Marcelina Starok

Pascal Preira

Muriel Vayssade

Karsten Haupt

Laurence Salome

Claire Rossi

Laurence Salomé

EGFR inhibition by curcumin in cancer

Keywords: Epidermal growth factor receptor, curcumin, autophosphorylation, tyrosine kinase inhibition, human epidermoid carcinoma cells

come

INTRODUCTION

The epidermal growth factor receptor (EGFR), also known as ErbB1 or HER1, is one of the most studied members of the receptor tyrosine kinase superfamily. 1 EGFR is a central element for cellular signal transduction and a regulator of essential epidermal functions, as proliferation, survival, differentiation or migration. [2][3][4] EGFR plays also a critical role in a variety of pathophysiological phenomena, particularly in cancer development. [5][6][7] The EGF receptor is characterized by a modular structure consisting of an extracellular ligandbinding domain, a single hydrophobic transmembrane region, and an intracellular part including the tyrosine kinase domain. In the generally accepted model of EGFR activation, the oligomerization of protein monomers is considered as a key intermediate step between binding of the growth factor and activation of the tyrosine kinase. Ligand binding to the extracellular domain stabilizes the receptor in its extended conformation, which promotes its dimerization. [8][9][10] In this conformation, the proximity of the tyrosine kinase domains promotes the transphosphorylation of tyrosine residues on both monomers, which creates docking sites for enzyme substratesintracellular proteins participating in signal transduction. 11 Numerous reports based on different approaches are supporting this concept, such as the observation of cluster formation by single molecule tracking or FRET. 12 The immense interest in this receptor was inspired by the finding that its excessive signalling, related to overexpression, mutations or autocrine stimulation, disrupts equilibrium between cell growth and apoptosis, leading to the development of a variety of solid tumors. 5,7,13,14 The most aggressive forms of tumors, correlated with poor prognosis and reduced chemosensitivity, are usually associated with EGFR overexpression. 7,15 For that reasons EGFR has been a key target of the pharmaceutical industry since its first clinical trial performed in 1990. 13,16,17 Two major approaches have been developed for targeting EGFR which demonstrated benefits in clinical trials: monoclonal antibodies (mAbs), which interact with the extracellular domain of the protein making ligand binding and consecutive dimerization impossible, and tyrosine kinase inhibitors (TKIs), usually targeting the ATP binding site of the tyrosine kinase domain. Although a number of mAbs or TKIs are commercially available for the treatment of a variety of cancers, ongoing efforts are made by academic research and industrial pharmaceutical R&D to develop new drugs and/or new strategies in order to reduce side-effects or resistance development. 18,19 Natural substances are the source of promising molecules with anticancer potential, such as epigallocatechin gallate, delphinidin or butein. 20,21 Among the polyphenols that inhibit EGFR signaling, the most widely studied is curcumin, found in the rhizomes of Curcuma longa (turmeric). [22][23][24][25][26] Curcumin exhibits remarkable pharmacological activities, such as antiinflammatory, anti-neoplastic, anti-oxidant and chemopreventive effects, and has been shown to be pharmacologically safe even at high doses (12 g/day). [27][28][29] Curcumin action includes the regulation of signal transduction pathways and the inhibition of enzymes and key transcription factors. 30 After treatment with curcumin, cancer cells display several features typical of apoptotic cells. 31,32 Inhibitory effect of curcumin on ligand-induced activity of EGFR was proven by studies on living cells, on proteins reconstituted in liposomes and on isolated proteins. [23][24][25] However, the exact mode of action of this molecule is not fully understood. 33 The present work is scrutinizing the mechanism of early curcumin action on EGFR autophosphorylation by different approaches: the effect of curcumin on EGFR activation was studied in vitro on separate EGFR domains and on human epidermoid carcinoma cells. The impact of curcumin on the fluidity of the lipid bilayer, which constitutes the direct environment of EGFR, was investigated as well. The results indicate a dual mode of action of curcumin, and thus contribute to our understanding of the interaction of curcumin with the cell membrane, and of receptor activity modulation.

MATERIALS AND METHODS

Cell culture. Human epidermoid carcinoma cell line A-431 was purchased from American Type Culture Collection (ATCC ® CRL-1555) and cultured in high glucose (4500 mg/L) Dulbecco's Modified Eagle's Medium (DMEM) (D6429 Sigma, France) supplemented with 10 % (v/v) fetal bovine serum (FBS) (Gibco, France) and 1 % (v/v) penicillin/ streptomycin (Gibco, France).

Routinely cells were kept in a humidified incubator at 37˚C with 5 % CO2. The culture medium was changed three times a week. Subculturing was performed once a week, using a standard trypsinization protocol with 0.25 % trypsin-EDTA (1X) (Gibco). For standard culture, cells were seeded into 75 cm 2 flasks (Falcon) at 1 500 000 cells per flask. For the ELISA based assay cells were cultured in 96-well black cell culture plates with clear bottom (BD Falcon) at 35 000 cells/well unless other indicated. Since curcumin stock solution was prepared in DMSO, control experiments were performed with DMSO (<0.04 % v/v), no DMSO effect on EGFR phosphorylation could be observed (data not shown).

