Discovery of Myricetin as a Potent Inhibitor of Human Flap

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Cite This: J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Discovery of Myricetin as a Potent Inhibitor of Human Flap Endonuclease 1, Which Potentially Can Be Used as Sensitizing Agent against HT-29 Human Colon Cancer Cells Long Ma,*,∥ Xiuqi Cao,∥ Haiyue Wang,∥ Kui Lu,∥ Ying Wang, Chunhao Tu, Yujie Dai, Yuanyuan Meng, Yuyin Li, Peng Yu, Shuli Man, and Aipo Diao*

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State Key Laboratory of Food Nutrition and Safety, Key Laboratory of Industrial Fermentation Microbiology (MOE), Tianjin Key Laboratory of Industrial Microbiology, School of Biotechnology, Tianjin University of Science & Technology, Tianjin 300457, China S Supporting Information *

ABSTRACT: Human flap endonuclease 1 (hFEN1) is instrumental in DNA replication and repair. It is able to cleave the 5′ single-stranded protrusion (also known as 5′ flap) resulting from strand displacement reactions. In light of its crucial functions, hFEN1 is now deemed as a nontrivial target in the DNA damage response system for anticancer drug development. Herein, we report that myricetin and some natural flavonoids are able to inhibit hFEN1. Structure−activity relationship, inhibitory mechanisms, molecular docking, and cancer cell-based assays have been performed. Our original findings expand the activity of flavonoids and may pave the way for flavonoid-assisted targeted cancer therapy. KEYWORDS: human flap endonuclease 1 (hFEN1), myricetin, flavonoids, inhibitor, colon cancer treatment, structure−activity relationship analysis, molecular docking, sensitizing effect researched.7 Nevertheless, there is no new class of inhibitors reported and fully studied since then. Flavonoids are defined as a group of natural substances belonging to polyphenols.8 They are widely distributed in fruit, vegetables, grains, bark, roots, stems, flowers, tea, and wine. These natural products have long been known for their beneficial effects on human health, and more than 4000 members of them have been identified.8 In the current research, we set out to hunt small molecular inhibitors of hFEN1. We focused on bioflavonoids, including flavones, flavonols, flavanones, flavanols, flavanonols, isoflavonoids, and flavan-3-ols. All these compounds were investigated for their inhibitory effects on hFEN1. Myricetin (3,3′,4′,5,5′,7hexahydroxyflavone) was found to be the strongest hFEN1 inhibitor among all bioflavonoids tested. A preliminary study that combined myricetin with paclitaxel (PTX) was also carried out to test its potential sensitizing effect.

1. INTRODUCTION Human flap endonuclease 1 (hFEN1), as a prototypical member of the 5′-nuclease superfamily, is a metal iondependent and structure-specific nuclease. It is tasked for removal of 5′ single-stranded protrusions, termed as flaps, resulting from strand displacement reactions in either lagging strand synthesis of DNA replication or long-patch base excision repair (BER).1 The oligonucleotide cleavage performed by hFEN1 generates a 5′ phosphorylated end suitable for ligation, thereby ensuring genome stability. Critically, human malignancies are characterized by fast and limitless DNA replication, which requires overly activated DNA repair system to restore the errors accumulated in this process. In fact, hFEN1 is overexpressed in various human cancers such that hFEN1 could be considered as a biomarker (Table SI-1).2 In some cases, up-regulated hFEN1 enhances cancer susceptibility, aggravates the malignancies, and lowers the survival rates of cancer patients. In addition, a number of DNA-damaging anticancer treatments may lead to the compensatory activation of the DNA damage response (DDR) pathway, which accounts for the chemotherapy and radiotherapy resistances.3 This also applies to hFEN1.4 In addition, hFEN1 inhibition could be explored in terms of synthetic lethality-based anticancer treatment. This strategy is rooted in the principle that for genetically defective cancer cells, specific inhibition of synthetic lethal gene partners results in selective cancer cell killing.5 In this regard, hFEN1 could potentially be a druggable target, as it is in partnership with various genes that frequently mutate in specific cancers.6 More than a decade ago, Tumey et al. reported that some Nhydroxyurea compounds had hFEN1 inhibitory activity, and subsequently, this type of inhibitor has been heavily © XXXX American Chemical Society

