Article pubs.acs.org/molecularpharmaceutics
Recombinant Receptor-Binding Domain of Diphtheria Toxin Increases the Potency of Curcumin by Enhancing Cellular Uptake Ashok Kumar,† Gopal Das,‡ and Biplab Bose*,† †
Department of Biotechnology and ‡Department of Chemistry, Indian Institute of Technology Guwahati, Guwahati, 781039 India S Supporting Information *
ABSTRACT: Diphtheria toxin (DT) binds to a specific cell surface receptor, gets internalized, and causes cytotoxicity through its catalytic domain. The toxicity of DT is used in several therapeutic molecules. Here, we have exploited the receptor-binding ability of DT to increase cellular uptake of curcumin, a hydrophobic molecule with low bioavailability and cellular uptake. We have expressed only the receptor-binding domain of DT (RDT) in Escherichia coli. Purified RDT binds to the receptor with an affinity equivalent to that of full-length DT. It also binds to curcumin forming a curcumin−RDT complex, and this increases the fluorescence intensity and fluorescence lifetime of curcumin. The curcumin−RDT complex binds to the receptor and associates with human glioblastoma cells (U-87 MG) expressing the receptor. The cellular uptake of curcumin is higher for the curcumin−RDT complex than curcumin alone. This increase in uptake enhances the antiproliferative effect of curcumin and induces apoptosis of these cells even at a lower dose. KEYWORDS: diphtheria toxin, curcumin, cellular uptake
1. INTRODUCTION The potency of a drug can be enhanced by the synergistic effect of another drug or by making a formulation that can increase stability/solubility/delivery of the drug to target cells. Various approaches from encapsulation in liposomes1 to protein-based nanocarriers2 have been used to enhance delivery of therapeutic agents. Biomolecules and biomimetic molecules are also used to enhance cellular uptake of drugs. For example, cell-penetrating peptides, derived from natural sequences or designed one, increase the cellular uptake of therapeutic molecules by endocytosis or by passive translocation through plasma membrane.3 Serum albumin binds to several drugs and accumulates preferentially at the site of inflammation and in tumors, due to leaky blood vessels, leading to enhanced drug delivery.4 A ligand−receptor system like transferrin and its receptor has also been explored to enhance cellular uptake and potency of drugs.5 Curcumin is a potential drug for several diseases including some cancers.6 However, hydrophobicity, poor bioavailability, and low cellular uptake of this molecule limits its utilities.7 Chemical derivatization has been extensively used to create curcumin analogues without these limitations.8 Safavy et al.9 have conjugated curcumin to PEG to increase its solubility in water. They have observed that such an increase in aqueous solubility leads to enhanced cellular uptake and cytotoxicity of curcumin. Formulations using cyclodextrin10 and polymeric nanoparticles11 have also been developed to increase the potency of curcumin. These carrier systems increase the potency by increasing the cellular uptake of curcumin. © 2013 American Chemical Society
One can exploit ligand−receptor interaction to achieve enhanced cellular delivery of curcumin. Ligands to cell surface receptors overexpressed in disease related cells are usually used for such purpose. Heparin-binding EGF-like growth factor (HB-EGF) is one such cell surface molecule. HB-EGF is overexpressed in several types of cancers and is involved in tumor growth, metastasis, and angiogenesis.12 HB-EGF is expressed as a membrane anchored molecule, which subsequently gets released through ectodomain shedding.13 As a membrane anchored molecule, HB-EGF participates in juxtacrine signaling.14 Interestingly, HB-EGF is also a cell surface receptor for diphtheria toxin (DT). DT has three domains: an N-terminal catalytic domain (C-domain) which is involved in cellular toxicity, a region that facilitates the delivery of catalytic domain across the cell membrane (T-domain), and a C-terminal region that binds to the EGF-like domain of HBEGF (R-domain).15 On binding to cell surface HB-EGF, DT is internalized through receptor-mediated endocytosis.15 In the present work, we have expressed the recombinant receptor-binding domain of DT (RDT) that retains binding to cell surface HB-EGF. This recombinant molecule binds to curcumin. Curcumin provided as a curcumin−RDT complex has a higher cellular uptake and cytotoxicity. Received: Revised: Accepted: Published: 208
July 1, 2013 October 10, 2013 November 13, 2013 November 13, 2013 dx.doi.org/10.1021/mp400378x | Mol. Pharmaceutics 2014, 11, 208−217
Molecular Pharmaceutics
Article
