RESEARCH ARTICLE Open Access

Flavonoids modulate multidrug resistancethrough wnt signaling in P-glycoproteinoverexpressing cell linesS. Mohana1, M. Ganesan1, N. Rajendra Prasad1*, D. Ananthakrishnan2 and D. Velmurugan2,3

Abstract

Background: Wnt signaling has been linked with P-glycoprotein (P-gp) overexpression and which was mainlymediated by β-catenin nuclear translocation. Flavonoids have already been reported as modulators of the Wnt/β-catenin pathway and hence they may serve as promising agents in the reversal of P-gp mediated cancer multidrug resistance (MDR).

Methods: In this study, we screened selected flavonoids against Wnt/β-catenin signaling molecules. The bindinginteraction of flavonoids (theaflavin, quercetin, rutin, epicatechin 3 gallate and tamarixetin) with GSK 3β wasdetermined by molecular docking. Flavonoids on P-gp expression and the components of Wnt signaling in drug-resistant KBCHR8–5 cells were analyzed by western blotting and qRT-PCR. The MDR reversal potential of theseselected flavonoids against P-gp mediated drug resistance was analyzed by cytotoxicity assay in KBCHR8–5 andMCF7/ADR cell lines. The chemosensitizing potential of flavonoids was further analyzed by observing cell cyclearrest in KBCHR8–5 cells.

Results: In this study, we observed that the components of Wnt/β-catenin pathway such as Wnt and GSK 3β wereactivated in multidrug resistant KBCHR8–5 cell lines. All the flavonoids selected in this study significantly decreasedthe expression of Wnt and GSK 3β in KBCHR8–5 cells and subsequently modulates P-gp overexpression in this drug-resistant cell line. Further, we observed that these flavonoids considerably decreased the doxorubicin resistance inKBCHR8–5 and MCF7/ADR cell lines. The MDR reversal potential of flavonoids were found to be in the order oftheaflavin > quercetin > rutin > epicatechin 3 gallate > tamarixetin. Moreover, we observed that flavonoidspretreatment significantly induced the doxorubicin-mediated arrest at the phase of G2/M. Further, thecombinations of doxorubicin with flavonoids significantly modulate the expression of drug response genes inKBCHR8–5 cells.

Conclusion: The present findings illustrate that the studied flavonoids significantly enhances doxorubicin-mediatedcell death through modulating P-gp expression pattern by targeting Wnt/β-catenin signaling in drug-resistantKBCHR8–5 cells.

Keywords: Wnt/β-catenin signaling, Flavonoids, Molecular docking, P-glycoprotein, Multidrug resistance, GSK 3β

* Correspondence: drprasadnr@gmail.com1Department of Biochemistry and Biotechnology, Annamalai University,Annamalai Nagar, Tamil Nadu 608 002, IndiaFull list of author information is available at the end of the article

© The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link tothe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Mohana et al. BMC Cancer (2018) 18:1168 https://doi.org/10.1186/s12885-018-5103-1

RETRACTED ARTIC

LE

BackgroundMultidrug resistance (MDR) is a mechanism throughwhich several cancer subtypes exhibit resistance to anti-cancer drugs resulted in the chemotherapy failure [1].This MDR phenomenon is mainly associated with over-expression of membrane-bound molecular “pumps”that dynamically efflux out structurally and functionallydifferent anticancer drugs from the tumor cells. TheP-glycoprotein (170 kDa), belongs to ATP-binding cas-sette transporters family (ABC), confer resistance tovarious chemotherapeutic drugs [2]. Thus, inhibition ofits drug transport function or modulation of its expres-sion in cancer cells will be a novel strategy to overcomecancer multidrug resistance.Existing data illustrated that natural flavonoids pos-

sess significant modulatory effects on drug resistancein cancer [3]. Recently, we systematically screened fla-vonoids against P-gp drug efflux function usingcalcein-AM drug efflux system and further observedflavonoids such as quercetin and rutin reverse MDRseveral folds in KBCHR8–5 cell lines [4]. Shtil et al.,1994 demonstrated modulation of P-gp overexpressionat the molecular level to overcome MDR in cancercells [5]. The activation of Wnt/β-catenin signalingmolecules leads to overexpression of P-gp which con-tributed to clinical MDR [6]. In the canonicalpathway, β-catenin is phosphorylated and activated bya set of proteins which includes GSK-3β, axin andAPC. Stabilized cytoplasmic β-catenin translocatesfrom the cytoplasm to the nucleus and activatesT-cell factor (TCF) transcription factors then subse-quently activates ABCB1 overexpression [7]. There-fore, downregulation of Wnt/GSK 3β/β-cateninpathway possibly will reduce the P-gp expression andinduce chemosensitization in drug-resistant cells. TheGSK 3β is an important factor of Wnt/β-catenin sig-naling and pharmacological inhibition or modulationof GSK 3β expression might reverse the MDR indrug-resistant cells.Numerous reports illustrate that flavonoids could able

to modulate the Wnt pathway thereby increases to theirantitumor effect against cancer cells [8, 9]. Kitagawa et al.,(2004) illustrated the reversal potential of flavonoids onthe function of P-gp in KB-C2 cells using daunorubicinand rhodamine-123 [10]. Herein, we investigated the che-mosensitizing efficacy of selected flavonoids like theafla-vin, rutin,, quercetin, epicatechin 3-gallate and tamarixetinin colchicines-selected KBCHR8–5 cell lines through tar-geting Wnt/GSK 3β/β-catenin pathway. To determinewhether these flavonoids modulate P-gp mediated MDR,we carried out cell-based assays, transcriptome analysisand Wnt proteins expression in the presence or absenceof these flavonoids in KB 3–1 and colchicine-selectedKBCHR8–5 cell lines.