Phospho-EGFR cell-based ELISA assay.

Cell-based immunoassay allows for simultaneous quantification of the phosphorylated EGFR (Y1068) and total EGFR (pan-protein) which is used for normalization of the results. The method does not require cell lysis and the assay was performed directly in the culture plate after cell fixation and permeabilization. A431 cells were seeded at 35 000 cells/well in the black cell culture 96-well plates with clear bottom (BD Falcon). After 24 h of growth, complete growth medium was replaced by the serum free medium for the following 24 h, preceded by a single wash with PBS. Next, serum free medium containing different concentrations of curcumin was introduced into the culture for 4 h and then 100 ng/ml of EGF (Sigma-Aldrich) in serum free medium was added for an additional hour in the dark. Both incubation steps were performed at 37ºC in 5 % CO2 atmosphere. Afterwards the cells were fixed with paraformaldehyde and immediately treated as described in the Human Phospho-EGFR/ ErbB1 (Y1068) cell-based ELISA protocol (R&D Systems). The fluorescence intensity was read at indicated wavelengths using a Cary Eclipse Fluorescence Spectrophotometer (Agilent Technologies) equipped with a plate reader. The phosphorylation levels are expressed as normalized relative fluorescence units (RFU).

Tyrosine kinase assay. EGFR tyrosine kinase domain (SRP5023, Sigma) was diluted from the stock at 0.1 µg/ml to the concentration 140 pM with Kinase Reacting Buffer provided in the Universal Tyrosine Kinase Assay Kit (Takara, Ozyme). Curcumin was added at the desired concentration to the solution and the reaction was initiated by addition of ATP. The phosphorylation reaction was performed in the dark in order to prevent curcumin degradation and was stopped after 30 min. Within this time frame, less than 5% of curcumin decomposed (as judged from UV/Vis spectra). Further steps were exactly as in the manual provided by manufacturer.

Absorbance was measured using a spectrophotometer equipped with a plate reader (Dynex MRX Microplate Reader). The data were analyzed based on a calibration curve prepared with the standard included in the set.

Biacore-based competition assay. Surface plasmon resonance (SPR) binding experiments were performed on a BIAcore T100 instrument (GE Healthcare) at 25°C in 10 mM HEPES buffer pH 7.4, containing 150 mM NaCl, 3.4 mM EDTA and 0.005 % Tween-20. Human recombinant EGF (R&D Systems) was coupled to a CM5 BIAcore sensor chip as follows: the chip was activated with a freshly prepared 1:1 (v/v) mix of 0.4 M 1-Ethyl-3-(dimethylaminopropyl) carbodiimide (EDC) with 0.1 M N-hydroxysuccinimide (NHS) for 7 min at 10 µl/min. EGF (200 µg/ml in 10 mM sodium acetate pH 4.0) was then injected on the activated surface at 10 µl/min for 10 min. An EGF-free control surface was prepared with the same immobilization procedure, but injecting buffer instead of EGF. Recombinant human EGFR Fc chimera covering the sequence of the extracellular domain of the protein, Met1-Ser645 (R&D Systems) was injected simultaneously over the EGF and the control surfaces at a series of concentrations (0 to 400 nM) with a flow rate 5 µl/min, for 7 min, time during which binding reached a plateau in each case. The final sEGFR binding sensogram was obtained by subtracting the bulk shift using the control sensogram. The equilibrium binding constant KD was determined to be 11 nM using the Biacore T100 Evaluation software.

Competition assays were performed by injecting sEGFR with curcumin or free EGF on the immobilized EGF in the same conditions. Mixtures were prepared 20 min prior to injection with a final concentration of 600 nM for sEGFR, 30 µM for curcumin and 6 µM for EGF. The surface was regenerated between sEGFR injections with 5 mM NaOH during 1 min at 5 µl/min.

Measurement of lipid bilayer fluidity.

Liposome formation and characterization. Lipids were dissolved in chloroform and mixed in a glass vial with the fluorescent probes at the desired ratio. Chloroform was evaporated under gentle nitrogen flow and the resulting film was stored in a dessicator in the dark and under vacuum for minimum 2 h to remove organic solvent residues. The dry film was then rehydrated to a final lipid concentration of 1 mg/ml in HEPES Buffer saline solution (HBS) (HEPES-Na 20 mM pH 7.4, NaCl 150 mM) by 5 min vortexing. Vesicles were produced from the multilamellar vesicles suspension by ultrasonic treatment (4 cycles of 3 min at 100 W each, separated by a 3 min pause) during which the suspension was kept in an ice bath, with a tip sonicator (Ultrasonic Processor Vibra Cell, Sonics Materials). The obtained suspension of small unilamellar vesicles (SUV) was filtered immediately through a 0.2 µm filter (Acrodisc). SUV size average and distribution were determined by dynamic light scattering (DLS) using a ZetaSizer Nano ZS (Malvern Instruments), yielding a rather homogenous size with a diameter of 58 ± 7 nm.