using Chimera.18 Clusters where curcumin was docked on the receptor-binding face of RDT were rejected. 2.7. Spectroscopic Studies. Fluorescence spectra were measured by a single beam fluorescence spectrometer (LS55, Perkin-Elmer), using a 10 mm path length quartz cell. The emission spectra were recorded from 450 to 700 nm with an excitation wavelength of 430 nm. The excitation and emission slit widths were 2.5 nm and 15 nm, respectively. The time-resolved fluorescence decay of curcumin, alone or in complex, was recorded using a LifeSpec II fluorescence lifetime spectrometer (Edinburgh Instruments). Samples were excited at 405 nm, and the decay was measured in a time scale of nanoseconds per channel. The decay curves were analyzed by FAST Version 3.4.1 (Edinburgh Instruments). Exponential components analysis (with tail fitting) was performed. Fittings with the best χ2 value are reported. Average fluorescence lifetimes were calculated from the fitted data. 2.8. Fluorescence Microscopy. U-87 MG cells in 96-well plates were fixed, blocked, and incubated with RDT (200 ng/ well) for 2 h at 37 °C. Subsequently, bound RDT was detected using mouse anti-His antibody (Calbiochem) followed by fluorescein isothiocyanate (FITC) tagged goat antimouse antibody (BD Biosciences). Similarly, fixed cells were treated with mouse anti-HB-EGF antibody (R&D Systems) followed by goat antimouse−FITC conjugate to detect HB-EGF. U-87 MG cells in 6-well plates were incubated with curcumin and curcumin−protein complexes for 4 h in the presence of serum. Subsequently, cells were washed with PBS and fixed. In all cases, the nucleus was counter-stained with 4',6-diamidino-2phenylindole (DAPI). Double-stained cells were imaged using a fluorescence microscope (Nikon Eclipse TiU). A filter for green fluorescence (B-2E/C, Nikon) was used for FITC and curcumin. DAPI was detected using a UV-2E/C filter (Nikon). 2.9. HPLC to Detect Cellular Uptake of Curcumin. Cells were treated with curcumin or protein−curcumin complexes for 2 h in the presence of serum. Treated cells were washed thoroughly with PBS and harvested by trypsinization. HB-EGF, BSA, and RDT have multiple cleavage sites for trypsin. Therefore, thorough washing followed by trypsinization would have removed most of curcumin bound to cell surface either alone or as protein−curcumin complex. Subsequently, trypsinized cells were washed with PBS, and internalized curcumin was extracted from these cells with methanol.19 The methanolic extract was analyzed by high-performance liquid chromatography (HPLC) to quantify the amount of curcumin.20 Perkin-Elmer Series 200 HPLC equipped with a silica-based C18 column (Brownlee Analytical, Perkin-Elmer) was used. 2.10. Assay for Cell Viability. Cells in a 96-well plate were treated with curcumin or curcumin−RDT complex in the absence of serum. At a specific time point, the cell viability was measured by a 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide (MTT) based colorimetric assay.21 The percentage cell viability was calculated relative to cells treated with an equivalent amount of ethanol in PBS. 2.11. Flow Cytometric Analysis. Cells treated, in the absence of serum, were harvested, stained with propidium iodide (PI), and analyzed by flow cytometry for the cell cycle assay.22 The signal for PI was measured in FL2 channel in log mode, and doublet discrimination was achieved using FL2 as a DDM parameter. Data were analyzed by Modfit LT (Verity Software).
2. MATERIALS AND METHODS 2.1. Cell Culture. Human glioblastoma cell line U-87 MG was procured from the National Centre for Cell Science (India) and was maintained in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) (PAA). 2.2. Cloning and Expression. Mutated DT cloned in pET-22b was procured from Addgene (Plasmid 11081) and was used as a template for amplification of N-terminal truncated DT, corresponding to 382nd to 536th amino acid of DT (NCBI GenBank sequence AAV70486). The amplified region corresponds to the receptor-binding domain of DT and was cloned into pET-22b (Novagen). The recombinant protein was expressed in Escherichia coli BL21 (0.5 mM IPTG, 8 h at 28 °C). It was purified using His-Trap FF affinity column (GE Healthcare) as per the manufacturer’s protocol, and the residual imidazole was removed by dialysis against phophate-buffered saline (PBS). Purified protein was characterized by sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDSPAGE) and Western blot analysis using mouse anti-His antibody. 