2. MATERIALS AND METHODS 2.1. Materials and Sample Preparation. Myricetin (≥96%), dihydromyricetin, quercetin (≥98%), quercetin (≥95%), luteolin (≥98%), fisetin (≥98%), kaempferol (≥97%), galangin (≥98%), apigenin (≥98%), genistein (≥97%), isoliquiritigenin (≥98%), morin (≥95%), gallic acid (≥97%), rhamnetin (≥97%), and (−)-epicatechin (≥98%) were purchased from Sigma-Aldich (U.S.). Taxifolin (≥98%) and (+)-catechin (≥97%) were purchased from Aladdin Chemistry Co., Ltd. (China). Isoquercetin (≥95%) was purchased from J&K Scientific Ltd. (China). Rutin (≥95%) was purchased from Solarbio Received: October 5, 2018 Revised: January 2, 2019 Accepted: January 7, 2019

A

DOI: 10.1021/acs.jafc.8b05447 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

temperature. To investigate the interaction with hFEN1, 100 μM myricetin was used. 2.6. Fluorogenic Assay for IC50 Measurement. This was based on previous publication with slight modifications. Simply, the DNA substrate containing a double flap region used in the assay was prepared from the following DNA strands with a 1:1:1 ratio: quencher strand (5′-CACGTTGACTACCGCTCAATCCTGACGAACACATC-BHQ1), flap strand (5′-FAM-GATGTCAAGCAGTCCTAACTTTGAGGCAGAGTCCGC), and template strand (5′GCGGACTCTGCCTCAAGACGGTAGTCAACGTG-3′) by a standard annealing procedure in buffer containing 50 mM Tris-HCl (pH 8.0), 100 mM KCl, and 5 mM MgCl2. The annealed DNA substrates were then stored at −20 °C as 1 μM stock. The hFEN1 fluorogenic assay was carried out in a 100 μL reaction mixture. First, 80 μL of either hFEN1 at 200 nM or buffer (as nonenzyme control) was pipetted into a 96-well plate. Subsequently, 10 μL of inhibitor at appropriate concentration was added. Finally, 10 μL of 100 nM substrate was added to start the reaction. Assays were carried out at 37 °C, and kinetic data were recorded over 5 min in a Tecan Infinite 200 Pro plate reader (Tecan, Switzerland) with excitation and emission wavelengths at 484 and 519 nm, respectively. All data were processed and fitted using nonlinear regression by GraphPad Prism 5 software (San Diego, CA, U.S.), and the IC50 values were subsequently acquired. 2.7. Gel Electrophoresis for hFEN1 DNA Cleavage Test. Simply, the DNA substrate containing a double flap region used in the assay was prepared as described in section 2.6; the only difference was the flap strand was labeled with a Cy5 rather than a FAM dye. In addition, a synthetic free 5′ flap sequence (5′-Cy5-GATGTCAAGCAGTCCTAACTT-3′) was adopted. The hFEN1 cleavage assay was performed in a 100 μL reaction mixture containing 50 mM Tris-HCl (pH 8.0), 10 mM MgCl2, 1.0 mM DTT, 0.01% Tween-20. Briefly, 500 nM hFEN1 (or cell extract) and small molecule compounds at different concentrations were incubated at room temperature for 15 min, followed by the addition of 500 nM annealed DNA substrate labeled with Cy5 dye. The mixture was incubated for a further 30 min at 30 °C. Each reaction was terminated by the addition of 10 μL of stop solution containing 50 mM EDTA and 4% SDS. After incubation for an additional 10 min at room temperature, the reaction was separated on a 4% agarose gel electrophoresis. A LI-COR Odyssey Infrared Imaging System was used to visualize the gel. 2.8. Molecular Modeling. The hFEN1-product complex (PDB: 3Q8K) was obtained from the RCSB Protein Data Bank. All bound waters and the inhibitor molecules were removed from the protein, and optimization for protein and ligand was performed. The molecular docking calculation was performed using Libdock program of Discovery Studio 2.5 software (Accelrys Inc., U.S.). The figures of the docked model shown were generated using Discovery Studio 2017 (NeoTrident Technology Ltd., China). 2.9. Overexpression and Knockdown of hFEN1 in HT-29 Colon Cancer Cells. Total RNA was isolated from HT-29 cells, and the RNA was reversely transcribed with oligo (dT) primers using a reverse transcription system. The target gene hfen1 was amplified by PCR. Forward primer: 5′-CCGCTCGAGATGGGAATTCAAGGCCTGGCCAAACT-3′; reverse primer: 5′-TCCCCCCGGGTTATTTTCCCCTTTTAAACTTCCCTGCT-3′. The target gene and vector pLVX-AcGFP1-N1 were ligated to obtain a recombinant plasmid pLVX-hFEN1. Lentiviral vectors expression hFEN1 shRNAs (shRNA1 and shRNA2) and the scamble shRNA control vector were also prepared. Lentivirus packaging was performed using HEK293T cells according to the manufacturer’s manual. HT-29 cells were infected with lentiviruses for 4 days prior to the experiments. 2.10. Cell Viability Assay. Cell viability was determined using a MTT assay. HT-29 cells were seeded into 96-well plates at a density of 8000 cells/well overnight and left to adhere overnight. HT-29 cells were cultured using Dulbecco’s Modified Eagle Medium (DMEM) and Ham’s F-12 Nutrient Mixture (F) (DMEM/F12 1:1 mixture) in a humidified atmosphere containing 5% CO2. On the following day, the cells were exposed to tested compounds at 37 °C. Prepared MTT (5