MethodsMolecular dockingInduced-fit docking was carried out to predict theaflavin,quercetin, rutin, epicatechin 3 gallate and tamarixetinbinding interaction in the GSK 3β using Glide and primemodules [11]. Ligprep 2.3 module (Schrodinger) wasused for the preparation of theaflavin, quercetin, rutin,epicatechin 3 gallate and tamarixetin. The 3D GSK 3β(PDB: 5HLN) structure was obtained from the PDB(http://www.rcsb.org). The Schrodinger software wasused for GSK 3β preparation as per the procedure de-scribed previously [12].

Chemosensitizing effect of flavonoids by MTT assayWe have analyzed the chemosensitizing potential of fla-vonoids by MTT assay [13]. KB 3–1, KBCHR8–5,MCF-7 and MCF-7/ADR cells (1X104 cells/ well) wereinitially seeded in 96 well plates and kept incubated for24 h. Further, cells were preincubated with or withoutthe different concentration of flavonoids (1–10 μM perwell) for 2 h, consequently, various concentrations ofdoxorubicin were added into the designated wells for 72h. Then, MTT solution (4 mg/ml) was added and incu-bated for 4 h. Further, 100 μL of DMSO was added andthe absorbance of formazan solution was measured at570 nm using a multimode reader (Tecan, Austria).

Western blot analysisWe have done western blot analysis to find out flavo-noids mediated alteration of protein expression inKBCHR8–5 cells. The KBCHR8–5 cells (5 × 106 cells)were lysed using RIPA buffer. The protein concentrationwas estimated using nanodrop spectrophotometer(Thermo Scientific Inc.). Proteins were separated by 12%SDS-PAGE then blotted to nitrocellulose membrane.Then, the blotted membranes were treated with 5% BSAat for 1 h. The membranes were then kept incubated at4 °C overnight with monoclonal antibodies for P-gp(1:1000), Wnt (1:1000), GSK 3β (1:1000) (Santa Cruz,USA). Then, the membrane was incubated for 1 h withthe horseradish peroxidase conjugated secondary anti-bodies. Then, the protein expressions were detectedusing chemiluminence western blot detection kit(Biorad, USA).

qRT-PCR analysis of LRP6, FZD1, APC and axin expressionThe mRNA expression of LRP 6, Frizzled (FZD) 1, ad-enomatous polyposis coli (APC) and axin, in KBCHR8–5 cells was analyzed using real-time PCR. cDNA wassynthesized using 100 ng total RNA by RT2 First strandkit. Complimentary DNA was amplified (20 μL) usingSYBR green master mix and 0.5 μM of the specificprimers. Real-time PCR was carried out on Eppendorfmaster cycler (Eppendorf, Thermocycler, USA). The

Mohana et al. BMC Cancer (2018) 18:1168 Page 2 of 11

RETRACTED ARTIC

LE

Fig. 1 a) Pymol observation of ligand binding domain (LBD) of GSK 3β with theaflavin. b) Ligplot representation illustrates hydrophobic andhydrogen bondig of GSK 3β with theaflavin

Fig. 2 a and b. Wnt, GSK 3β and β-catenin mRNA and protein expression pattern in KB3–1 and KBCHR8–5 cell lines. Protein (Western blot) andmRNA (qRT-PCR) expression status of Wnt, GSK 3β, LRP6, FZD1, APC and axin in KBCHR8–5 cells. (c) theaflavin, (d) quercetin, (e) rutin, (f)epicatechin 3 gallate (E3G) and (g) tamarixetin. The protein levels were quantified by LI-COR Image Studio tool. The data denote means ± SDfrom three experiments. The protein expressions were normalized to the β-actin expression level. Gene expression was normalized with 18S anddepicts quantification of three independent experiments (means ± S.D). Symbols not sharing a common symbol vary significantly atp≤ 0.05 (DMRT)

Mohana et al. BMC Cancer (2018) 18:1168 Page 3 of 11

RETRACTED ARTIC

LE

gene expression levels were normalized to 18S mRNAexpression in each sample. The mean cyclic threshold(Ct) of each gene expression was accounted to measurethe relative gene expression by employing the formula2-ΔΔCt.

Cell cycle analysisAfter treatment with flavonoids and/or doxorubicin cells(1X106cells/well) were trypsinized and washed with PBS.Then, the treated cells were fixed using cold 70% ethanoland incubated for overnight at 4 °C. After a single wash

with PBS, the cells were incubated using 50 pg/ml ofpropidium iodide and 0.1 mg/ml of RNaseA for 30min.After that, cells were kept incubated for 30 min in dark.The DNA content in each phase of the cell cycle wasthen analyzed using a FACS (BD Aria III, BD Biosci-ences) [14].