2.3. Formation of Curcumin−Protein Complexes. The curcumin (Himedia) stock solution (1 mg/mL) was prepared in ethanol. A required amount of curcumin was added to a required amount of protein (RDT or BSA) solution in PBS (pH 7.4) to achieve desired molar ratios and incubated for 2 h at 4 °C to allow complexation. Curcumin diluted in PBS, with the required concentration and solvent ratio, was used as a control. 2.4. Receptor Binding Assays. For the solid-phase enzyme-linked immunosorbent assay (ELISA), a microplate was coated with recombinant human HB-EGF (R&D Systems). After blocking, RDT was added and incubated for 2 h at room temperature. Bound RDT was detected using mouse anti-His antibody (Calbiochem) followed by goat antimouse-HRP conjugate (Sigma). ELISA was developed by using orthophenylenediamine (OPD), and the absorbance was measured at 492 nm. A similar procedure was used to detect the binding of the curcumin−RDT complex to HB-EGF. For cell-based ELISA, cells in a 96-well plate were fixed with 10% formalin in PBS for 45 min at 4 °C. After blocking, RDT was added and incubated for 2 h. Bound RDT was detected as described above. 2.5. Surface Plasmon Resonance (SPR). Binding kinetics was measured using Biacore X100 (GE Healthcare). Recombinant human HB-EGF (R&D systems) was immobilized on a CM5 sensor chip using manufacturer’s protocol (∼400 RU). Binding was measured using single-cycle kinetics, with sequential injection (25 °C, flow rate of 30 μL/min) of five samples of each analyte in increasing order of concentration (from 300 to 1500 nM). Data were fitted to a 1:1 binding model using BIAevaluation Software (version 2.0). 2.6. Molecular Docking. SwissDock16 was used for docking. The structure of RDT was derived from the X-ray crystal structure of DT (PDB ID: 1F0L) after trimming C- and T-domains. This corresponds to Ser381−Ser535 of 1F0L. Four structures of curcumin collected from ZINC database17 were used for docking (ZINC ID: 00899824, 17255287, 20230445, and 31261437). Dockings were performed as blind, with default parameter values in “Accurate” mode with no flexibility for the protein. Clusters were ranked based on a Fullfitness score, and the top ten clusters of each experiment were further analyzed 209
dx.doi.org/10.1021/mp400378x | Mol. Pharmaceutics 2014, 11, 208−217
Molecular Pharmaceutics
Article
Figure 1. RDT binds to HB-EGF. (a) Solid-phase ELISA and (b) cell ELISA to detect binding of RDT to HB-EGF. Each bar represents the mean of four different wells. (c) Fluorescence imaging to show the binding of RDT to U-87 MG cells. Cells equivalently treated, without RDT, are shown as (-)ve control. Images were taken at 10× magnification. (d) Sensogram of one representative SPR experiment for RDT is shown with residual values of data fitting in the lower panel. Average kinetic parameters for RDT and DT, calculated from three experiments, are shown in the table.
at the C-terminal and some additional amino acids at the Nterminal originating from the vector (Supporting Information, Supplementary Figure S1a). The purified protein was further characterized by Western blot using anti-His antibody (Supplementary Figure S1b). 3.2. Functional Characterization of RDT. Experiments were performed to check the receptor binding ability of RDT. As shown in Figure 1a, RDT binds to recombinant HB-EGF coated on an ELISA plate in a dose-dependent fashion. Binding was observed also in cell ELISA. For cell ELISA, U-87 MG cells that express HB-EGF were used. Expression of HB-EGF in U87 MG cells was confirmed by reverse transcription polymerase chain reaction (RT-PCR) and immunofluorescence imaging (Supplementary Figure S2a,b). As shown in Figure 1b, RDT binds to U-87 MG cells in a dose-dependent fashion. Subsequently, fluorescence imaging was used to reconfirm that RDT binds to cell surface HB-EGF on U-87 MG cells (Figure 1c). In this experiment, RDT was allowed to bind to cell surface HB-EGF in U-87 MG cells, and bound RDT was detected using an FITC-tagged antibody. SPR was used to determine the kinetic parameters of RDT binding to HB-EGF (Figure 1d). It was observed that the binding affinity of RDT is equivalent to that of the full-length DT and close to the
Cells were stained using an Annexin V-FITC Apoptosis Detection Kit I (BD Biosciences) as per the manufacturer’s protocol and analyzed by flow cytometry to detect apoptosis. Signals of FITC and PI were measured in FL1 and FL3 channel in log mode, respectively. Data were analyzed using FCS Express 4 (De Novo Software). All experiments were performed using a FACSCalibur (BD Biosciences) flow cytometer. 2.12. Data Analysis. SigmaPlot was used for statistical analyses and to generate graphs. Means of multiple data points are plotted. Error bars represent standard deviations. One way and two-way analysis of variance (ANOVA) were performed with a pairwise comparison.
