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European Journal of Medicinal Chemistry 58 (2012) 390e395

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European Journal of Medicinal Chemistry

journal homepage: http: / /www.elsevier .com/locate/ejmech

Short communication

Hesperetin: An inhibitor of the transforming growth factor-b (TGF-b) signalingpathway

Yong Yang a,*, Joy Wolfram a, Haifa Shen a, Xiaohong Fang b, Mauro Ferrari a,*aDepartment of Nanomedicine, The Methodist Hospital Research Institute, Houston, TX 77030, USAbBeijing National Laboratory for Molecular Sciences, Institute of Chemistry, Key Laboratory of Molecular Nanostructures and Nanotechnology, Chinese Academy of Sciences,Beijing 100190, PR China

a r t i c l e i n f o

Article history:Received 16 May 2012Received in revised form4 September 2012Accepted 16 October 2012Available online 25 October 2012

Keywords:HesperetinTGF-b inhibitionAtomic force microscopySingle-molecule fluorescence imaging

* Corresponding authors. Tel.: þ1 7134414117.E-mail addresses: yyang@tmhs.org (Y. Yang), mfer

0223-5234/$ e see front matter � 2012 Elsevier Mashttp://dx.doi.org/10.1016/j.ejmech.2012.10.028

a b s t r a c t

We have identified a previously unknown function of the natural compound, hesperetin. Here, wedemonstrate that this small molecular agent is able to inhibit the transforming growth factor-b (TGF-b)signaling pathway. Single-molecule force measurements and single-molecule fluorescence imaging showthat hesperetin interferes with ligandereceptor interactions. Furthermore, by Western blot analysis, itwas confirmed that hesperetin also inhibits the phosphorylation of Smad3, a down-stream target of theTGF-b pathway. In addition we demonstrated that this compound hinders TGF-b1-induced cancer cellmigration and invasion. These results suggest a potential future application for hesperetin as a TGF-b inhibitor in cancer therapy.

� 2012 Elsevier Masson SAS. All rights reserved.

1. Introduction

Hesperetin (40-methoxy-30,5,7-trihydroxyflavanone), a naturalpredominant flavanone found in citrus fruits and used in Chinesemedicine, has previously been shown to have anti-cancer activityby inhibiting proliferation or inducing apoptosis [1e5], inhibitingangiogenesis [2,6] and by acting as an anti-oxidant [7e9]. Here weexamine the effect of hesperetin on the transforming growthfactor-b (TGF-b) pathway. TGF-b signaling is initiated by thebinding of a ligand to TGF-b receptors, which thereby form a het-eromeric signaling complex. This leads to the phosphorylation ofa subset of downstream signaling molecules, the receptor-activatedSmads, which regulate the transcription of target genes [10,11].

We have previously shown that naringenin, another citrus bio-flavonoid, is able to block the TGF-b pathway [12]. In this study, wedemonstrate that hesperetin likewise inhibits TGF-b signaling, andfurthermore we show that this inhibition blocks the metastaticproperties of cancer cells. To date, there are several syntheticinhibitors of the TGF-b signaling pathway that are at the pre-clinicalor clinical level [13e16]. These agents are powerful tools forunderstanding and targeting signal transductionpathways involvedin pathological processes. Hesperetin and naringenin represent

rari@tmhs.org (M. Ferrari).

son SAS. All rights reserved.

a new group of agents that can suppress the TGF-b pathway, andcould potentially have several advantages compared to theirsynthetic counterparts (e.g. lower toxicity, unique mechanism ofinhibition and multiple advantageous effects).

2. Results and discussion

2.1. TGF-b ligandereceptor binding

To determine whether hesperetin affects ligandereceptorinteractions we performed single-molecule force spectroscopy inliving cells, measuring the binding force and binding probability ofTGF-b1 and TGF-b type II receptor (TbRII). The ligand was attachedto the atomic force microscopy (AFM) tip and then inserted ontoHeLa cells, which naturally express TbRII (Fig.1a and b). The ruptureforce was measured by moving the tip in and out of contact withthe cell surface. The rupture force was identified as a suddendeviation in the smooth force curve [17]. To confirm that themeasured forces were specific to the ligandereceptor pair ofinterest, the extracellular domain of TbRII (TbRII-ECD) was added asa control (Fig. S1a). Force distribution histograms were shown todisplay a single maximum peak with a Gaussian fit, indicating thatsingle-molecule forces were measured (Fig. S1b and c). Thefrequency of adhesion events was low, thereby, ensuring that theforce was mediated by a single ligandereceptor pair (<30%