𝑟 = 𝐼 0 -𝐺𝐼 90 𝐼 0 + 2𝐺𝐼 90 (1)
where I0 is the fluorescence intensity measured with polarizer in parallel orientation (0º) and I90

the intensity in perpendicular orientation (excitation 0º and emission 90º). G is the correction factor derived from the ratio of emission intensity at 0 and 90º with the excitation polarizer at 90º and is taking into account the different sensitivity of the detection system for vertically and horizontally polarized light (Equation 2):

𝐺 = 𝐼 90 𝐼 0 (2)
Fluorescence Recovery After Photobleaching (FRAP). The diffusion coefficient of lipids was determined using FRAP. Experiments were carried out in a confocal laser-scanning system (LSM 710, Carl Zeiss) based on an AxioObserver Z1 inverted microscope (Carl Zeiss). Observations were done with a 63 x immersion objective. The duration of the recordings was set to 20 s.

The trajectories of all QD of a video sequence were determined using the Mutiple Target Tracing program developed by Sergé and collaborators. 36 Only trajectories of more than 5 s were further analyzed with a homemade program written in Visual Basic (VBA Excel, Microsoft) computing the Mean Square Displacement as a function of time interval, MSD(  n t), using the formula (Equation 3):

                 n N i i i n i i n y y x x n N t n MSD 1 2 2 1 ) ( ( 3
)
where n is the number of time intervals, t is the time interval between two successive frames (40 ms), N is the total number of frames, in our case N ≤ 500 frames, x(t) and y(t) are the QD coordinates at time t.

To obtain the most accurate and precise values of the diffusion coefficients, we estimated the short term diffusion coefficient D1-2 obtained from only the first two MSD(  n t) points:

D1-2 = [MSD(2t) -MSD(t)] / (4t)

RESULTS

Curcumin decreases the EGF induced phosphorylation level of EGFR in A431 cells.

We investigated the short-term effect of curcumin on the EGF-induced EGFR phosphorylation in human epidermoid carcinoma cells (A431) at low polyphenol concentrations. The choice of this cell line was motivated by the high expression level of EGFR, which was estimated to be up to 2-3 x 10 6 receptors per cell. 38 The fluorescence signal due to the presence of phosphorylated EGFR (Y1068) was measured in whole A431 cells and normalized to the fluorescence signal related to the total EGFR (pan-protein) content using a cell-based immunoassay. The Y1068 residue belongs to the group of the first tyrosine residues to be autophosphorylated. 39 As the ligand incubation time applied for phosphorylation studies was found to vary between 2 min and 1 h in the literature. [40][41][42] We first examined the EGFR phosphorylation yield as a function of the time of cell exposure to 100 ng/mL EGF (Figure S1). The highest phosphorylation yield was obtained for 1 h with a 4-fold increase compared to the basal level in the absence of EGF treatment. Therefore this exposure duration was used in the following experiments and the EGF induced phosphorylation degree was further taken as our reference to determine inhibition levels. Based on the previously published results 25 , curcumin pre-incubation time before EGF treatment was set to 4 h. Curcumin does not affect EGF binding to EGFR extracellular domain.

One hypothesis for curcuma action on ligand induced activation of EGFR is that this polyphenol interfers in the ligand binding to the extracellular domain that enhances dimer stability and as a consequence promotes trans-autophosphorylation. We used surface plasmon resonance spectroscopy to investigate the ability of curcumin to compete with ligand binding to EGFR. EGF was immobilized on a BIAcore CM5 sensor chip. The immobilization level of EGF yielded 295 ± 54 RU (three independent immobilizations) which is in the range of previously published data. 43 The soluble extracellular domain of EGFR (sEGFR) was passed at different concentrations over these surfaces and the maximum binding response (Rmax) was plotted against sEGFR concentration (Figure S2). The best fit of the generated binding curve was obtained using a twosites Langmuir model. We chose this model since it yielded a much better fit than the one-site model, and because sEGFR from human origin does not dimerize in solution, thus ruling out the cooperativity (Hill) model that has been suggested for sEGFR from other species. 44 The fit leads to the determination of two equilibrium constants KD1 = 0.97 ± 0.10 nM and KD2 = 60.76 ± 5.17

nM with equivalent maximum number of binding sites (BMAX1: 109.0 ± 4.3 and BMAX2: 166.2 ± 3.8, respectively). These two affinity levels for EGF may be due to different conformations that sEGFR can adopt in solution. There are two populations of EGFR regarding affinity towards binding EGF correlated with the conformational changes of the receptor. [45][46][47][48] However this does not constitute a limitation for our competition studies since in both case sEGFR bound strongly to the immobilized EGF and the KD are low enough for competitive assays.