3. RESULTS 3.1. Expression of Recombinant Receptor-Binding Domain of DT (RDT). Diphtheria toxin is a Y-shaped molecule with three independent domains. The toxin binds to its receptor, HB-EGF, through the C-terminal receptorbinding domain (NCBI CDD: 65145). This receptor-binding domain (155 amino acids) was cloned and expressed in E. coli with a His-tag at the C-terminal. The matured protein, purified from E. coli cell lysate, is approximately 20 kDa having His-tag 210
dx.doi.org/10.1021/mp400378x | Mol. Pharmaceutics 2014, 11, 208−217
Molecular Pharmaceutics
Article
reported value for full-length DT.23 However, kinetic parameters, association and dissociation rate constants, for RDT vary by one order from those of DT. RDT binds with a higher association rate constant (ka) but dissociates faster than DT. RDT is smaller in size, and this may be the reason for the faster kinetics. Taken together, results shown in Figure 1 prove that, though expressed as an independent domain, RDT retains HB-EGF binding ability. 3.3. RDT Binds to Curcumin. As RDT has a receptor binding ability, it can be used for the targeted delivery of drugs to cells overexpressing HB-EGF. For targeted delivery, drugs are usually linked to homing molecules through a covalent linker. However, proteins like serum albumin and transferrin noncovalently bind to drugs and deliver those to cells.4,24 Even proteins forming higher order structures can bind and carry drugs.25 Therefore, we inquired whether RDT can bind to any therapeutic molecule. Curcumin is a potential chemotherapeutic agent. It is a fluorochrome, and its fluorescence intensity depends upon the polarity of the environment with substantially higher fluorescence yield in a nonpolar environment than in an aqueous environment.26 Curcumin binds to proteins like BSA and HSA at hydrophobic sites.26 When incubated with these proteins, curcumin forms curcumin−protein complexes and shows enhanced fluorescence in these complexes.27,28 Therefore, protein−curcumin interaction can be easily monitored using fluorescence spectroscopy. Molecular docking was performed to explore the possibility of binding of curcumin to RDT. RDT has several patches of hydrophobic regions on the surface. Multiple potential binding sites for curcumin were observed in our docking experiments. One such potential binding pose is shown in Figure 2a. In this pose, curcumin remains in an extended conformation and binds to a hydrophobic groove (Figure 2b). This binding site is made up of 11 amino acids, five of which are hydrophobic and three have aromatic side chains (T386, Q387, P388, F389, L390, W398, V401, S404, L529, F530, and E532 of 1F0L). The interaction of curcumin with hydrophobic and aromatic residues of proteins has been reported in several studies.29 The extended conjugated aromatic ring of the curcumin at one end rests on a hydrophobic patch provided by P388, F389, and L390 (Supplementary Figure S3). Binding of curcumin in this pose is further facilitated via formation of a hydrogen bond between a keto group and the side chain of Q387 (Supplementary Figure S3). Such a hydrogen bond between the keto−enol group of curcumin and the side chains of proteins has been reported in other docking studies.29 To generate a curcumin−RDT complex, curcumin was incubated with RDT in PBS at 4 °C for 2 h. Subsequently, fluorescence spectroscopy was used to detect formation of the curcumin−RDT complex. In our experiments, when excited at 430 nm, curcumin in PBS showed two low-intensity broad peaks near 510 and 570 nm. However, when incubated with RDT, the fluorescence emission of curcumin increased almost 4-fold with maximum at 510 nm (Figure 3a). RDT in itself does not have any significant fluorescence in this range when excited at 430 nm (Supplementary Figure S4). A similar change in fluorescence emission was observed when curcumin was incubated with BSA (Figure 3a). The fluorescence emission spectrum shown in Figure 3a indicates that curcumin binds to RDT which provides a hydrophobic environment, thereby enhancing the fluorescence of curcumin. The behavior of curcumin−RDT complex formation was further investigated by
Figure 2. Docking of curcumin to RDT. The ribbon diagram (a) shows the orientation of a potential binding pose (score = −970.3). (b) Surface rendering of the same binding site. Color code for protein surface: blue = hydrophilic and orange/red = hydrophobic.