(China). SYPRO Orange were purchased from Sigma-Aldrich (U.S.). Paclitaxel was purchased from MedChemexpress Co., Ltd. (U.S.). Commercial antibodies were purchased from the following sources: Antibody against hFEN1 (SC-13051) was purchased from Santa Cruz Biotechnology (U.S.). Antibodies against GFP and GAPDH were purchased from Sungene Biotech Co., Ltd. (Tianjin, China). Antibody against γH2AX (#9718) was purchased from Cell Signaling Technology (U.S.). HRP-conjugated secondary antibodies were purchased from Invitrogen (U.S.). All DNAs were chemically synthesized and purified by Generay Biotech Co., Ltd. (China). All chemicals and DNAs were used without further purification. Deionized water was prepared using a Milli-Q Ultrapure water system. All other chemicals used were purchased from Aladdin Chemistry Co., Ltd. (China) or Sigma-Aldrich (U.S.) unless otherwise stated. DNA concentrations were quantified at 260 nm absorbance using a UV-1800 ultraviolet−visible (UV−vis) spectrophotometer (Shimadzu, Japan), and the extinction coefficients were calculated using the online IDT OligoAnalyzer 3.1. 2.2. Protein Expression and Purification. The plasmid of hFEN1 was a kind gift from Prof. Jane Grasby (University of Sheffield). The plasmids were transformed into E. coli BL21(DE3) using the heat shock method. Briefly, a mixture of chemically competent bacteria and plasmids were placed at 42 °C for 90 s and then placed back on ice. E. coli BL21(DE3) cells, transformed with the hFEN1 plasmids, were grown in Luria−Bertani (LB) medium supplemented with 100 mg/mL kanamycin and incubated at 37 °C with shaking (150 rpm). Isopropylb-D-1-thiogalactopyranoside (IPTG) was added to a final concentration of 0.2 mM to initiate protein expression when cell optical density (OD600) reached 0.4−0.6. The cultures were then continued for an additional 30 h at 16 °C and then harvested by centrifugation at 8000 rpm for 20 min at 4 °C. The cell pellets were resuspended in lysis buffer (50 mM Na−P, 300 mM NaCl, 10 mM imidazole, pH 8.0) supplemented with protease inhibitors and lysozyme for 30 min on ice, followed by sonication. Cell debris was removed by centrifugation (13 000 rpm, 15 min, 4 °C). The supernatant was filtered through a Millipore membrane and filled into a 5 mL nickel Sepharose 6 Fast Flow column, and the column was washed with 5 column volumes of washing buffer (50 mM Na−P, 300 mM NaCl, 10 mM imidazole, pH 8.0). The target protein was eluted with a stepwise gradient of 50, 100, 200, and 250 mM imidazole. The eluted protein was dialyzed against buffer (50 mM Na−P, 300 mM NaCl, 10 mM imidazole, pH 8.0) and concentrated using a Millipore concentrator. The protein concentration was measured on the basis of the Lambert−Beer law, and the extinction coefficient was determined using the ExPAsy ProtParam tool. 2.3. Cell Culture. Nonmalignant human colon epithelial cells NCM460 were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium. Human colon cancer HT-29 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) and Ham’s F-12 Nutrient Mixture (F) (DMEM/F12 1:1 mixture). All experiments were carried out in complete medium containing 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 0.1 mg/mL streptomycin. All cells were incubated at 37 °C in a humidified atmosphere with 5% CO2. 2.4. Differential Scanning Fluorimetry. The stability of purified hFEN1 in different buffer systems was assessed as a function of inhibitor concentration by differential scanning fluorimetry (DSF). Simply, final volumes of 20 μL containing 5 μM hFEN1 in 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10 mM MgCl2 or CaCl2 where necessary, 1 × SYPRO Orange and various concentrations of compound myricetin or LK238 were mixed in PCR tubes, respectively. The tubes were put into an Applied Biosystems StepOne Real-Time PCR (Life Technologies) instrument for thermal denaturation. The ROX channel was chosen for fluorescence recording from 25 to 95 °C at a scan rate of 1 °C/min. 2.5. Circular Dichroism. Circular dichroism was executed with hFEN1 concentration at 10 μM buffered with 50 mM Na−P (pH 8.0), 150 mM NaCl, and 5 mM Ca2+ or Mg2+ where necessary using Chirascan spectrometer (Applied Photophysics, U.K.) at ambient B