Samples of 600 nM of sEGFR containing increasing concentrations of curcumin were injected over the EGF-derivatized sensor surface. No influence on the equilibrium SPR response of sEGFR binding could be observed even with up to a 50-fold molar excess of curcumin (Figure 2). A positive control was performed using a 1:10 mixture of sEGFR and free EGF, the binding of sEGFR to immobilized EGF was fully blocked in this case. This results shows that curcumin does not act as a ligand binding inhibitor.

Inhibitory effect of curcumin on EGFR tyrosine kinase domain activity.

The tyrosine kinase inhibitory effect of curcumin on a recombinant, 695-end, active human EGFR domain (EGFR-TK) was evaluated (Figure 3 The inhibition at 1 µM curcumin was equal to 26.8 ± 2.6 %. This effect increased slightly with the augmentation of the curcumin concentration and reached 30.8 ± 0.9 % at 5 µM. A partial inhibition of the EGFR kinase had already been observed with other inhibitor compounds using life cell kinase assays, 49 and was confirmed for curcumin by our results with A431 cells in Figure 1. To speculate on a potential reason for this partial inhibition of EGFR-TK, the above observation of high and low affinity binding of EGF to EGFR, leading us to postulate two populations of sEGFR, might in turn indicate the existence of two possible conformations of the whole protein and thus also of the EGFR-TK domain, with different affinities for the inhibitor.

Curcumin is thus able to act on the EGFR phosphorylation via a direct action on the tyrosine kinase domain as expected for a small and hydrophobic molecule. Nevertheless, the inhibition of tyrosine kinase activity by curcumin observed here appears to be slightly lesser than the inhibition of EGFR phosphorylation on the Y1068 site determined in A431 cells (Figure 1) which reached an average of 40 % for 5 µM of curcumin. This is all the more surprising given that in the TK assays the experiment was performed in an environment deprived of other factors than the tested agent, while in cells the total curcumin concentration is not available for interacting with the TK domain since it is located in different membrane compartments, i.e. incorporated into plasma membrane or nucleus. 50 This led us to consider the involvement of an additional mechanism in curcumin mediated EGFR inhibition at the cellular level. Curcumin is known to interact with the plasma membrane. 31,32,51 The membrane is the natural environment of EGFR and plays an important role in the receptor diffusion, which in turn is essential for its dimerization resulting in activation. The question was therefore whether curcumin could affect the membrane properties to an extent that would alter the EGFR lateral diffusion? To answer this question, analyses allowing to better understand the curcumin effect on the membrane fluidity were performed.

Curcumin decreases the lipid bilayer fluidity

Local membrane viscosity changes were assessed by steady-state fluorescence anisotropy studies providing a sensitive indicator of the motional freedom of a fluorescent probe inserted in a membrane. 52 The partitioning of two fluorescent probes, TMA-DPH and DPH, has been employed

to monitor the influence of curcumin on the lipid dynamics properties in different membrane regions. The polar region of TMA-DPH is anchored at the lipid-water interface and DPH locates in the hydrophobic core of the lipid bilayer. 53 The fluorescence anisotropy values are inversely proportional to membrane fluidity in the probe environment. 54 We performed the anisotropy measurements on pure DPPC vesicles. This strategy allowed us to determine the influence of curcumin on lipid fluidity at two different gel/liquid-crystalline phospholipid order, as DPPC has a phase transition at about 41.5°C. The effect of curcumin on the local fluidity along graded depths in the phospholipid bilayer is presented Figure 4. The anisotropy values were increasing with increasing curcumin concentrations over the whole temperature range for both fluorescent probes. This rise was observed in both the solid gel and liquid-crystalline phases, as well as around the melting transition. These results demonstrate a strong influence of curcumin on the fluidity of the lipid bilayer both in the interfacial layer and in the hydrophobic core. Interestingly, the critical concentration of curcumin at which the first significant changes appear was lower for TMA-DPH (1 µM) than for DPH (1.5 µM). If we consider the location of these probes in the lipid bilayer, we can assume that the external leaflet was more sensitive to the curcumin triggered changes than the inner hydrophobic compartment.