Fig. 1. Measurements of binding between TGF-b1 and TbRII in living cells. (a and b) Schematic diagram of single-molecule force measurements in living cells with a ligand-modifiedAFM tip in the absence (a) or presence (b) of the extracellular domain of TbRII (TbRII-ECD). The tips were positioned directly above a cell expressing the desired receptors. (c)Binding probabilities derived from AFM force curves. Cells were treated with vehicle (0.1% DMSO), hesperetin (100 nM) or the extracellular domain of TbRII (TbRII-ECD). Data isexpressed as mean � SD. *P < 0.05. (d) Fluorescence intensity by flow cytometry using biotinylated TGF-b1 and avidin-FITC. Groups comprise vehicle (0.1% DMSO), hesperetin(100 mM) and negative control reagent.

Y. Yang et al. / European Journal of Medicinal Chemistry 58 (2012) 390e395 391

adhesion events: >85% probability of single-molecule forcemeasurement) [18e20]. The results indicate that the addition ofhesperetin to cell culture lowers the probability that the ligand willbind to the receptor (vehicle: 23.4% � 2.5%, hesperetin:10.5% � 1.7%) (Fig. 1c). When binding does occur, however, therupture force is similar to that of the control (Fig. S1b and c). Tofurther confirm that hesperetin inhibits the interaction betweenthe ligandereceptor pair, flow cytometry was performed usingbiotinylated TGF-b1 and avidin-FITC. Fig. 1d demonstrates that thefluorescence signal decreases in the presence of hesperetin.

2.2. TGF-b receptor dimerization

It is known that the TGF-b receptors exist as monomers at lowexpression levels, and follow the general ligand-induced receptordimerization model for activation upon TGF-b1 stimulation [21,22].Here we have used total internal reflection fluorescence microscopy(TIRFM) to image individual GFP-tagged TbRII molecules on the cellmembrane, in order to examine the effect of hesperetin on dimerformation. As shown in a typical TIRFM image of resting HeLa cells(Fig. 2a andMovie S1), TbRII-GFPmolecules appear as well-disperseddiffraction-limitedfluorescent spots (5� 5 pixels, 800� 800 nm). Thefluorescence intensities of these spotswereusuallymaintained for lessthan 5 s after dropping back to background level, indicating that theyare single TbRII molecules. The fluorescence intensity distribution ofthe spots exhibited a sum of two Gaussian distributions (Fig. S2a). Thefirst population, representing TbRII monomers, had a peak intensityclose to that of a single GFP molecule (Fig. S2c), while the secondpopulation, corresponding to TbRII dimers, had a peak value ofapproximately twice this size (Fig. S2a). Uponadditionof hesperetin toligand-stimulatedcells thepercentageofdimersdecreased from40.2%to19.9% (Fig. 2bandc). In theabsenceofTGF-b1,hesperetinwasunableto affect receptor dynamics (Fig. S2a and b). An alternative way to

examine dimer formation is by utilizing the bleaching-step countingmethod, where one-step photobleaching spots represent monomersand two-step spots represent dimers (Fig. S3) [21e23]. With thistechnique we show that hesperetin lowers the frequency of dimers(control: 39.9 � 2.5%, hesperetin: 19.5 � 1.5%) (Fig. 2d).

Supplementary video related to this article can be found athttp://dx.doi.org/10.1016/j.ejmech.2012.10.028.

2.3. TGF-b downstream signaling

In order to confirm that hesperetin inhibits TGF-b signalingtransduction, protein levels of a down-stream molecule, phos-phorylated Smad3, were investigated. The result demonstrates thathesperetin effectively reduces the TGF-b1 induced phosphorylationof Smad3 inHeLa cells (Fig. 3a). Furthermore, in the presence of TGF-b1 hesperetin administration significantly decreases the nucleartranslocation of Smad3 (Fig. 3b). To further investigate the effect ofhesperetin on TGF-b1 downstream signaling we also examined theexpression of p21, an inhibitor of cell cycle progression. Previousreports have shown that TGF-b1 stimulation leads to an increase inthe expression of p21 [24]. Our results demonstrate that hesperetinsignificantly inhibits TGF-b1 stimulated expression of p21 (Fig. 3c).