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Figure 1. Identification of hFEN1 is remarkably up-regulated in colon cancer. (A) hFEN1 expression is significantly higher in colon tumors than in noncolon ones (t test was performed, and P < 0.001 tumor vs normal). (B) Correlation of hFEN1 expression and colon cancer stages. (C) Kaplan− Meier analysis of survival of patients with colon cancer. (D) Network of interactions of hFEN1 with other proteins. (E and F) Western blot result for revealing hFEN1 level in NCM460 and HT-29 cell lines and the statistical analysis (***P < 0.005 HT-29 vs. NCM460). mg/mL, in pH 7.4 PBS buffer) was added to each well, and the plates were incubated at 37 °C for 4 h. Dimethyl sulfoxide (DMSO) was added to each well to dissolve the formazan dye. Subsequently, the absorbance of each well was measured at 490 nm by a microplate reader (TECAN Infinite M200 PRO, Switzerland). The cell viability was calculated as follows: cell viability (%) = (average A490 of the treated group − average A490 of the blank group)/ (average A490 of the control group − average A490 of the blank group) × 100%. The cell viability of the control group was assigned a value of 100%. The experiments were repeated in triplicate. 2.11. Colony-Forming Assay. HT-29 cells were seeded (800/ well) in triplicate with 2 mL growth medium in 3 cm culture dishes. Cells were refed with 10% fresh medium containing 1 μg/mL puromycin. After 2 weeks, colonies were stained with 0.1% crystal violet. Excess dye was removed by washing for 5 min with PBS, and colonies were counted and pictured. The cell colony-forming efficiency was obtained by calculating percentages in terms of the numbers of colonies relative to the control (defined as 100%). 2.12. Western Blot. The harvested cells were washed by ice-cold PBS twice and then lysed with RIPA buffer supplemented with 1 mM PMSF (phenylmethylsulfonyl fluoride) and protease inhibitors (Pierce protease Inhibitor Tablets, Thermo Fisher Scientific) and phosphatase inhibitors where necessary. Protein samples were extracted and then resolved by 12% sodium dodecyl sulfate− polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a methanol-activated PVDF membrane. The membrane was then blocked in PBST buffer containing 5% nonfat dry milk for 1 h at room temperature. Blots were then incubated overnight at 4 °C with primary antibodies. HRP-conjugated secondary antibody was

incubated for 2 h at room temperature. Finally, the chemiluminescence (ECL) for visualization was conducted by a Sage Creation MiniChemki imaging system. 2.13. Statistical Analysis. The values obtained in the experiments were expressed as the mean ± standard deviation (SD) and analyzed by Student’s t test where necessary. All statistical analyses were performed using SPSS 17.0 software, and P < 0.05 was considered significant.

3. RESULTS AND DISCUSSION 3.1. Relationship of hFEN1 with Colon Cancer. To begin, we investigated The Cancer Genome Atlas (TCGA) gene expression difference between colon cancer and normal healthy samples. As shown in Figure 1A, hFEN1 expression was highly up-regulated in the cancer group compared with the normal one. Besides, hFEN1 was elevated during the American Joint Committee on Cancer (AJCC) stages of cancer, indicating its potential roles in cancer progression and development (Figure 1B). This suggested that the malignancy of colon cancer was correlated with hFEN1 overexpression. In support of this correlation, patients with high expression of hFEN1 had poorer prognoses than those with low levels of hFEN1 (Figure 1C). Also, hFEN1 is a hub intertwined in a complex molecular network, which may afford some synthetic lethal interactions for cancer treatment (Figure 1D). For certainty, we performed a Western blot experiment to check the hFEN1 level of NCM460 (human normal colonic C


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Figure 2. Discovery of myricetin and some natural flavonoids as FEN1 inhibitors. (A) Cleavage pattern of hFEN1. (B) Fluorogenic FEN1 assay. (C) One N-hydroxyurea reference compound LK238 and the proposed double metal cations binding model of N-hydroxyurea species compounds (upper panel); the chemical structure of myricetin (lower panel). (D) Results of fluorogenic hFEN1 assay. Green line and axis, addition of myricetin in DNA substrate without hFEN1. (E) Myricetin dose-dependently inhibited purified hFEN1. (F) Myriectin inhibited hFEN1 in whole cell extract of HT-29 cells. Different concentrations of myricetin were incubated with HT-29 cells for 24 h; after this the cells were disrupted to make cell extract. Subsequently, the DNA cleavage test was carried out accordingly using gel electrophoresis. Myr, myricetin; Sub, DNA substrate; CE, cell extract.