That implies that the molecule is gradually embedded into membrane structure, at low concentration it stands exclusively in the interface and when the concentration increases, it enters deeper into the hydrophobic core. Such a mechanism of the interaction of the curcumin with the membrane has been already described by Barry and collaborators. 55 Furthermore, the curcumin effect on the lateral diffusion of phospholipids in the lipid bilayer was quantified using the fluorescence recovery after photobleaching (FRAP) technique. This assay was performed on an EggPC/NBD-DPPE bilayer supported on a glass surface (SLB). The florescence recovery of a bleached area of the SLB containing fluorescent lipids was monitored after incubation with increasing concentrations of curcumin added in the buffer reservoir above the bilayer. The lipid diffusion coefficient (D) was plotted as a function of curcumin concentration in Figure 5. The value of the lateral coefficient in the absence of lipids is consistent with the previously published data for SLB of the same lipid composition. 56,57 Curcumin significantly decreases lipid lateral diffusion at concentration above 0.4 µM. Together with the fluorescence anisotropy data, these findings confirm the proposed scenario of the rigidification of the membrane by curcumin. In membrane models, this effect is significant already at low curcumin concentrations. Therefore one may conclude that it should also impact the diffusion of the membrane proteins. Nevertheless, all of the aforementioned tests were conducted in biomimetic membrane models. These structures, despite having several advantages for practical reasons, do not exactly mirror the complex cell membrane properties. In order to confirm our hypothesis that receptor diffusion is impacted by the membrane rigidification caused by curcumin, we studied the alteration of EGFR mobility by curcumin in the plasma membrane of A431 cells in single particle tracking experiments. In fact, the EGFR diffusion coefficient is in the 10 58,59 , which is at the limit of detection of FRAP. 60 The evaluation of its decrease caused by curcumin required thus a more sensitive technique.

Single particle tracking of quantum dot labelled EGFR in the plasma membrane of A431 cells treated with curcumin

We employed the single particle tracking technique to measure the diffusion coefficient of the EGFR in the A431 cells treated or not with curcumin. The conditions of cells treatment were exactly the same as those applied for studies of the EGFR phosphorylation level in cells.

Functionalisation of quantum dots (QD) was optimised in a two-step procedure. Firstly, binding affinity of biotinylated EGFR antibodies purchased from three different suppliers were tested to choose the one with the highest affinity. Secondly, QD conjugated to Ab at different ratios were prepared and 20 short trajectories of 5 s acquired to rapidly evaluate the diffusion coefficient. The best condition was chosen as the one giving the highest value of the diffusion coefficient, corresponding to a QD:Ab molar ratio of 1:10. 37 Prior to SPT experiments, the specificity of the labelling was verified by counting the average number of QD bound per cell after incubation of the cells with QD conjugated to EGFR antibodies in comparison to bare QD. The fraction of unspecific binding was found to be 6.5%.

SPT data show variations in the short term diffusion coefficient D1-2 obtained in the different conditions. EGF addition to the cells starved in serum-free medium prior to the measurements caused significant elevation of the average diffusion coefficient of the EGFR, D1-2 = 0.034 ± 0.002 µm 2 s -1 , compared to that measured in cells not stimulated with the ligand, D1-2 = 0.025 ± 0.002 µm 2 s -1 . Curcumin addition to the cells medium before stimulation with the ligand reduced the EGF effect. The presence of the polyphenol at 1 µM in the sample pre-treated with the ligand did not affect significantly the measured diffusion coefficient, but at 5 µM curcumin the mean D1-2 = 0.025 ± 0.001 µm 2 s -1 , was reduced to its value in non-stimulated cells (Figure 6 and S3). Once established that curcumin affects the diffusion coefficient of the ligand activated EGFR in A431 cells, we further tested if the effect was depending of the EGF-mediated activation of the receptor. For that, the experiment was repeated for 5 µM curcumin without the addition of EGF.

As presented in Figure 6, the mean value of D1-2, compared to the one in the absence of curcumin (and EGF) also decreased in this case (D1-2 = 0.015 ± 0.001 µm 2 s -1 ). These findings suggest that curcumin reduces the EGFR diffusion in A431 plasma membrane independently of the ligand induced receptor activation.

DISCUSSION

In the present work, we aimed to investigate the mode of action of curcumin on the EGFR phosphorylation. We first evaluated the effect of curcumin on EGFR autophosphorylation in human epithelial cancer cells (A431). The curcumin treatment of A431 was already found to affect the autophosphorylation activity of the EGFR tyrosine kinase in a dose-and time-dependent manner. 25 In the cited study, the highest inhibition level was obtained for a 4 h pre-treatment of cells, and the lowest concentration tested was 10 µM. Most of the studies being conducted on the inhibition of EGFR kinase activity by curcumin in cells have been performed using high concentrations of curcumin up to 5 µM. 24,[61][62][63] However, such concentrations cannot be achieved by oral administration. Due to its poor bioavailability, the curcumin levels found in human serum are very low, mostly in the 10-100 nM range 64 ; the highest value reported was 1.77 +/-1.87 µM after taking 8 g of curcumin. 65 For these reasons, we restricted the range of the curcumin concentrations applied on cells and membranes models to up to 5 µM.

The results obtained on A431 cells indicate that short-term treatment with curcumin affects the phosphorylation of the Y1068 residue of EGFR. Curcumin significantly inhibits the EGF-mediated tyrosine kinase activity starting at 1 µM concentration after 4 hours of cell exposure to curcumin.