2.4. Cell invasion and migration

There is a controversy surrounding the role of the TGF-b pathway in cancer progression. Although the TGF-b signalingcascade suppresses tumor growth in early carcinogenesis, mainlythrough the ability of TGF-b to inhibit the cell cycle, it paradoxicallyfavors tumor progression during late-stage metastasis [25]. Tobetter understand the inhibitory effect of hesperetin on TGF-b signaling propagation we investigated the effects of this agent onTGF-b1-induced cell invasion and scattering. A scratch motility

Fig. 2. Measurements of TbRII monomers and dimers. (a) A typical single-molecule fluorescence image of TbRII-GFP molecules on a HeLa cell membrane. Scale bar: 4 mm (b and c).Fluorescence intensity distribution of individual TbRII-GFP spots on cells incubated with vehicle (0.1% DMSO, N ¼ 336) (b) or 100 mM hesperetin (N ¼ 347). (c) Solid curves show thefitting of the Gaussian function with the arrowheads indicating peak positions and numbers in parentheses are the respective fractions. (d) Frequency of one- and two-stepbleaching events of TbRII-GFP under various conditions. Data is expressed as mean � SD. *P < 0.05.

Y. Yang et al. / European Journal of Medicinal Chemistry 58 (2012) 390e395392

assay demonstrates that starved untreated HeLa cells exhibit onlylimited wound closure activity, while TGF-b1 treated cells show anacceleration of wound closure. However, when adding hesperetin(100 mM, 24 h), the cell migration capacity was reduced (Fig. 4a andb). The ability of hesperetin to prevent TGF-b1 induced invasionwas similarly examined. By using the Boyden chamber assay it wasconfirmed that hesperetin also suppresses the cells ability to invadeacross a collagen matrix (Fig. 4c). Furthermore, we show thathesperetin has low cellular toxicity when used at the sameconcentration as in the migration assays (Fig. S4).

3. Conclusion

In summary, we have discovered a novel TGF-b signalingantagonist and verified its function by single-molecule fluores-cence microscopy and force spectroscopy. Our results demonstratethat hesperetin reduces the binding probability of TGF-b1 to itsreceptor TbRII, thereby reducing receptor dimerization and acti-vation. We further demonstrate that the downstream signaltransduction event of Smad3 phosphorylation is inhibited. More-over, cell scattering and invasion experiments reveal that hesper-etin has the potential to become an anticancer agent for theinhibition of TGF-b induced tumor progression.

4. Experimental protocols

4.1. Cell culture

HeLa cells were cultured with Dulbecco Modified Eagle Medium(DMEM) (Gibco) supplemented with 10% fetal bovine serum (FBS)(HyClone). Cells were maintained at 37 �C and 5% CO2.

4.2. Atomic force microscopy (AFM)

Atomic force microscopy (AFM) was used to measure thebinding force between transforming growth factor-b1 (TGF-b1) andTGF-b type II receptor (TbRII). The AFM tip (type: NP, from Veeco,Santa Barbara, CA, USA) with TGF-b1 was prepared as previouslyreported [18,20]. The force was measured with a PicoSPM II,a PicoScan 3000 controller, a large scanner (Molecular Imaging,Tempe, AZ) and an inverted fluorescence microscope (OlympusIX71, Japan). The loading rate of force measurements was1.0 � 104 pN/s. The force curves were recorded and analyzed byPicoScan 5 software (Molecular Imaging, Tempe, AZ). All forceswere measured in living HeLa cells with contact mode at roomtemperature. Cells were treated with vehicle (0.1% dimethyl sulf-oxide (DMSO)), hesperetin (100 mM) or the extracellular domain ofTbRII for 1 h.

4.3. Flow cytometry

Flow cytometry was used to measure the binding of TGF-b1 toTbRII. TGF-b was labeled using the Fluorokine TGF-b-biotin kit(R&D Systems) according to manufacturer’s instructions. HeLa cellswere incubated with vehicle (0.1% DMSO) or hesperetin (100 mM),in the presence of biotinylated TGF-b1 for 1 h.