Table 1. Summary of Inhibitory Activities of Selected Flavonoids and the SAR Study compound

2−3 DBa

4C = O

myricetin dihydromyricetin quercetin taxifolin isoquercetin luteolin fisetin kaempferol galangin apigenin morin rhamnetin genistein rutin (−)-epicatechin isoliquiritigenin (+)-catechin gallic acid LK238

+ − + − + + + + + + + + + + −

+ + + + + + + + + + + + + + −

3 OH OH OH OH Glub H OH OH OH H OH OH

5

7

2′

3′

4′

5′

IC50 (μM)

OH OH OH OH OH OH H OH OH OH OH OH

OH OH OH OH OH OH OH OH OH OH OH OCH3

H H H H H H H H H H H H

OH OH OH H OH OH OH H H H OH OH

OH OH OH OH OH OH OH OH H OH H OH

OH OH H OH H H H H H H OH H

H H

H H

OH OH

OH OH

0.69 ± 0.05 4.26 ± 0.30 5.45 ± 0.59 9.42 ± 0.49 17.40 ± 1.18 7.26 ± 0.55 9.75 ± 0.56 46.10 ± 14.34 >50 >100 8.66 ± 0.32 21.63 ± 19.68 >100 >50 >100 >100 >100 22.15 ± 7.20 0.17 ± 0.01

Isoc Rha-Glu OH

d

OH OH

OH OH

% inhibitation at 100 μM 99.1 99.2 99.0 93.2 55.8 86.4 95.7 73.3 55.6 33.2 68.2 56.2 40.2 58.1 9.8 31.9 18.4 98.3 99.7

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.2 0.2 0.3 4.5 2.6 3.8 1.1 4.5 2.9 2.3 2.0 4.5 4.7 1.6 4.8 1.3 5.2 1.1 0.2

2−3 double bond. bGlu, glucose. cIso, isoflavone. dRha-Giu, α-L-rhamnopyranosyi-(1 → 6)-β-D- glucopyranose.

a

3.2. Identification of Myricetin and Some Natural Flavonoids as Inhibitors of hFEN1. After screening some small molecule libraries using a well-established fluorescencebased assay followed by subsequent validation by agarose gel

epithelial cell line) and HT-29 (human colon carcinoma cell line), as shown in Figure 1E,F. Clearly, the hFEN1 level of the former was much lower than the latter (P < 0.005), which agreed well with the bioinformatics analysis. D


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Figure 3. Fitted IC50 data of fluorogenic assay (A) and structure−activity relationship (SAR) summary (B).

Figure 4. CD spectra and DSF assay. (A and B) CD spectra to monitor the interactions of hFEN1 with myricetin in the presence of Ca2+ and Mg2+. (C) The binding of LK238 and myricetin with hFEN1 measured by DSF assay.

electrophoresis,9 we found that myricetin was able to inhibit hFEN1 activity with a 690 ± 50 nM IC50 value. Myricetin is a member of the flavonoid class and commonly derived from vegetables, fruits, nuts, berries, tea, and red wine. Figure 2A shows the cleavage pattern of hFEN1, in which hFEN1 cut the phosphodiester bond between the +1 and −1 nucleotides in the junction at 5′ flap end. Figure 2B depicts fluorogenic hFEN1 assay used in inhibitor screening. Figure 2C describes the N-hydroxyurea compound (named LK238, which was synthesized by the authors to serve as a “reference” compound) and the double metal cations binding model proposed by Tumey et al.7 The N-hydroxyl group played a critical role in coordination of two divalent metal ions needed for hFEN1 catalysis. More strikingly, myricetin (structure drawn in Figure 2C) displayed a comparable inhibitory effect evidenced by fluorogenic assay (Figure 2D). It was noted that myricetin did not suppress the fluorescence of DNA substrate (green line, Figure 2D), indicating that myricetin did not quench the FAM dye by itself. This dose-dependent inhibition of myricetin on hFEN1 was also proved by gel electrophoresis with purified hFEN1 (Figure 2E). After searching the literature, we found that myricetin aglycone was able to transport into HT-29 cells and 70% of original myricetin can be retained;10 in addition, myricetin concentration-dependently inhibits MMP-2 enzyme activity in various colorectal carcinoma cell lines including HT-29, indicating a considerable uptake of myricetin by HT-29. We thus proceeded to incubate myricetin with HT-29 cells for 24 h to allow it to pass through. We then tested the inhibition effect of endogenous hFEN1 by myricetin after cell disruption; interestingly, hFEN1 can be inhibited dose-dependently, as shown in Figure 2F, indicating a fine transmembrane transport of myricetin. 3.3. Structure−Activity Relationship Study. We screened some natural flavonoids (Figure SI-1) with structures