To our knowledge, this is the first time that an effect at such a low dose is reported. As Korutla and Kumar already indicated, the early effect of curcumin on EGFR phosphorylation suggests a direct action of curcumin on the membrane receptor at the molecular level. The author's assumption is that curcumin may interfere with the ligand induced activation of EGFR. 25 The molecular mechanism of the short-term inhibition of EGFR kinase activity by curcumin remains however unknown.

To elucidate the mechanism by which curcumin directly acts on EGFR, we performed in vitro experiments on separated recombinant EGFR domains. Surface competition assays were carried out in order to determine the ability of curcumin to compete the soluble EGFR extracellular domain binding to immobilized EGF. No effect on the SPR response of sEGFR binding to immobilized EGF could be observed even for concentrations corresponding to a 50-fold molar excess of curcumin. ELISA-based assays were employed to investigate the inhibitory effect of curcumin on tyrosine kinase activity of EGFR intracellular domain. 66 The results demonstrate that curcumin at 5 µM significantly lowered the EGFR-TK activity by 30 %. Thus, the direct action of curcumin on the receptor is on its intracellular domain.

However, these in vitro experiments on soluble EGFR domains reflect only partially the molecular mechanism of the short-term action of curcumin in cells. Like the majority of the tyrosine kinase inhibitors, curcumin is a highly hydrophobic molecule with a logP of 3.28, causing its accumulation in tissues and more specifically in the cellular membranes. 32, [67][68][69][70] The higher curcumin inhibitory level in cellulo than in solution indicates that an additional molecular mechanism may be involved in the early effect of curcumin on EGFR phosphorylation, related to an accumulation of the polyphenol in the membrane. Considering that curcumin has been suggested to change the properties of the cell membrane and to affect membrane-bound proteins in an indirect manner 71 , we hypothesized that EGF receptor autophosphorylation was also impacted by modulation of the membrane physical properties resulting from the curcumin interaction with the EGFR surrounding.

As a first-line study, we investigated the effect of curcumin on the lipid bilayer fluidity in biomimetic models. Cell membranes are composed of a lipid bilayer intercalated with other constituents such as proteins. The complexity of native membranes and their interactions with the intra-and extracellular environment makes the precise effect of the studied polyphenol on membrane properties difficult to track and to interpret. 72 As an illustration, although a decrease in the lateral mobility of lipids in membranes had already been reported for human cells treated with 10 µM of curcumin, this could not be clearly linked to the physical insertion of curcumin in membrane and was attributed to the induction by curcumin of reactive oxygen species production. 73 In this study, liposomes and planar lipid bilayers with different lipid compositions were employed to mimic the organization of the cell membrane matrix in a simplified and controlled manner. The measurements of TMA-DPH and DPH fluorescence anisotropy indicated that curcumin affects the bilayer fluidity in the region where the fluorescent probes were located.

A significant fluidity decrease was observed at the interphase between the hydrophilic and the hydrophobic parts of the bilayer starting 1 µM and in the hydrophobic region starting 1.5 µM, independent of the state of the lipid phase. The interaction mode of curcumin with the lipid bilayer has been described to be a two-step process. 74,75 Our results are consistent with the concentration threshold of 1.5 µM, at which curcumin begins to intercalate into the bilayer's hydrophobic core, as found by Chen and collaborators. The lateral fluidity of lipids in planar bilayer models is also strongly decreased by curcumin insertion. First significant changes occur starting at 0.4 µM of curcumin. As FRAP analyses were performed on supported lipid bilayers and not on liposomes, a parallel regarding the curcumin concentration threshold could not rigorously be drawn. Moreover, the relative fragility of the lipid assembly in the SLB model may lead to amplified curcumin effects. All together these results allow us to conclude that curcumin, at lower µM concentrations, causes a pronounced dose-dependent rigidifying effect on the lipid bilayer, which is only based on its insertion in the bilayer and which strongly affects the lateral mobility of the lipids. We can logically assume that by modifying the membrane fluidity properties, curcumin may affect the spatiotemporal dynamic of EGFR in the membrane and, as a consequence, its activation resulting from dimer formation.