PCR arrayThe total RNA was isolated using RNAeasy kit (Qiagen,India). The relative mRNA expression (RQ) pattern of 9genes involved in drug resistance, cell cycle, apoptosis,

Fig. 3 Theaflavin, quercetin, rutin, epicatechin 3 gallate (E3G) and tamarixetin and/or doxorubicin on MDR-linked gene expression pattern inKBCHR8–5 cells. The mRNA expression levels of 9 genes involved in drug resistance and Wnt/β-catenin signaling were detected using qPCR.Clustergram was constructed using the SA Biosciences online tool using three independent experiments

Fig. 4 Effect of flavonoids on doxorubicin-induced cell cycle arrest. Theaflavin, quercetin, rutin, epicatechin 3 gallate (E3G) and tamarixetintreatment potentiates G2/M arrest in doxorubicin-treated KBCHR8–5 cells. The cells were exposed to 7 μM of doxorubicin alone or in combinationwith 10 μM of theaflavin, rutin, epicatechin 3-gallate, quercetin and tamarixetin for 24 h. Different cell cycle phases were monitored byflow cytometer

Mohana et al. BMC Cancer (2018) 18:1168 Page 4 of 11

RETRACTED ARTIC

LE

Fig. 5 Chemosensitizing effect of the selected flavonoids in drug-resistant KBCHR8–5 cell lines. Concentration dependent curves of doxorubicinwith or without flavonoids (1, 5 and 10 μM) in parental KB 3–1 and KBCHR8–5 cell lines were constructed. The IC50 values of KBCH

R8–5 cell lineswere equaled with parental KB 3–1 cells (Table 1). Data with error bars show the mean ± S.E.M of four experiments, each done in triplicate

Mohana et al. BMC Cancer (2018) 18:1168 Page 5 of 11

RETRACTED ARTIC

LE

and Wnt/β-catenin pathway were investigated by PCRarray by SYBR Green PCR master mix (Qiagen,qRT-PCR array) on Eppendorf realplex PCR instrument.The gene expression in fold changes was plotted as clus-tergram using PCR data analysis.sabiosciences.com/pcr/arrayanalysis.php.

Results and discussionThe P-glycoprotein (ABCB1/MDR1) serves as key regu-lators in the efflux of chemotherapeutic agents [15]. Sev-eral recent findings indicate the link between Wnt/β-catenin pathway and the ABC transporters overex-pression [16, 17]. Previously, it has been reported thatWnt5A regulates ABCB1 expression pattern through thenon-canonical PKA/β-catenin pathway in drug resistantcancer cells [18]. Flavonoids have been reported to in-hibit ABCB1 transporters that contribute to the develop-ment of MDR [19]. In this study, we investigated thereversal of P-gp mediated MDR via targeting the Wnt/β-catenin pathway by selected dietary flavonoids whichshow chemosensitizing property in our preliminary stud-ies [4]. In this study, induced-fit docking reveals that fla-vonoids inhibit GSK 3β directly by interacting to theATP binding site of the protein. Among the flavonoidsstudied theaflavin effectively interact with Ligand Bind-ing Domain (LBD) of GSK 3β (− 85.58 kcal/mol) (Fig. 1;Additional file 1: Figure S1 and Table S1). The bindinginteraction of theaflavin against GSK 3β was comparedwith its cocrystal 65C (6-[(2-{[4-(2, 4-dichlorophenyl)-5-(4-methyl-1H-imidazole-2-yl) pyrimidin-2-yl] amino}ethyl) amino] pyridine- 3-carbonitrile) and VAL-135 wasfound to be contributed in the common hydrogen-bondinteractions. The ATP-competitive GSK 3β inhibitorsbind with GSK 3β by hydrogen bonding to the carbonyland amino groups of the valine 135 amino acid and alsoto the carbonyl oxygen of Asp133 within the hinge areaof the ATP-binding pocket [20]. Shin et al. (2007) estab-lished a hydroxyl group at C7 of the benzimidazole togenerate hydrogen bonds to the amino group of Val 135and the carbonyl group of the Asp 133 residue [21].Additionally, Coffman et al. (2011) developed severalGSK-3β inhibitors and these compounds interacts withinthe GSK-3β ATP site. Similarly, in our study, we foundflavanoids binds within the ATP site of GSK-3β particu-larly with the Val 135 and Asp 133 residues of the hingeregion [22]. Sivaraman (2015) screened GSK 3β inhibi-tors against flavonoids which shows Val 135 to be themajor active amino acid which is present in all thedocked compounds [23]. Johnson et al., (2011) showedmolecular docking of citrus flavonoids with GSK-3β andfound that quercetin effectively inhibits GSK-3β activity[24]. Moreover, Iftikhar and Rashid (2014) showed apharmacophore model of flavonoids to generate potentinhibitors for targeting Wnt signaling pathway [25].

Therefore, the present results suggest that the flavonoidscould interact with GSK-3β backbone amino acidsAsp133 and Val135. Our in vitro findings along withfindings of other investigators trigger us to experimen-tally prove the role of dietary flavonoids on the role offlavonoids Wnt/GSK-3β pathway to overcome MDR incancer.We observed that the Wnt and GSK-3β were activated

in the colchicine-selected KBCHR8–5 cell lines. Activationof the Wnt signaling elements might lead to the overex-pression of membrane P-gp. We found that β-catenintranslocation into the nucleus in drug resistant KBCHR8–5 cells. This substantiates the role of Wnt/β-catenin inABCB1 overexpression in the KBCHR8–5 cells (Fig. 2;Additional file 1: Figure S2). In this present study, flavo-noids also decreased the expression of Wnt and GSK 3βin KBCHR8–5 cells. Further, flavonoids treatment pre-vented the translocation of β-catenin to the nucleus in thedrug-resistant cells. Thereby, flavonoids downregulateP-gp overexpression in KBCHR8–5 cells; this was noticedin a dose-dependent manner (Fig. 2). Similarly, Park andChoi reported that binding of Tcf complexes with specificDNA binding sites has been suppressed by flavonoidsthrough diverse mechanisms in colorectal cancer [26]. It