similar to myricetin and found out some were also active in terms of hFEN1 inhibition. However, it seemed that neither chalcones nor isoflavones were inhibitors of hFEN1. The IC50 values of all flavonoids are listed in Table 1. The data and curve fittings of some flavonoids (IC50 < 20 μM) are listed in Figure 3A. We next performed a structure−activity relationship (SAR) investigation that is summarized in Figure 3B. Simply, the presence of A and C rings was crucial for inhibitory activity, because gallic acid also afforded three hydroxyl groups (similar to ring B of myricetin) but displayed far weaker inhibitory activity. The combination of 3′, 4′, 5′ −OH was crucial, and at least two hydroxyl groups were requested for notable inhibition. Galangin, a compound with no hydroxyl groups on ring B but otherwise exactly the same as myricetin, had little inhibitory activity, suggesting the importance of the combination of hydroxyl groups on the B ring. This deduction can be also supported by the decreased activity of myricetin < quercetin < kaempferol that had three, two, and one −OH on ring B, respectively. By comparison of myricetin and quercetin with their respective dihydroxylated form, it was revealed that the 2−3 double bond contributed to the inhibitory activity. Double bonding was considered to increase the rigidity of the molecules; thus, a more planar conformation was possibly favored in this regard. With respect to the 5-OH on ring A, the effect mattered, which can be best mirrored by the fact that fisetin and quercetin had much higher IC50 values. Luteolin was devoid of 3-OH compared with quercetin, and the latter was marginally better than the former, indicating that 3-OH was less important. Rhamnetin, different from quercetin only by a 7-methoxyl substitution on ring A, was far less active than quercetin, suggesting the important role of 7-OH. 3.4. Circular Dichroism Spectroscopic Study Proves Mg2+ Is Required for the Binding of Myricetin to hFEN1. We used circular dichroism (CD) spectroscopy to probe the E


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Figure 5. Molecular insight of myricetin as an inhibitor of hFEN1. (A) Normalized initial reaction rates vs substrate at various concentrations of myricetin and noncompetitive fittings. (B) Docked pose of myricetin bound to hFEN1−product complex (PDB: 3Q8K). (C) Binding model of myricetin interacted with hFEN1−product complex. (D) Schematic representation of proposed interactions of myricetin with relevant amino acid residues of hFEN1 (Note: red, purple, green, and blue dotted lines denote π−π stacking, conventional hydrogen binding, π−anion, and metalcoordination interaction, respectively). (E) Proposed structure of hFEN1−product complex superimposed with myricetin (green). Note: hFEN1 is not shown; −1 and −2 are the two terminal nucleotides of the DNA product (PDB: 3Q8K).

conformation change of hFEN1 in the presence of myricetin. As shown in Figure 4A, in the presence of Ca2+ buffer, the addition of myricetin did not change the CD signal of hFEN1, indicating no evident binding with hFEN1. In contrast, in Mg2+-containing buffer (Figure 4B), it seemed that myricetin physically interacted with hFEN1, as evidenced by the clear signal shift in the spectrum. Thinking further, our CD result demonstrated that myricetin was capable of free hFEN1 binding, which was in a good agreement with the non-

competitive kinetic model. We also tested the binding of hFEN1 with small molecule by differential scanning fluorimetry (DSF) using SYPRO Orange,11 and data are shown in Figure 4C. It was shown there was significant temperature increase with the addition of myricetin in Mg2+-containing solution rather than the Ca2+-containing one. This result corresponded well with the CD measurement, in which the presence of Mg2+ rather than Ca2+ was prerequisite for the interaction of myricetin with hFEN1. Similarly, N-hydroxyl F