In order to confirm the possible curcumin impact on EGFR mobility in the cell membrane, the motion of single EGF receptors, labeled with quantum dots, was tracked in the membrane of curcumin-treated A431 cells. Starting 5 µM concentration, curcumin decreases significantly the average diffusion coefficient of EGFR in the plasma membrane of A431 cells, confirming our hypothesis that the molecular mechanism of curcumin inhibition of EGFR autophosphorylation is dual and combines an action on the tyrosine kinase activity together with an effect on the EGFR lateral mobility in the membrane. It is known that EGFR dimerization and its resulting autophosphorylation is strongly dependent on the receptor diffusion governed by the fluidity of its surrounding membrane. This influence of membrane fluidity on the receptor dimerization and its consecutive activation was already demonstrated by studying the influence of the cholesterol content in membrane models and plasma membranes. 76,77 Cholesterol depletion from the membrane results in enhanced EGFR dimerization and causes an increase in receptor autophosphorylation, and vice versa. The cholesterol effect on EGFR activity is rather related to the physical effects on membrane properties than direct interactions with the receptor. 78 However, the comparison of the action of curcumin on fluidity and the cholesterol content cannot be further extended since cholesterol plays a complex role, not yet completely elucidated, in EGFR clustering in cholesterol and phosphatidic acid-enriched nanodomains. 76,79,80 The effect of curcumin to slow down the lateral movement of EGFR in the plane of the membrane was observed upon ligand stimulation as well as upon ligand-independent activation. This result indicates that the action of curcumin on the membrane is not specifically related to the organization of EGFR in nanoclusters, which is driven by changes in lipid properties in response to EGF-induced activation of the receptor. 59,79 CONCLUSION The main goal of this study was to improve our understanding of the mode of action of curcumin in EGFR inhibition. We demonstrated that the overall short-term inhibition of EGFR phosphorylation by curcumin, observed in A431 cells, is a combined effect of the direct curcumin action on tyrosine kinase domain and alterations of the physical plasma membrane properties influencing receptor dimerization. Indeed, in addition to the direct inhibition of the enzymatic activity, we identified an additional mechanism based on the curcumin interaction and accumulation in the plasma membrane. Transposing the curcumin concentration range employed in our study to human organism relies on higher local curcumin concentration in membranes than in plasma. A contribution of the membrane in the mode of action of curcumin on EGFR allows to better take into account the extremely poor solubility of curcumin in plasma and cytosol. Curcumin insertion into the membrane leads to a lipid bilayer rigidification and a receptor lateral mobility decrease, which is correlated to receptor dimerization and modulation of autophosphorylation. By this dual mode of action, curcumin is able to affect receptor activity in cells at low micromolar concentrations. Curcumin therefore appears to be a promising compound that may act on both ligand-induced and ligand-independent EGFR activation and their related cancer types. Beyond EGFR, the results of this study highlight the importance of the interaction of active molecules with membranes in cancer treatment.

TABLE OF CONTENTS GRAPHICS

Fluorescence

  anisotropy measurements. Steady-state fluorescence emission anisotropy (r) was measured with a Cary Eclipse Fluorescence Spectrophotometer (Agilent Technologies) equipped with a thermostated cuvette holder. SUVs made of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC, Sigma-Aldrich) and containing 1,6-diphenyl-1,3,5-hexatriene (DPH) or 1-(4trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate (TMA-DPH) fluorescent probes (Sigma-Aldrich) were diluted in HBS solution to the final concentration of 200 µM DPPC and 5 µM fluorescent probe. Different volumes of curcumin stock solution in DMSO (27 mM) were added directly in the quartz cuvette to obtain the desired final concentration and were incubated with the SUVs for 30 min in the dark prior to fluorescence measurement. Fluorescence intensities were collected at 435 nm for DPH and 428 nm for TMA-DPH with excitation wavelengths 357 and 358 respectively. Anisotropy was automatically calculated by the software of the spectrophotometer according to Equation 1:

For

  fluorescence measurements, a lipid fluorescent probe (1,2-dipalmitoyl-sn-glycero-3phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) triethylamine salt, DPPE-NBD (Molecular Probes)) was added into the vesicles at a 2 % molar ratio. Supported lipid bilayer (SLB) formation was performed by deposition of EggPC/DPPE-NBD vesicle suspension (1 mg/ml) into a 400 µl measurement cell, obtained from the top half of a spectrophotometer cuvette (internal diameter 1 cm) glued onto ultraclean glass cover slide. After 1 hour, the cell was carefully flushed with HEPES buffer. Concentrated solution of curcumin pre-diluted in HEPES buffer was added directly in the cell and incubated with the SLB 20 min prior each FRAP measurement.In a typical experiment, the NBD fluorescent probe was excited by an argon ion laser (488 nm, 25 mW) and the emitted corresponding image (square of 225 µm x 225 µm) was collected by a detector LSM-P-PMT (Carl Zeiss) during a 1 s scan. A smaller area (square of 45 µm x 45 µm) was bleached using full laser power (100 %) in scanning mode for 4 s. The fluorescence recovery was recorded with a low laser power (2 %). Images were acquired every 4 s via the integrated Zen software (Carl Zeiss). Quantitative analysis of the gray levels of the bleached area was performed to evaluate the fluorescence recovery using ImageJ software. Diffusion coefficient and the mobile fraction of the lipids were determined from the fit of the experimental normalized fluorescence intensity recovery curve to the theoretical one established according to the calculations described previously.34,35 Single Particle Tracking (SPT) experiments. Two days before the experiments, cells were plated on coverslips. Streptavidin-coated Quantum Dots (QD) (655 nm, Q10121MP, Molecular Probes, Life Technologies) were pre-coupled to a biotinylated anti-hEGFR antibody (R&D systems) by a 15 min incubation in PBS with BSA 1 % and D-glucose 2% (PBS+) at a 1:10 molar ratio QD : antibody. Prior to microscopy observations, the coverslips were mounted in a chamber (25 mm inner diameter) and the EGF receptors were then labelled for 30 min with 0.03 nM conjugated QD in PBS+ (100 µL). Unbound QD were then washed away by replacing the solution three times with 3 mL PBS+.Tracking of the QD at the cell surface was performed at room temperature (22 ± 1 °C) on an Axioplan 2 microscope (Zeiss) equipped with a Cascade II 512 EM-CCD camera (Roper Scientific) operating at 25 Hz acquisition frequency. QD were illuminated by means of an X CITE 120 light source containing a metal halide vapour short arc lamp and observed through a Fluar 100X/1.30 oil UV objective associated to a 1.6 X multiplier tube lens connected to the camera.