4.4. Single-molecule fluorescence microscopy

Single-molecule fluorescence microscopy was performed tostudy TbRII dimerization. A TbRII-GFP expression plasmid wascreated by subcloning the gene into the HindIII and BamHI sites ofpEGFP-N1 (Clontech) [12,21,22]. Cells were cultured in 35-mm

Fig. 3. Effects of hesperetin on TGF-b downstream signaling. (a) Western blot analysisof phosphorylated Smad3 (p-Smad3) in HeLa cell lysates. (b) Immunofluorescenceimaging of Smad3. In the presence of hesperetin the nuclear translocation of Smad3 issuppressed. Scale bar: 20 mm. (c) Western blot analysis of p21 in HeLa cell lysates.

Y. Yang et al. / European Journal of Medicinal Chemistry 58 (2012) 390e395 393

glass-bottom dishes (MatTek Corporation) and transfected with0.2 mg/ml plasmids in phenol red-free DMEM using lipofect-amine2000 (Invitrogen). To achieve low-level protein expressioncells were incubated with the plasmid for 4 h. Cells received eithervehicle (0.1% DMSO) or 100 mM hesperetin (Sigma Aldrich) for 1 hbefore imaging. For ligand stimulation the transfected cells wereincubated for an additional 15 min with 10 ng/ml TGF-b1 (R&D).The fluorescence intensity measurements were performed onliving cells with a CO2 incubation system (INU-ZIL-F1, TOKAI HIT),while the photobleaching study was done on fixed cells (4% para-formaldehyde/PBS solution, 10 min). The intensity of single GFPmolecules was measured to confirm that it corresponded to that ofTbRII monomers. GFP proteinwas purified from Escherichia coli anddissolved in a high salt buffer (600mMNaCl,150mMPBS buffer, pH7.4) to prevent dimer formation and then immobilized on cover-slips through a biotin coupled GFP antibody (Clontech), as previ-ously reported [12,21,22].

Single-molecule fluorescence imaging was carried out using aninverted microscope (IX 71, Olympus, Japan) with a total internalreflective fluorescence illuminator, a 100X/1.45NA Plan Apochro-mat TIR objective (Olympus, Japan) and a 14-bit back-illuminated

electron-multiplying charge-coupled device (EMCCD) (AndoriXon DU-897 BV) [12,21,22]. A 488-nm argon laser with the powerof 5 mW (Melles Griot, Carlsbad, CA) was used to excite the GFP inepi-fluorescence mode. Before being directed into the EMCCD(gain: 300) the fluorescent signals were passed through two filters,BA510IF and HQ 525/50 (Chroma Technology). To insure homoge-neous illumination only the central quarter of the chip(256 � 256 pixels) was used for imaging analysis. MetaMorphsoftware (Molecular Device) was used to acquire and analyzemovies (200 frames for each sample at a frame rate of 10 Hz). Thebackground fluorescence was first subtracted from each frameusing the rolling ball method in Image J software (National Instituteof Health). The first frame of each movie was used for the selectionof fluorescent spots and the threshold was set at four times that ofthe mean intensity of an area lacking fluorescent spots. The imagewas then filtered again with a user-defined program in Matlab.

4.5. Western blotting

Western blotting was performed in order to study downstreamsignaling of the TGF-b pathway. For detection of Smad3, the starvedHeLa cells were treated with vehicle (0.1% DMSO) or hesperetin(100 mM) for 1 h in the absence or presence of TGF-b1. For thedetection of p21, the starved HeLa cells were treated with vehicle(0.1% DMSO) or hesperetin (100 mM) for 24 h in the absence orpresence of TGF-b1. Cells were resuspended in 200 ml of 0.9% salinewith protease inhibitors (1 mM phenylmethylsulfonyl fluoride,10 mg/ml aprotinin, 10 mg/ml leupeptin, and 10 mg/ml pepstatin A)and mixed with 200 ml double-strength Laemmli buffer (100 mMTriseHCl, pH 6.8, 200mMdithiothreitol, 4% SDS, 0.2% bromophenolblue, and 20% glycerol). Cell lysates were denatured for 10 min at95 �C. Cell lysates were separated by SDS gel electrophoresis (7.5%SDS gels) and transferred to polyvinylidene fluoride (PVDFmembranes) with 0.192 M glycine, 0.025 M Tris and 20% methanol.Membranes were saturated for 1 h at 4 �C in TBS, pH 7.4, containing5% nonfat milk and probed for 2 h at room temperature withprimary antibodies for p-Smad3 (Cell Signaling, dilution 1:1000),Smad3 (Cell Signaling, dilution 1:1000) or p21 (Cell Signaling,dilution 1:1000). After washing the blots were probed for 1 h atroom temperature with peroxidase-conjugated secondary anti-bodies. Antibody binding was detected by enhanced chem-iluminescence (Amersham Life Sciences, Amersham, UK).