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Figure 6. Effects of hFEN1 and myricetin on HT-29 colon cancer cells. (A) Knockdown and overexpression of hFEN1 in HT-29 cells, and the relationship of hFEN1 level with the expression of phosphorylated γH2AX. Lenti-hFEN1, overexpression of hFEN1; shFEN1, knockdown of hFEN1. (B) Cell viability of hFEN1 silenced and overexpressed HT-29 cells using MTT assay. (C) Representative images of colony-forming assay of hFEN1 silenced and overexpressed HT-29 cells. (D) Statistical analysis of the results derived from colony-forming assay (panel C). (E) MTT assay of myricetin-treated HT-29 cells. (F) Western blot image and statistical analysis of phosphorylated γH2AX levels in HT-29 cells with different treatments after 48 h. Myricetin and PTX used were 32 μM and 100 nM, respectively. (G and H) Co-treatment of PTX and myricetin (32 μM) for 48 and 72 h, respectively, to test the combined synergistic effect. *P < 0.05; **P < 0.01; ***P < 0.005 vs control. N.S., not significant; Myr, myricetin; PTX, paclitaxel; Ctrl, control.

that the +1 nucleotide has been cleaved during hydrolysis) in hFEN1-product complex (PDB: 3Q8K) in order to let myricetin enter into the active site of hFEN1. As shown in Figure 5D, the ring A of myricetin stretched toward the two divalent metal ions in this case; the 4-keto and 5-OH were crucial as they were supposed to engage with Arg100 and Lys93 via hydrogen bonds. The two amino acid residues were instrumental to hFEN1’s activity, and previous study showed the mutation of each site caused a drastically reduced activity.1b Asp34 and Glu160 seemed to form hydrogen bonding with 7-OH. Ring B faced to Tyr40, where a T-shaped π−π interaction possibly took place. Metal-coordination interaction could be formed between divalent metals and the hydrogen atoms of 5- and 7-hydroxyls. Asp179 and −2 guanosine of DNA product contributed to π−anion interactions. Superimposing of myricetin with hFEN1− product complex revealed that myricetin possibly shared the same pocket with the terminal nucleotide of the DNA product in the hFEN1 protein (Figure 5E). As proposed previously, hFEN1 uses a unified threading and double nucleotide

urea compounds were also proven to bind hFEN1 in the presence of Mg2+ but not Ca2+.7b As shown in Figure 4C, this was also confirmed by our LK238 compound (N-hydroxyl urea reference compound). It is known that Ca2+ ions facilitate accommodation of the substrate DNA, but they do not support catalysis even though they possibly occupied similar sites on the protein with Mg2+ ions. 3.5. Molecular Docking Study. Next, we addressed the mechanism of myricetin inhibition using in vitro kinetic analysis. By comparing four available models, including competitive, uncompetitive, noncompetitive, and mixed inhibition, it was displayed that hFEN1 was best fitted using the noncompetitive model (Figure 5A). The Ki value was 1.287 ± 0.096 nM. This model suggested that myricetin was able to bind to DNA-free and DNA-bound forms of hFEN1 and assumed both complexes had equivalent ligand dissociation constants (Kd). In silico molecular docking was performed, and in combination with SAR analysis, we proposed a model for the binding of myricetin to hFEN1 (Figure 5B,C). We first manually delete −1 nucleotide (note G