Figure 1 .

 1 Figure 1. Box plot presenting EGF induced phosphorylation level of EGFR on Y1068 in A431 cells after treatment with assigned agents at specified concentrations added to serum free culture media. The phosphorylation levels were normalized to the EGFR level and expressed in normalized relative fluorescence units (RFU). Whiskers: from min to max values (n = 8). Asterisks indicate significant differences in phosphorylation levels in treated and control cells (Mann-Whitney U test): *, p ≤ 0.05; **, p ≤ 0.01; ****; p ≤ 0.0001.

Figure 2 .

 2 Figure 2. Surface Plasmon resonance competition analysis showing the effect of curcumin (30 µM) and free EGF (6 µM) upon the binding of 600 nM sEGFR to immobilized EGF. Mixture of sEGFR and curcumin or ligand.

Figure 3 .

 3 Figure 3. Inhibitory effect on EGFR tyrosine kinase domain by curcumin. The results were normalized by dividing the tyrosine kinase activity of EGFR-TK in the presence of curcumin by the one measured in its absence. Average and standard deviations were calculated from 3 independent experiments.

Figure 4 .

 4 Figure 4. Average values of steady-state fluorescence anisotropy for TMA-DPH (A) and DPH (B) probes in DPPC vesicles (200 µM) with different concentration of curcumin (1 to 3 µM) indicated in the legend in the temperature range. Averages were calculated from 3 independent experiments. For clarity, only the anisotropy plots corresponding to curcumin concentrations that lead to significant anisotropy changes are shown.

Figure 5 .

 5 Figure 5. Lateral diffusion coefficient of NDB-DPPE in EggPC SLB upon treatment with indicated concentration of the curcumin (n = 6). Asterisks indicate significant differences in diffusion coefficients obtained in the presence and in the absence of curcumin (Mann-Whitney U test): **, p ≤ 0.01.

Figure 6 .

 6 Figure 6. Mean diffusion coefficient determined by SPT for the EGFR labelled with QD in A431 cells cultured in SFM for 24 h prior to measurement and treated with indicated solutions, curcumin (curc) was introduced 4 h before the measurement, EGF at 100 ng/ml was added in parallel with QD-Ab; bars represent the standard error; 109 ≤ n ≤ 173. Asterisks indicate significant differences in EGFR diffusion coefficients compared to the control condition (EGF treatment) (Mann-Whitney U test): **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001.

  independently of the diffusion mode of the particles.

37

Statistical analysis. All statistical evaluations were performed using GraphPad InStat software and were based on Mann-Whitney U test, where: *, p ≤ 0.05, significant; **, p ≤ 0.01, very significant; ***, p ≤ 0.001, extremely significant; ****, p ≤ 0.0001, extremely significant (threshold and nomenclature as in GraphPad Software). The number of experiments is indicated in each figure caption.

ASSOCIATED CONTENT

Supporting Information. Phosphorylation level of EGFR on Y1068 in A431 cells as a function of EGF exposure time. Surface Plasmon Resonance analysis of curcumin inhibition of the sEGFR binding to immobilized EGF. Frequency histograms of D (log scale), the diffusion coefficient of EGFR as determined from SPT measurements on the A431 cells treated with curcumin. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION

Corresponding Author * Tel.: 0033 3 44 23 45 85. E-mail: claire.rossi@utc.fr.

Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENT

This research was supported by grants from the Centre National de la Recherche Scientifique (CNRS), the European Union (co-funding of equipment within the CPER 2007-2013, and Marie Curie Research Training Network CHEBANA, FP7 ITN 2010-264772). We thank F. Oudet and all his collaborators for their help and advices. We thank Pascale Vigneron for her precious technical assistance.


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