4.6. Immunofluorescence imaging

HeLa cells were cultured in 35 mm dishes for a period of 24 h,followedbywashingwithPBS, and incubationwith fresh serum-freemedium for another 24 h. The nuclear translocation of Smad3 wasassessed by treating cells with 0.1% DMSO (vehicle) or hesperetin(100 mM) for 1 h, in the absence or presence of TGF-b1. Cells werethen fixed with 4% paraformaldehyde and permeabilized with 0.1%Triton X-100. 1% BSA in PBST (PBS containing 0.1% Tween-20) wasused to block nonspecific binding sites. The fixed cells were thenincubated with Smad3 monoclonal rabbit primary antibody over-night (Cell Signaling,1:200 dilution), followed bywashing and a 1 hincubation with Alexa 488-conjugated anti-rabbit IgG secondaryantibody (Invitrogen, 1:500 dilution). All antibodies were diluted inPBST solution containing 1% BSA. Fluorescence imaging was per-formed with a confocal microscope (Olympus IX81).

4.7. Scratch motility and invasion assay

Scratch motility and invasion assays were performed to inves-tigate TGF-b1-induced cell migration. Cells were grown overnightto confluency in serum-containing DMEM and then starved for

Fig. 4. Evaluation of hesperetin on TGF-b1-stimulated scattering migration (a and b) and invasion in HeLa cells (Boyden chamber assay) (c). The data represent mean � SD fromthree independent analyses. *P < 0.05.

Y. Yang et al. / European Journal of Medicinal Chemistry 58 (2012) 390e395394

24 h. For the scratch motility assay 1 �106 cells were seeded in a 6-well plate. The monolayer was scratched with a pipette tip andwashed with PBS to remove floating cells. The cells then receivedeither vehicle (0.1% DMSO) or hesperetin (100 mM) with or withoutTGF-b1 for 24 h. Five randomly selected fields in the scratched areawere photographed and the mean amount of cells/field wascalculated. For the invasion assay the cells (1 � 106 cells/ml) weresuspended in serum-free media and treated with vehicle andhesperetin, as mentioned above. The assay was performed in trip-licates using QCMTM 24-Well Collagen-Based Cell Invasion Assay(Millipore), according to the manufacturer’s instructions.

4.8. Cell proliferation assay

HeLa cells were seeded in 96-well plates (1 � 104 cells/well)with complete medium for 24 h, followed by treatment withvehicle (0.1% DMSO) or 0e200 mM of hesperetin for 24 h. Cellularproliferation was quantified using the CellTiter 96� AQueous Non-Radioactive Cell Proliferation Assay (Promega Corporation),according to the manufacturer’s instructions.

4.9. Statistical analysis

The data is expressed as the mean and standard deviation (S.D).Statistical significance was measured with the Student’s t-test(unpaired and two-tailed) (Microsoft Excel).

Conflicts of interest

All authors declare no competing financial interests.

Acknowledgments

The authors acknowledge supports from the following fundingsources: Ernest Cockrell Jr. Distinguished Endowed Chair (M.F.), USDepartment of Defense (W81XWH-09-1-0212) (M.F.), National

Institute of Health (U54CA143837, U54CA151668) (M.F.), NationalBasic Research Program of China (2007CB935601) (X.F.), NationalNatural Science Foundation of China (Nos 90713024, 20821003)(X.F.), Chinese Academy of Sciences (X.F.) and Nylands NationFinland (J.W.).

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.ejmech.2012.10.028.

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