incubating curcumin with RDT in different molar ratios. As shown in Figure 3b, the intensity of fluorescence emission at 510 nm increases with the increase in the amount of RDT, as more and more curcumin moves from an aqueous environment to the hydrophobic environment provided by RDT. Fluorescence spectra of this experiment are shown in Supplementary Figure S5. Formation of the curcumin−RDT complex was substantiated by time-resolved fluorescence analysis (Figure 3c). As evident from Figure 3c, the fluorescence decay of curcumin in the curcumin−RDT complex is slower than that of curcumin alone in PBS. The data were fitted to multicomponent exponential models, and the fittings with best χ2 values are reported. As per the model fitting, average lifetimes of fluorescence decay of curcumin are 0.86 ns and 1.04 ns for curcumin alone and for the curcumin−RDT complex, respectively. It is well-known that solvent can quench the fluorescence of a fluorophore and complexation with a protein can reduce such quenching, thereby increasing the fluorescence lifetime.30 Water quenches the fluorescence of curcumin.31 Therefore, complexation with RDT may have reduced such quenching and increased the fluorescence lifetime of curcumin. 3.4. RDT as a Carrier of Curcumin. Complexation with curcumin may affect the receptor binding ability of RDT. Therefore, solid-phase ELISA was performed to detect the 211
dx.doi.org/10.1021/mp400378x | Mol. Pharmaceutics 2014, 11, 208−217
Molecular Pharmaceutics
Article
Subsequently, we enquired whether this complex can deliver curcumin to cells expressing HB-EGF. U-87 MG cells were incubated with curcumin, curcumin−RDT, and curcumin−BSA complexes. After thorough washing, fluorescence microscopy was used to detect the presence of curcumin. Green fluorescence of curcumin was not observed in cells treated with curcumin alone, as it has very low fluorescence in aqueous environment and most of the curcumin must have washed-off (Figure 4a, lower panel). Curcumin in complex with BSA has a high and detectable fluorescence (as shown in Figure 3a). However, no fluorescence was observed in cells treated with this complex (Figure 4a, middle panel). This indicates that the curcumin−BSA complex does not get attached to U-87 MG cells and may have got removed by washing. This is expected as BSA does not have any specific binding to U-87 MG cells. On the other hand, fluorescence of curcumin was detected and found to be colocalized with U-87 MG cells when provided as the curcumin−RDT complex (upper panel of Figure 4a). This clearly indicates that RDT can act as a carrier to specifically deliver curcumin to these cells. The presence of cell surface HB-EGF in U-87 MG cells must have allowed the curcumin− RDT complex to get associated with these cells. Subsequently, an HPLC-based method was used to detect the amount of curcumin internalized by these cells. Data of the experiment are shown in Figure 4b. It was observed that there is a basal level of internalization of curcumin when given alone or in complex with BSA. However, the amount of curcumin internalized by these cells is substantially higher when given as a complex with RDT. 3.5. Curcumin−RDT Complex Has a Higher Potency than Curcumin. RDT binds curcumin and delivers it to cells expressing HB-EGF, enhancing cellular uptake. This should lead to an increase in potency of curcumin. Curcumin is known to inhibit proliferation of wide varieties of cancer cells and often triggers apoptosis.6 Therefore, U-87 MG cells were treated with curcumin either alone or in complex with RDT, and the viability of these cells was measured after 72 h of treatment. IC50 of curcumin for U-87 MG cells is high and approximately 40 μM.32 In our experiment, curcumin was used at suboptimal doses. Curcumin did not show a considerable effect on the viability of these cells (Figure 5a). However, the curcumin− RDT complex reduced the viability of U-87 MG cells in a dosedependent fashion, and the extent of inhibition is much higher than what is achieved by curcumin alone (Figure 5a). This may have been achieved as RDT acts as a carrier to increase accumulation of curcumin in and around the cells, thereby increasing the effective potency of curcumin. An increase in efficacy of curcumin may also happen due to the synergistic effect of RDT. RDT may block signaling through HB-EGF, thereby damping the canonical survival signals through MAPK and PI3K pathways. Therefore, an MTT assay was performed where curcumin and RDT were added sequentially. RDT was added first, so that it can bind and downregulate the signaling of HB-EGF, and curcumin was added after one hour. As shown in Figure 5b, sequential addition of RDT and curcumin does not increase the effect of curcumin. This ruled out the possible synergistic effect of RDT when given with curcumin. Subsequently, we looked into the mechanism behind the antiproliferating effect of the curcumin−RDT complex. Curcumin is known to induce cell cycle arrest.6 A flow cytometry based experiment was used to understand the effect of curcumin−RDT on the cell cycle of U-87 MG cells. Treatment with curcumin−RDT complex reduced cells in the
Figure 3. Fluorescence spectroscopy to detect binding of curcumin to RDT: (a) Fluorescence emission spectra of curcumin alone or in complex with RDT or BSA in PBS. The concentration of curcumin was 10 μM. The molar ratio of curcumin to protein was 10:1. (b) Linear enhancement of fluorescence intensity at 510 nm with an increase in the amount of RDT. Here, 10 μM curcumin was incubated with different amounts of RDT (0−2 μM). (c) Time-resolved fluorescence decay of curcumin alone or in complex with RDT in PBS. The concentration of curcumin was 10 μM. The molar ratio of curcumin to protein was 10:1.