Table 1 The concentration of flavonoids necessary for 50%inhibition (IC50 values). Data of mean ± SEM are from four analyticalexperiments, each conducted in triplicate. bFR: fold-drug resistancewas derived by dividing the IC50 concentration for doxorubicin ofKB 3–1 and KBCHR8–5 cells in the absence/presence of flavonoidsby IC50 value for doxorubicin of KB 3–1 cells

Compound KB 3–1 KB ChR 8–5

IC50 ± SEMα (μM) FRb IC50 ± SEMα (μΜ) FRb

Doxorubicin 0.04 ± 0.01 [1.0] 7 ± 0.20 [175.0]

+ 1 μM Theaflavine 0.03 ± 0.01 [0.8] 3 ± 0.04 [75.0]

+ 5 μM Theaflavine 0.02 ± 0.01 [0.5] 0.6 ± 0. 03 [15.0]

+ 10 μM Theaflavine 0.02 ± 0.01 [0.5] 0.3 ± 0.02 [7.5]

+ 1 μM Quercetin 0.02 ± 0.01 [0.5] 4 ± 0.05 [100.0]

+ 5 μM Quercetin 0.03 ± 0.01 [0.8] 1 ± 0.01 [25.0]

+ 10 μM Quercetin 0.02 ± 0.01 [0.5] 0.6 ± 0.01 [15.0]

+ 1 μM Rutin 0.03 ± 0.01 [0.8] 6 ± 0.05 [150.0]

+ 5 μM Rutin 0.02 ± 0.01 [0.5] 3 ± 0.01 [75.0]

+ 10 μM Rutin 0.02 ± 0.01 [0.5] 0.7 ± 0.01 [17.5]

+ 1 μM E3G 0.02 ± 0.01 [0.5] 5 ± 0.04 [125.0]

+ 5 μM E3G 0.03 ± 0.01 [0.8] 3 ± 0.02 [75.0]

+ 10 μM E3G 0.02 ± 0.01 [0.5] 1 ± 0.02 [25.0]

+ 1 μM Tamarixetin 0.03 ± 0.01 [0.8] 6 ± 0.03 [150.0]

+ 5 μM Tamarixetin 0.02 ± 0.01 [0.5] 4 ± 0.02 [100.0]

+ 10 μM Tamarixetin 0.02 ± 0.01 [0.5] 2 ± 0.01 [50.0]

Mohana et al. BMC Cancer (2018) 18:1168 Page 6 of 11

RETRACTED ARTIC

LE

Fig. 6 Chemosensitizing potential of the selected flavonoids in drug-resistant MCF-7/ADR cell lines. Concentration-dependent curves ofdoxorubicin with or without flavonoids (1,5 and 10 μM) in parental MCF-7 and MCF-7/ADR cell lines were constructed. The IC50 values of MCF-7/ADR cell lines were compared with parental MCF-7 cells. Data denotes the mean ± S.E.M of four experiments, each done in triplicate

Mohana et al. BMC Cancer (2018) 18:1168 Page 7 of 11

RETRACTED ARTIC

LE

has been well established that the down-regulation of thecanonical Wnt/GSK-3β/β-catenin pathway is known todownregulates the P-gp expression in various cancer sub-types [27–31]. Previously, it has been reported that quer-cetin binds with β-catenin thereby block bindinginteraction between β-catenin and TCF [32].In this study, flavonoids prevent the translocation

β-catenin, thereby downregulates P-gp expression inKBCHR8–5 cells. The phosphorylation-dependent deg-radation of β-catenin prevented nuclear translocationand binding on the mdr1 promoter which downregulatesP-gp by temozolomide acting like a Wnt-pathway inhibi-tor [33]. Wnt/β-catenin acts as a potential target to over-come resistance in cholangiocarcinoma [6]. Further, theFZD1 silencing significantly downregulated cytoplasmicand nuclear β-catenin expression levels and down-regu-lates the expression of MDR1/P-gp, thereby restoredsensitivity to chemotherapy drugs [34]. Flavonoids arereported to block different components of Wnt signalingthereby reverses MDR [35]. Previously, it was illustratedthat quercetin could regulates wnt signaling by affectingtheir pathway components in colon cancer cells, SW480cells, leukemia and lymphoma cells [36]. Isoquercitrininhibits glioblastoma proliferation through Wnt/β-ca-tenin pathway [37]. Recently, Chen et al., (2018) showedquercetin enhances the efficacy of chemotherapeuticdrugs in ABCB1, ABCC1 and ABCC2-overexpressingcells by regulating the FZD7/β-catenin signaling [16].The Wnt/β-catenin signaling has been found to be

related to the overexpression of ABC transporters [38,39]. The β-catenin was found to be released from theAPC/axin complex which activates transcription ofthe mdr1 gene. We observed that overexpression ofmRNA patterns of ABCB1 and Wnt/β-catenin path-way components such as LRP 6 and GSK 3β inKBCHR8–5 cells (Fig. 2; Additional file 1: Figure S3).Flavonoids pretreatment also augmentdoxorubicin-induced apoptosis in KBCHR8–5 celllines. Doxorubicin mediate apoptotic cell death bymodulating signaling elements [40]. Previous reportstate that flavonoids augment cell cycle arrest in dis-tinct phases of cancer [41]. Flavonoids significantlydownregulate the mRNA expression of CDK2,BCL-XL and upregulate p53, CDKN1A, BAX inKBCHR8–5 cells (Fig. 3). The MDR1 promoter hasalso been affected by p53 which affects endogenousMDR1 expression [41]. In this study, we found thatthe studied flavonoids sensitize doxorubicin and up-regulate p53 expression which subsequently inducesapoptotic events in drug-resistant cells. Moreover, weobserved that flavonoids pretreatment significantly aug-ment the doxorubicin-mediated arrest at the G2/M phaseof the cell cycle (Fig. 4). Flavonoids significantly enhancedoxorubicin efficacy in drug-resistant KBCHR8–5 cells.