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Journal of Agricultural and Food Chemistry unpairing mechanism to achieve accurate incision, whereby the targeted phosphodiester bond is presented to the active site metal ions.12 Thus, our model implied that myricetin may be able to block the base unpairing of DNA substrate, thus inhibiting the activity of hFEN1, in which the catalytic metals were not accessible for hydrolysis reaction. This proposed mechanism was reminiscent of the action of the HIV integrase inhibitor raltegravir. Raltegravir similarly obstructs access of the reacting phosphodiester bond to the catalytic metals.13 3.6. hFEN1 Promotes Colon Cancer Cell Survival, and Myricetin Sensitizes Cancer Cells to the Treatment of Paclitaxel. Generally speaking, flavonoids can undergo significant metabolism via deglycosylation, converting flavonoid glycosides to aglycones. The uptake and absorption of aglycones take place in small and/or colonic intestine epithelial cells.14 In this regard, we deliberately designed our experiment and chose human intestinal epithelial HT-29 cells to test the bioactivity of myricetin, because we thought that myricetin would be effectively absorbed by HT-29 before degradation and thus the hFEN1 inhibition activity would remain. As shown in Figure 6A, silencing and overexpression of hFEN1 resulted in increased and decreased levels of phosphorylated γH2AX, respectively. It is generally considered that phosphorylated γH2AX is a hallmark for DNA double strand breaks (DSBs), that is a type of severe DNA damage leading to cell apoptosis. Similarly, in terms of cell viability, hFEN1 silencing caused it to decrease (P < 0.005); in contrast, overexpression of hFEN1 gave rise to enhanced cell viability (P < 0.01). Collectively, these two experiments indicated that it was reasonable to promote cancer cell apoptosis by pharmacological inhibition of hFEN1. Moreover, the colony-forming assay (Figure 6C,D) showed that overexpression of hFEN1 induced colony formation, while the down-regulation of endogenous hFEN1 reduced colony formation efficiency. This suggested the function of hFEN1 in promoting the proliferation of HT-29 colon cancer cells, corroborating the importance of hFEN1 in cancer. Figure 6E indicates that a single treatment with myricetin was almost ineffective to HT29 cells. Paclitaxel (PTX) is a microtubule-stabilizer that selectively arrests cells in the G2 or mitotic phases of the cell cycle. PTX is one fine example of the most widely used chemotherapeutic agents from a natural origin and is used to test if myricetin, as a DNA repair inhibitor, could sensitize cancer cells to chemo-compounds which induce DNA lesions in cancer cells. There is growing evidence indicating PTX is able to induce DNA lesions.15 These facts make us consider the combination of PTX with hFEN1 inhibitor myricetin for treatment to obliterate colon cancer cells. As shown in Figure 6F, the expression level of phosphorylated γH2AX, a wellestablished molecular marker of DNA damage, was detected. It can be seen that myricetin induced marginally increased phosphorylated γH2AX expression compared with the control. While cotreatment of 32 μM myricetin with 100 nM PTX caused statistically significant increase in phosphorylated γH2AX level compared to PTX separate treatment, implying that at nontoxic dosage, myricetin was able to markedly potentiate the action of PTX. Additionally, MTT assay was employed to confirm the synergistic effect of combined myricetin with PTX (Figure 6G,H). Taken together, all these preliminary cell-based tests suggested that the sensitizing effect of myricetin may come from its hFEN1 inhibitory effect. In conclusion, flavonoids are widely distributed in nature, having multifaceted biochemical and physiological roles.16

Some of them are well-proven as potential therapies against cancers.17 Flavonoids are reported to be inhibitors of a series of enzymes and transporters.18 The current study confirms some flavonoids are capable of inhibiting DNA-repairing enzyme hFEN1. The plasma concentrations of flavonoids are usually less than 10 μM, which means that myricetin and some other flavonoids such as dihydromyricetin, quercetin, luteolin, fisetin, taxifolin, etc. are able to inhibit hFEN1 significantly at physiological relevant concentrations. Moreover, in the light of the importance of hFEN1 as a crucial target in oncotherapy, we envision the usefulness of its inhibitors in cancer treatment. Myricetin is one type of naturally occurring bioflavonoid. Myricetin acts as a promising cancer chemopreventive agent in some cancer models,19 including colorectal cancer, the third most common cancer for incidence and the fourth one for mortality worldwide. In addition, myricetin displays weak anticancer activity against human colon cancer cells via proliferation inhibition and apoptosis induction.20 In summary, our report not only expands the reach of natural flavonoids in terms of bioactivity at the enzyme level but also potentially paves the way for developing flavonoid-assisted targeted cancer treatments. In addition, there is huge scope for studying myricetin’s effect on the 5′ nuclease family and the combination of myricetin with other drugs to target DDR for cancer treatment.21

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.8b05447. Experimental procedures (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] or [email protected]. *E-mail: [email protected]. ORCID

Long Ma: 0000-0001-8479-2663 Kui Lu: 0000-0002-9685-2691 Author Contributions ∥

L.M., X.C., H.W., and K.L. contributed equally to this work and should be regarded as joint first authors.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work is supported by National Natural Science Foundation of China (Nos. 81503086 and 21672161); Foundation of Key Laboratory of Industrial Fermentation Microbiology of Ministry of Education and Tianjin Key Lab of Industrial Microbiology (2017YC003); Tianjin Municipal Science and Technology Committee (118PTSYJC00140); and opening research funding from Tianjin State Key Laboratory of Modern Chinese Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin China. We thank Prof. Jane Grasby and Dr. L. David Finger (University of Sheffield, U.K.) for their kind gift of hFEN1 plasmid. We thank Profs. Nan Wang and Xuegang Luo (Tianjin University of Science and Technology) for providing NCM460 cells. H


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Discovery of Myricetin as a Potent Inhibitor of Human Flap

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