binding of curcumin−RDT complex to HB-EGF. It was observed that the curcumin−RDT complex binds to HB-EGF coated on an ELISA plate (Supplementary Figure S6). 212
dx.doi.org/10.1021/mp400378x | Mol. Pharmaceutics 2014, 11, 208−217
Molecular Pharmaceutics
Article
Figure 4. RDT enhances cellular uptake of curcumin. (a) U-87 MG cells were incubated with curcumin (2 μM) and curcumin−protein complexes (molar ratio 10:1). Fluorescence microscopy at 20× magnification. (b) Internalized curcumin was measured by HPLC. Cells were treated with curcumin (1 μM) and curcumin−protein complexes (molar ratio 10:1) for 2 h. One-way ANOVA with pairwise comparison: *no significant difference between these two (p = 0.143), **significantly different from others (p < 0.001).
for DT. Such enhanced uptake potentiates the antiproliferative effect of curcumin. Cell surface HB-EGF is the receptor for diphtheria toxin. However, the primary role of HB-EGF is to act as a growth and survival factor. It is overexpressed in several types of cancer and is involved in proliferation and migration of cancer cells, angiogenesis, and development of drug resistance.12 It can be targeted either to block signaling or to deliver a cargo to cells overexpressing HB-EGF. CRM197, a mutated full-length DT, blocks neoplastic signaling through HB-EGF.34 It provides a synergistic effect when used in combination with paclitaxel by blocking the HB-EGF signaling.35 Using CRM197 conjugated to horseradish peroxidase (HRP), Gaillard et al.36 have shown that CRM197 may be used for delivery to cells expressing HBEGF. Our work shows that RDT may be used for the same purposes. Being smaller than the full-length DT, RDT is expected to have better tissue penetration and lesser immunogenicity.37,38 We have cloned and expressed RDT using an E. coli expression system. This protein retains receptor binding ability and binds to cells expressing HB-EGF. Some proteins can bind to certain drugs through hydrophobic interactions.39,40 Such a protein−drug complex can be used for drug delivery. We have explored the possibility of using RDT for similar purposes.
G1 phase (Figure 6). A slight increase in cells in S and G2/M phase was observed, but the change was statistically insignificant. Additionally, it increases the sub-G0/G1 population from 2.9 ± 1.7% in untreated cells to 21.0 ± 1.3%. Treatment with curcumin (20 μM) or RDT (0.1 μM) alone has no effect on cell cycle profile and the size of the sub-G0/G1 population. Karmakar et al.33 have earlier shown that curcumin induces apoptosis of U-87 MG cells. Therefore, flow cytometry was used to detect apoptotic population using double staining with annexin-V-FITC and PI. The result of the flow cytometry experiment is shown in Figure 7. Cells in the lower and upper right quadrants represent early and late apoptotic cells. As shown in this figure, treatment with curcumin−RDT induces apoptosis, thereby increasing the apoptotic cell population to ∼30%. At the same concentration, curcumin or RDT alone does not induce apoptosis of these cells.
4. DISCUSSION Diphtheria toxin (DT) has been used to create immunotoxins. The catalytic function of DT is utilized in those recombinant molecules to kill target cells. However, in this work we have used the nontoxic receptor-binding domain of DT to enhance cellular uptake of a therapeutic molecule. We have shown that RDT binds to curcumin and can be used to deliver an enhanced amount of curcumin to cells expressing HB-EGF, the receptor 213
dx.doi.org/10.1021/mp400378x | Mol. Pharmaceutics 2014, 11, 208−217
Molecular Pharmaceutics
Article
Figure 5. Effect of curcumin−RDT on viability of U-87 MG cells. (a) MTT assay to measure cell viability. The concentration of RDT was 0.1 μM, and the concentration of curcumin was varied. (b) MTT assay to detect synergism. Here, C: curcumin (20 μM), C-RDT: curcumin− RDT complex (20 μM:0.1 μM), RDT+C: RDT (0.1 μM) followed by curcumin (20 μM), RDT: RDT (0.1 μM), Un: untreated. Cells were treated for 72 h. One-way ANOVA with a pairwise comparison was used. **Significantly different from other treatment groups (p < 0.001).
Figure 6. Effect of curcumin−RDT on the cell cycle. Cells were treated with curcumin (20 μM), RDT (0.1 μM), or curcumin−RDT complex (20 μM:0.1 μM) or left untreated for 48 h and analyzed by flow cytometry after staining with PI. (a) Histograms for PI intensity in different treatment groups in a representative experiment. (b) Percentage of cells in different phases of the cell cycle in different treatment groups as calculated from three independent experiments. One-way ANOVA with a pairwise comparison: **p < 0.001, *p = 0.002.