Hence, we stated that downregulation of ABCB1 and sub-sequent modulation of doxorubicin-mediated cell cycle ar-rest and apoptotic signaling may be the reason for thechemosensitizing property of the studied flavonoids inP-gp overexpressing oral carcinoma cell lines. Thus, flavo-noids enhanced doxorubicin efficacy through Wnt/β-ca-tenin signaling and subsequently downregulates ABCB1expression thereby promotes doxorubicin-induced G2/Marrest and apoptosis in multidrug-resistant KBCHR8–5cells.The P-gp overexpressing KBCHR8–5 cells ex-

hibit 175-fold drug resistance to doxorubicin, comparedto KB 3–1 cell line (Fig. 5). We observed that flavonoidsconsiderably decreased doxorubicin resistance inKBCHR8–5 cell line (Table 1). We performed cell-basedcytotoxic assays in the MCF-7 and MCF-7/ADR celllines in the presence or absence of flavonoids (Fig. 6).MCF-7/ADR cell lines exhibit 27 fold resistances todoxorubicin, when compared to the parental MCF-7 celllines (Table 2). Flavanoids considerably decreased thedoxorubicin resistance in MCF-7/ADR cells when com-pared to the control MCF-7 cell lines. It has been found

Table 2 The concentration of flavonoid necessary for 50%inhibition (IC50 values) for cell viability was revealed. Mean ±SEM are from four analytical experiments which wereperformed in triplicate. bFR: fold-drug resistance was derived bydividing the IC50 concentration for doxorubicin of MCF-7 andMCF-7/ADR cells in the absence/presence of theaflavin,quercetin, rutin, epicatechin 3 gallate and tamarixetin by theIC50 concentration for doxorubicin of MCF-7 cells

Compound MCF-7 MCF-7/ADR

IC50 ± SEMα (μM) FRb IC50 ± SEMα (μΜ) FRb

Doxorubicin 0.32 ± 0.01 [1.0] 8.57 ± 0.23 [26.78]

+ 1 μM Theaflavine 0.32 ± 0.02 [1.0] 5.31 ± 0.04 [16.57]

+ 5 μM Theaflavine 0.29 ± 0.01 [0.9] 3.12 ± 0. 03 [9.75]

+ 10 μM Theaflavine 0.27 ± 0.01 [0.8] 0.8 ± 0.02 [2.5]

+ 1 μM Quercetin 0.31 ± 0.02 [0.9] 5.9 ± 0.05 [18.43]

+ 5 μM Quercetin 0..29 ± 0.02 [0.9] 3.41 ± 0.01 [10.65]

+ 10 μM Quercetin 0.28 ± 0.01 [0.8] 1.1 ± 0.01 [3.43]

+ 1 μM Rutin 0.32 ± 0.03 [1.0] 6.8 ± 0.05 [21.25]

+ 5 μM Rutin 0.30 ± 0.01 [0.9] 4.5 ± 0.01 [14.06]

+ 10 μM Rutin 0.28 ± 0.01 [0.8] 1.6 ± 0.01 [5.0]

+ 1 μM E3G 0.32 ± 0.02 [1.0] 6 ± 0.04 [18.75]

+ 5 μM E3G 0.31 ± 0.01 [0.9] 3.8 ± 0.02 [11.87]

+ 10 μM E3G 0.29 ± 0.02 [0.8] 1 ± 0.02 [3.12]

+ 1 μM Tamarixetin 0.32 ± 0.02 [0.8] 7.3 ± 0.03 [22.81]

+ 5 μM Tamarixetin 0.31 ± 0.02 [0.5] 4.9 ± 0.02 [15.31]

+ 10 μM Tamarixetin 0.30 ± 0.01 [0.5] 2.53 ± 0.01 [7.9]

Mohana et al. BMC Cancer (2018) 18:1168 Page 8 of 11

RETRACTED ARTIC

LE

that 10 μM of flavonoids significantly reverse the P-gpmediated MDR in KBCHR8–5 and MCF-7/ADR cells ascompared to other lower concentrations. Further, theMDR reversal potential of flavonoids was in the order oftheaflavin > quercetin > rutin > epicatechin 3-gallate >tamarixetin. Therefore, the studied flavonoids preventthe nuclear translocation β-catenin through interactingwith GSK 3β and different components of Wnt signalingpathway thereby downregulates P-gp overexpression indrug resistant oral carcinoma KB cells (Fig. 7).

ConclusionCollectively, flavonoids enhanced doxorubicin efficacythrough modulating Wnt/β-catenin signaling, downreg-ulating ABCB1 overexpression and augmenting doxorubicin-induced G2/M arrest and apoptosis in multidru-g-resistant KBCHR8–5 cells. Thus, flavonoids may beconsidered as an MDR reversal agent after confirming invivo chemosensitizing potential in preclinical animalmodels.

