Unfortunately, there has been a limited success due to the inherited side effects or unexpected pharmacokinetic interactions, which restricted the clinical application of these ABC transporter inhibitors (Bugde et al., 2017). Contemporarily, an increasing number of Chinese herbal medicines such as flavonoids, coumarins, terpenoids, and alkaloids were indicated to be an efficient way in MDR reversal, also according to their low cost and lower toxicity (Chen et al., 2016). In recent years, quercetin, a flavonoid compound, was reported to interact with ABC transporters in the development of MDR in HCC. Nevertheless, the function and molecular mechanisms of regulation of ABC transporters including ABCB1, ABCC1 and ABCC2 in the MDR have not been well elucidated. Some studies demonstrate that quercetin is a MDR modulator and thus a potential chemosensitizer in a plenty variety of cancers. It is
Quercetin inhibits the Rh123 and ADR efflux from BEL/5-FU cells
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and cytoplasmic β-catenin, ABCB1, ABCC1 and ABCC2 were declined compared to the control group (Q+sncRNA). Contemporary, quercetin could inhibit the FZD7, nuclear and cytoplasmic β-catenin, ABCB1, ABCC1 and ABCC2 expressions in the FZD7-lower expressed BEL-7402 cells. Therefore, we have presented that the ability of quercetin to reverse multidrug resistance may result from reduced ABCB1 and ABCC1/ 2 expressions via suppressing FZD7/β-catenin signaling pathway. Collectively, our study provides the first in vitro evidence that quercetin could enhance the efficacy of conventional chemotherapeutic agents in ABCB1, ABCC1 and ABCC2-overexpressing multidrug resistant BEL/5-FU cells, at least in part, by blocking the FZD7/β-catenin signaling pathway. Thus, quercetin may be used as an ABCB1 or ABCC1/2-mediated MDR reversal agent and is promising in the reversal of MDR for the clinic.
reported that all the hydrogen and hydrophobic interactions of quercetin with the amino acid residues present in the drug-binding pocket of P-gp/ABCB1 (Mohana et al., 2016). The same situation exhibited that quercetin interacted with MRP1/ABCC1 at the drug-binding site and mediated inhibition of ABCC1 function. It was also found that quercetin could inhibit the ATPase activity of purified reconstituted ABCC1 and ABCC4, suggesting quercetin is likely to be a substrate of both ABCC1 and ABCC4 (Wu et al., 2005). A study by van et al. showed that quercetin repressed ABCC1-mediated transport of dinitrophenyl glucuronide (DNP-SG) in ABCC1-transfected MDCKII cells to an extent similar to the typical ABCC inhibitor, MK571; whereas, similar inhibition was not obtained for ABCC2-mediated DNP-SG transport in ABCC2-transfected MDCKII. Moreover, quercetin affected ABCC1 but not ABCC2 mediated transport activity in a similar way as observed in the MCF7 cells (van Zanden et al., 2004). Based on the above, we confirmed to determine the potential role of quercetin and ABC transporter-mediated MDR in HCC. In our present study, the expression of ABCB1, ABCC1 and ABCC2 were all significantly high in BEL/5-FU cell line. Treatment with quercetin evidently increased the sensitivity of BEL/5-FU cells to chemotherapeutic drugs. Contemporary, we assessed the impact of quercetin exposure on the efflux function of ABCB1 by Rh123 transport assay (Qiu et al., 2017) and ABCC1/2 by ADR transport assay (Zhang et al., 2014) in the BEL/5-FU cells. Our results found that quercetin treatment distinctly increased the intracellular accumulation of substrate drugs Rh123 and ADR. Also, the mRNA and protein expressions of ABCB1, ABCC1 and ABCC2 were down-regulated after quercetin treatment. Consistent with these findings, these results demonstrate that quercetin is a potential MDR modulator in cancer chemotherapy. Considerable researches showed that Wnt/β-catenin signaling pathway acts essential roles in cell proliferation, differentiation, invasion and metastasis, and activation of the Wnt/β-catenin pathway results in many types of human cancers (Ahmad et al., 2017; Chen et al., 2017; Yang et al., 2017). Meanwhile, the Wnt/β-catenin signaling pathway was significantly linked to the expression of ABC transporters (Huang et al., 2017; Luo et al., 2016; Zhou et al., 2017). As far as we know, the canonical Wnt/β-catenin signaling cascade is commonly triggered through the binding of Wnt ligands to a seven-pass transmembrane Frizzled (FZD) receptor proteins and the lipoprotein receptor-related protein 5/6 (LRP5/6) co-receptor, and this interaction leads to the stabilization of cytosolic β-catenin which translocates into the nucleus, thus controlling key developmental gene expression. The FZD family contains ten members in humans (Li et al., 2017; McDonald and Silver, 2009). FZD7 located on human chromosome 2q33 is one of the most abundant Frizzled family proteins. FZD7 were markedly upregulated in various cancers including breast cancer, hepatocellular carcinoma and colon cancer, thereby activating Wnt signaling, and contributed to cancer malignancy by driving proliferation and invasion in cancer cells (Chakrabarti et al., 2014; Ueno et al., 2009; Wu et al., 2017; Yang et al., 2011). Lately, FZD7 has been reported to promote drug resistance of cancer cells through activation of Wnt signaling (Liu et al., 2017). Our previous study proved that FZD7 silencing promotes the sensitivity of cancer cells to 5-FU which related to downregulation of P-gp expression by blocking Wnt signaling. However, whether quercetin enhances the chemosensitivity of chemotherapeutic drugs by the correlation between ABC transporters expression and FZD7/β-catenin signaling pathway remains unknown. In this research, we further confirmed that knockdown of FZD7 with small interfering RNA significantly enhanced sensitivity to chemotherapeutic drugs, increased the cellular accumulation of ABCB1 substrate Rh123 and ABCC1/2 substrate ADR, and decreased the expression levels of the nuclear and cytoplasmic β-catenin, ABCB1, ABCC1 and ABCC2. Additionally, overexpression of FZD7 showed the inverse effect as expected, emphasizing the importance of FZD7 dysfunction in the development of MDR in HCC cells. What's more, in treatment with quercetin combined with siFZD7, the expression levels of FZD7, nuclear
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