Curcumin has a low solubility in aqueous solution. Binding to proteins through hydrophobic interactions increases its solubility and stability. Further, it has pronounced fluorescence only in a hydrophobic environment. Therefore, we selected curcumin to investigate the drug binding ability of RDT. Using fluorescence-based studies, we have proved that curcumin binds to RDT, forming a complex. In this complex, RDT provides a hydrophobic environment that causes enhancement in curcumin fluorescence and increases the average lifetime of fluorescence decay. Curcumin is known to bind proteins through hydrophobic interactions, and RDT has several hydrophobic patches on the surface. Our preliminary investigation using molecular docking shows that curcumin may bind to RDT at those hydrophobic patches. We observed that the curcumin−RDT complex binds to HBEGF and used this complex to deliver curcumin to human glioblastoma U-87 MG cells expressing HB-EGF. Mishima et al.41 have shown that HB-EGF is overexpressed in gliomas and induces proliferation of glioma cell lines in vitro. It has been shown that mutated DT, CRM197, increases the permeability of the blood−brain barrier and crosses the barrier.36,42 Therefore, DT or its derivatives can have a potential use in HB-EGF-targeted delivery in glioma. We have used fluorescence imaging to visualize delivery of curcumin to U-87 MG cells. Yadav et al.10 have used imaging to show the cellular uptake of curcumin. However, we have not observed curcumin fluorescence when U-87 MG cells were treated with curcumin
alone. This is expected as we have used a very low concentration of curcumin (2 μM), and it is known that water quenches the fluorescence of curcumin. As a control, we have used a curcumin−BSA complex in our experiment. The fluorescence signature of curcumin was not observed in cells treated with this complex too. BSA does not have any specific interaction with these cells. Therefore, any curcumin−BSA complex bound to cells would get removed by washing. Like curcumin alone, the curcumin−BSA complex can also get internalized through nonspecific fluid phase endocytosis. However, as the concentration of the complex was low, this process will not lead to any detectable signal in the imaging experiment. On the other hand, the fluorescence signal of curcumin was observed on and around the cells treated with the curcumin−RDT complex. Two aspects can be concluded from this observation: (1) the curcumin−RDT complex was retained even after washing as RDT has specific interaction with cell surface HB-EGF; this substantiates our expectation that RDT will be able to home the drug to cells expressing HB-EGF and 214
dx.doi.org/10.1021/mp400378x | Mol. Pharmaceutics 2014, 11, 208−217
Molecular Pharmaceutics
Article
Figure 7. Curcumin−RDT complex induces apoptosis in U-87 MG cells. Cells were treated with curcumin (20 μM), RDT (0.1 μM), or curcumin− RDT complex (20 μM:0.1 μM) or left untreated for 48 h and analyzed by flow cytometry. (a) Dot plots showing the data of a representative experiment. Cells in lower and upper right quadrants are in early and late apoptosis, respectively. (b) Percentage of apoptotic cells in different treatment groups as calculated from three independent experiments.
(2) a substantial portion of curcumin remains as curcumin− RDT complex that provides the required environment for enhanced and detectable fluorescence of curcumin. However, this experiment cannot reveal whether curcumin was internalized by the cells treated with the curcumin−RDT complex. DT is internalized by target cells through receptormediated endocytosis. It is not known whether RDT will have the same fate as rest of the domains of DT are absent. Even
then, one can expect that internalization of curcumin will be higher when cells are treated with the curcumin−RDT complex. Binding of RDT to cell surface HB-EGF would lead to accumulation of curcumin around the cells, thereby increasing the effective concentration. This will eventually lead to enhanced internalization even in absence of specific receptor-mediated endocytosis. We have used HPLC to measure internalized curcumin. When given as a complex 215
dx.doi.org/10.1021/mp400378x | Mol. Pharmaceutics 2014, 11, 208−217
Molecular Pharmaceutics
Article
complex binds to HB-EGF. This material is available free of charge via the Internet at http://pubs.acs.org.