Additional files

Additional file 1: Figure S1. A) Pymol outlook of ligand bindingdomain (LBD) of GSK 3β with quercetin. B) Ligplot image illustratehydrogen and hydrophobic bonding of GSK 3β with quercetin (ii). A)Pymol outlook of the ligand binding domain (LBD) of GSK 3β with rutin.B) Ligplot image indicates hydrogen bonding and hydrophobic

interactions of GSK 3β with rutin (iii). A) Pymol outlook of the ligandbinding domain (LBD) of GSK 3β with epicatechin 3 gallate. B) Ligplotview of hydrogen and hydrophobic bonding of GSK 3β with epicatechin3 gallate. (iv). A) Pymol image show of the ligand binding domain (LBD)of GSK 3β with tamarixetin. B) Ligplot image illustrate hydrogen andhydrophobic interactions of GSK 3β with tamarixetin. Figure S2. Wnt,GSK 3β and β-catenin mRNA and protein expression levels in KB3–1 andKBCHR8–5 cell lines. Expression levels were normalized with the expres-sion pattern ofβ-actin levels. Data are given as mean ± SEM of three independentexperiments. Data not sharing a similar marking (a, b…) differ significantlyat P < 0.05 vs. control (DMRT). Figure S3. Quantification of protein andRNA are depicted as graph. The densitometry values show means ± SDfrom three independent immunoblots. The relative density of protein ex-pression levels were normalized to the β-actin protein expression pattern.The mRNA expression pattern was normalized with 18S and the image il-lustrates quantification of three independent analysis (means ± S.D). Datanot showing a similar symbol differ significantly at p ≤ 0.05 (DMRT). TableS1. Induced-fit docking of flavonoids against GSK 3β. Docking analysiswas carried out for 5 flavonoids, which show glide energy, docking score,hydrogen bond interactions. The tested flavonoids exhibit strong inter-and intramolecular interactions with drug-binding pocket of GSK 3β.(DOC 808 kb)

AbbreviationsABCB1: ATP-binding cassette sub-family B member 1; APC: Adenomatouspolyposis coli; BAX: BCL2-Associated X Protein; BCL-XL: B cell lymphoma-extra large; CDK2: Cyclin-dependent kinase 2; CDKN1A: Cyclin-dependentkinase inhibitor 1A; FZD: Frizzled 1; GSK 3β: Glycogen synthase kinase 3β;LBD: Ligand Binding Domain; LRP: Lipoprotein receptor-related protein;MDR1: Multidrug resistance protein 1; MTT: 3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide; PBS: Phosphate buffered saline; PDB: ProteinData Bank; P-gp: P-glycoprotein; PI: Propidium iodide; qRT-PCR: Quantitativereal-time polymerase chain reaction; RCSB: Research Collaboratory forStructural Bioinformatics; Wnt: Wingless-related integration site

Fig. 7 Flavonoids downregulate mdr1 expression through wnt/β-catenin signaling pathway

Mohana et al. BMC Cancer (2018) 18:1168 Page 9 of 11

RETRACTED ARTIC

LE

FundingThis work was supported by a grant from the Science and EngineeringResearch Board, New Delhi, India (Ref. No. SB/EMEQ-504/2014). This work wasalso supported by the Indian Council of Medical Research, New Delhi in theform of Senior Research Fellowship to the first author S. Mohana (Ref. No.45/6/2014/HAE-BMS). No specific funding was received to design of the studyand collection, analysis, and interpretation of data and in writing themanuscript.

Availability of data and materialsThe data generated during the study are not publicly accessible becausethey were used in the current research program, but are accessible from thecorresponding author on rational request.

Authors’ contributionsSM, MG and NRP performed the molecular biology experiments andparticipated in the data acquisition and analysis. SM, DV and DA carried outthe molecular docking analysis. Chemosensitizing experiments, Western blotsand Cell cycle analysis were carried out by SM and MG. MG and NRP re-written and finalized the revised manuscript. NRP conceived and designedthe experiments and interpreted the data of the manuscript. All authors readand approved the final manuscript.

Ethics approval and consent to participateNot applicable.

Consent for publicationNot applicable.

Competing interestsThe authors declare that they have no competing interests.

Publisher’s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations.

Author details1Department of Biochemistry and Biotechnology, Annamalai University,Annamalai Nagar, Tamil Nadu 608 002, India. 2Bioinformatics InfrastructureFacility (BIF), University of Madras, Guindy Campus, Chennai, Tamil Nadu,India. 3CAS in Crystallography and Biophysics, University of Madras, GuindyCampus, Chennai, Tamil Nadu, India.

Received: 9 January 2018 Accepted: 16 November 2018

References1. Cancer multidrug resistance. Diseases. Nat Biotechnol. 2000;18:IT18–20.2. Kathawala RJ, Wang YJ, Shukla S, Zhang YK, Alqahtani S, Kaddoumi A,

Ambudkar SV, Ashby CR Jr, Chen ZS. ATP-binding cassette subfamily Bmember 1 (ABCB1) and subfamily C member 10 (ABCC10) are not primaryresistance factors for cabazitaxel. Chinese J of Can. 2015;34:1–6.

3. Abdallah HM, Al-Abd AM, El-Dine RS, El-Halawany AM. P-glycoproteininhibitors of natural origin as potential tumor chemo-sensitizers: a review. Jof Adv Res. 2015;6:45–62.

4. Mohana S, Ganesan M, Agilan B, Karthikeyan R, Srithar G, Beaulah Mary R,Ananthakrishnan D, Velmurugan D, Rajendra Prasad N, Ambudkar SV.Screening dietary flavonoids for the reversal of P-glycoprotein-mediatedmultidrug resistance in cancer. Mol BioSyst. 2016;12:2458–70.

5. Shtil A, Shushanov A, Moynova E, Stavrovskaya A. Frequency of metastasisin Syrian hamster tumor cells selected for low levels of “typical” multidrugresistance. Exp Toxicol Pathol. 1994;6:257–62.

6. Shen DY, Zhang W, Zeng X, Liu CQ. Inhibition of Wnt/β-catenin signalingdownregulates P-glycoprotein and reverses multi-drug resistance ofcholangiocarcinoma. Cancer Sci. 2013;104:1303–8.

7. MacDonald BT, Tamai K, He X. Wnt/b-catenin signaling: components,mechanisms, and diseases. Dev Cell. 2009;17:9–26.

8. Sarkar FH, Li Y, Wang Z, Kong D. Cellular signaling perturbation by naturalproducts. Cell Signal. 2009;21(11):1541–7.

9. Amado NG, Fonseca BF, Cerqueira DM, Neto VM, Abreu JG. Flavonoids:potential Wnt/beta-catenin signaling modulators in cancer. Life Sci. 2011;89(15–16):545–54.

10. Kitagawa S, Nabekura T, Kamiyama S. Inhibition of P-glycoprotein functionby tea catechins in KB-C2 cells. J Pharm Pharmacol. 2004;56(8):1001–5.

11. Friesner RA, Banks JL, Murphy RB, Halgren TA, Klicic JJ, Mainz DT, RepaskyMP, Knoll EH, Shelley M, Perry JK, Shaw DE, Francis P, Shenkin PS. Glide: anew approach for rapid, accurate docking and scoring. 1. Method andassessment of docking accuracy. J Med Chem. 2004;47:1739–49.

12. Suite S. Schrodinger. New York: LLC; 2009.13. Singh S, Prasad NR, Chufan EE, Patel BA, Wang YJ, Chen ZS, Ambudkar SV,

Talele TT. Design and synthesis of human ABCB1 (P-glycoprotein)inhibitors by peptide coupling of diverse chemical scaffolds on carboxyland amino termini of (S)-valine-derived thiazole amino acid. J MedChem. 2014;57:4058–72.

14. Muthusamy G, Balupillai A, Ramasamy K, Shanmugam M, Gunaseelan S,Mary B, Prasad NR. Ferulic acid reverses ABCB1-mediated paclitaxelresistance in MDR cell lines. Eur J Pharmacol. 2016 Sep 5;786:194–203.

15. Jaramillo AC, Saig FA, Cloos J, Jansen G, Peters GJ. How to overcome ATP-binding cassette drug efflux transporter-mediated drug resistance. CancerDrug Resist. 2018;1:6–29.

16. Huang GL, Song W, Zhou P, Fu QR, Lin CL, Chen QX, Shen DY. Oncogenicretinoic acid receptor gamma knockdown reverses multi-drug resistance Z.Chen et al. Phytomedicine 43 (2018) 37–45 44 of human colorectal cancervia Wnt/beta-catenin pathway. Cell Cycle. 2017;16:685–92.

17. Chen Z, Huang C, Ma T, Jiang L, Tang L, Shi T, Zhang S, Zhang L, Zhu P, Li J,Shen A. Reversal effect of quercetin on multidrug resistance via FZD7/β-catenin pathway in hepatocellular carcinoma cells. Phytomedicine.2018 Apr 1;43:37–45.

18. Hung TH, Hsu SC, Cheng CY, Choo KB, Tseng CP, Chen TC, Lan YW, HuangTT, Lai HC, Chen CM, Chong KY. Wnt5A regulates ABCB1 expression inmultidrug-resistant cancer cells through activation of the non-canonicalPKA/β-catenin pathway. Oncotarget. 2014 Dec;5(23):12273.

19. Miron A, Aprotosoaie AC, Trifan A, Xiao J. Flavonoids as modulators ofmetabolic enzymes and drug transporters. Ann N Y Acad Sci. 2017 Jun;1398(1):152–67.

20. Pandey MK, DeGrado TR. Glycogen synthase kinase-3 (GSK-3)-targetedtherapy and imaging. Theranostics. 2016;6(4):571.

21. Shin D, Lee SC, Heo YS, Lee WY, Cho YS, Kim YE, et al. Design and synthesisof 7-hydroxy-1H-benzimidazole derivatives as novel inhibitors of glycogensynthase kinase-3beta. Bioorg Med Chem Lett. 2007;17:5686–9.

22. Coffman K, Brodney M, Cook J, Lanyon L, Pandit J, Sakya S, et al. 6-amino-4-(pyrimidine-4-yl) pyridones: novel glycogen synthase kinase-3beta inhibitors.Bioorg Med Chem Lett. 2011;21:1429–33.

23. Sivaraman D, Panneerselvam P. Screening of potential glycogen synthasekinase -3β inhibitors from herbal Lead by in silico docking technique. Int JChemTech Res. 2015;8(6):834–42.

24. Johnson JL, Rupasinghe SG, Stefani F, Schuler MA, Gonzalez de Mejia E.Citrus flavonoids luteolin, apigenin, and quercetin inhibit glycogen synthasekinase-3β enzymatic activity by lowering the interaction energy within thebinding cavity. J Med Food. 2011;14(4):325–33.

25. Iftikhar H, Rashid S. Molecular docking studies of flavonoids for theirinhibition pattern against β-catenin and pharmacophore model generationfrom experimentally known flavonoids to fabricate more potent inhibitorsfor Wnt signaling pathway. Pharmacogn Mag. 2014;10(2):S264–71.

26. Park S, Choi J. Inhibition of β-catenin/Tcf signaling by flavonoids. J cellBiochem. 2010; 110(6):1376–85.Kim WK, bang MH, Kim ES, Kang NE,Jung KC, Cho HJ, park JH. Quercetin decreases the expression of ErbB2and ErbB3 proteins in HT-29 human colon cancer cells. J Nutr Biochem.2005;16:155–62.

27. Amado NG, Cerqueira DM, Menezes FS, da Silva JF, Neto VM, Abreu JG.Isoquercitrin isolated from Hyptis fasciculata reduces glioblastoma cellproliferation and changes β-catenin cellular localization. Anticancer Drugs.2009;20:543–52 Flahaut.

28. Meier R, Coulon A, Nardou KA, Niggli FK, Martinet D, Beckmann JS, JosephJM, Mühlethaler-Mottet A, Gross N. The Wnt receptor FZD1 mediateschemoresistance in neuroblastoma through activation of the Wnt / beta-catenin pathway. Oncogene. 2009;28:2245–56.

29. Correa S, Binato R, Du Rocher B, Castelo-Branco MT, Pizzatti L, Abdelhay E.Wnt/β-catenin pathway regulates ABCB1 transcription in chronic myeloidleukemia. BMC Cancer. 2012;12:303.

Mohana et al. BMC Cancer (2018) 18:1168 Page 10 of 11

RETRACTED ARTIC

LE

30. Pinzón-Daza ML, Salaroglio IC, Kopecka J, Garzòn R, Couraud PO, Ghigo D,Riganti C. The cross-talk between canonical and non-canonical Wnt-dependent pathways regulates P-glycoprotein expression in human blood-brain barrier cells. J Cereb Blood Flow Metab. 2014;34:1258–69.

31. Kim H, Seo EM, Sharma AR, Ganbold B, Park J, Sharma G, Kang YH, Song DK,Lee SS, Nam JS. Regulation of Wnt signaling activity for growth suppressioninduced by quercetin in 4T1 murine mammary cancer cells. Int J Oncol.2013;43:1319–25.

32. Riganti C, Salaroglio IC, Pinzòn-Daza ML, Caldera V, Campia I, Kopecka J,Mellai M, Annovazzi L, Couraud PO, Bosia A, Ghigo D, Schiffer D.Temozolomide down-regulates P-glycoprotein in human blood-brain barriercells by disrupting Wnt3 signaling. Cell Mol Life Sci. 2014;71:499–516.

33. Zhang H, Zhang X, Wu X, Li W, Su P, Cheng H, Xiang L, Gao P, Zhou G.Interference of frizzled 1 (FZD1) reverses multidrug resistance in breast cancercells through the Wnt/β-catenin pathway. Cancer Lett. 2012;323:106–13.

34. Aung TN, Qu Z, Kortschak RD, Adelson DL. Understanding the effectivenessof natural compound mixtures in cancer through their molecular mode ofaction. Int J Mol Sci. 2017;18(3):656.

35. Kawahara T, Kawaguchi-Ihara N, Okuhashi Y, Itoh M, Nara N, Tohda S.Cyclopamine and quercetin suppress the growth of leukemia andlymphoma cells. Anticancer Res. 2009;29(11):4629–32.

36. Shan BE, Wang MX, Li RQ. Quercetin inhibit human SW480 colon cancergrowth in association with inhibition of cyclin D1 and survivin expressionthrough Wnt/beta-catenin signaling pathway. Cancer Invest. 2009;27(6):604-12.

37. Luo K, Gu X, Liu J, Zeng G, Peng L, Huang H, Jiang M, Yang P, Li M, Yang Y,Wang Y, Peng Q, Zhu L, Zhang K. Inhibition of disheveled-2 resensitizescisplatin-resistant lung cancer cells through down-regulating Wnt/beta-catenin signaling. Exp Cell Res. 2016;347:105–13.

38. Zhou H, Lin C, Zhang Y, Zhang X, Zhang C, Zhang P, Xie X, Ren Z. miR506enhances the sensitivity of human colorectal cancer cells to oxaliplatin bysuppressing MDR1/P-gp expression. Cell proliferation. 2017;50:e12341.

39. Park EJ, Kwon HK, Choi YM, Shin HJ, Choi S. Doxorubicin inducescytotoxicity through upregulation of pERK-dependent ATF3. PLoS One.2012;7:e44990.

40. Chen H, Landen CN, Li Y, Alvarez RD, Tollefsbol TO. Epigallocatechin gallateand sulforaphane combination treatment induce apoptosis in paclitaxel-resistant ovarian cancer cells through hTERT and Bcl-2 down-regulation. ExpCell Res. 2013;319:697–706.

41. Thottassery JV, Zambetti GP, Arimori K, Schuetz EG, Schuetz JD. p53-dependent regulation of MDR1 gene expression causes selective resistanceto chemotherapeutic agents. Proc Natl Acad Sci U S A. 1997;94:11037–42.

Mohana et al. BMC Cancer (2018) 18:1168 Page 11 of 11

RETRACTED ARTIC

LE