with RDT, internalization of curcumin was substantially higher than the basal level. On the other hand, BSA failed to provide any significant change in uptake of curcumin. These observations established that, being a specific homing molecule, RDT can carry and deliver an enhanced amount of curcumin to these cells. Enhanced delivery of curcumin should eventually lead to an increased effect of curcumin on these cells. Curcumin inhibits multiple pathways, including NF-κB and AP-1 pathways, leading to inhibition of cell proliferation and apoptosis.43 The IC50 for curcumin is usually high. Poor solubility in water and low uptake by cells may be the causes of such high IC50. We have also observed that curcumin, in the concentration range of 1−20 μM, does not have considerable effect on the viability of U-87 MG cells. However, when used as the curcumin−RDT complex, the viability is reduced to ∼65%. We have also observed that the curcumin−RDT complex induces apoptosis in these cells. Curcumin (20 μM) alone does not show this effect. This increase in potency may have happened as RDT has targeted curcumin to U-87 MG cells and has increased its uptake. Such an increase in the effect of curcumin may also happen due to the synergistic effect of RDT. CRM197 is known to block signaling through HB-EGF. Though not investigated, RDT should also be able to block signaling through HB-EGF. That may lead to down regulation of PI3K and MAPK pathways. Curcumin modulates signaling through these two pathways,43 and blocking of HB-EGF by RDT may potentiate that effect. However, we have shown that sequential addition of RDT and curcumin does not enhance the effect of curcumin. Synergism may happen by another mechanism which has been observed between CRM197 and paclitaxel.35 Paclitaxel increases ectodomain shedding of HB-EGF in some ovarian cell line and thereby triggers pro-survival signaling through EGFR. When coadministered, CRM197 attenuates this signaling. RDT may also potentiate paclitaxel by this mechanism but is unlikely to show any such synergism with curcumin. Cleavage by metalloproteases leads to ectodomain shedding of HB-EGF.44 Curcumin is known to inhibit several metalloproteases.43 Therefore, one can expect that curcumin may decrease ectodomain shedding of HB-EGF. Considering these information and based on our observations, we believe that RDT is potentiating the effect of curcumin by increasing its delivery to target cells. This study demonstrates that one can utilize the receptor and drug binding ability of a bacterial toxin to create a system to enhance cellular uptake and potency of a drug. We can consider the curcumin−RDT complex as a proof-of-concept. Many other therapeutically active small molecules may bind to RDT, and RDT may be used to enhance their uptake in cells expressing HB-EGF. In some cases, apart from enhancing the cellular uptake, RDT may also potentiate the drug by simultaneously blocking signaling through HB-EGF.
■
■
AUTHOR INFORMATION
Corresponding Author
*Phone: +91-361-2582216. Fax: +91-361-2582249. E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS This work has been funded by research grant from Department of Biotechnology, India (project no. BT/01/NE/PS/08). We thank Dr. Chandra Chur Ghosh and Dr. Sudip Sen for valuable discussions and critical reading of the manuscript.
■
REFERENCES
(1) Schwendener, R. A. Liposomes in biology and medicine. Adv. Exp. Med. Biol. 2007, 620, 117−128. (2) Hawkins, M. J.; Soon-Shiong, P.; Desai, N. Protein nanoparticles as drug carriers in clinical medicine. Adv. Drug Delivery Rev. 2008, 60, 876−885. (3) Stewart, K. M.; Horton, K. L.; Kelley, S. O. Cell-penetrating peptides as delivery vehicles for biology and medicine. Org. Biomol. Chem. 2008, 6, 2242−2255. (4) Kratz, F. Albumin as a drug carrier: design of prodrugs, drug conjugates and nanoparticles. J. Controlled Release 2008, 132, 171− 183. (5) Qian, Z. M.; Li, H.; Sun, H.; Ho, K. Targeted drug delivery via the transferrin receptor-mediated endocytosis pathway. Pharmacol. Rev. 2002, 54, 561−587. (6) Epstein, J.; Sanderson, I. R.; Macdonald, T. T. Curcumin as a therapeutic agent: the evidence from in vitro, animal and human studies. Br. J. Nutr. 2010, 103, 1545−1557. (7) Anand, P.; Kunnumakkara, A. B.; Newman, R. A.; Aggarwal, B. B. Bioavailability of curcumin: problems and promises. Mol. Pharmaceutics 2007, 4, 807−818. (8) Anand, P.; Thomas, S. G.; Kunnumakkara, A. B.; Sundaram, C.; Harikumar, K. B.; Sung, B.; Tharakan, S. T.; Misra, K.; Priyadarsini, I. K.; Rajasekharan, K. N.; Aggarwal, B. B. Biological activities of curcumin and its analogues (Congeners) made by man and Mother Nature. Biochem. Pharmacol. 2008, 76, 1590−1611. (9) Safavy, A.; Raisch, K. P.; Mantena, S.; Sanford, L. L.; Sham, S. W.; Krishna, N. R.; Bonner, J. A. Design and development of water-soluble curcumin conjugates as potential anticancer agents. J. Med. Chem. 2007, 50, 6284−6288. (10) Yadav, V. R.; Prasad, S.; Kannappan, R.; Ravindran, J.; Chaturvedi, M. M.; Vaahtera, L.; Parkkinen, J.; Aggarwal, B. B. Cyclodextrin-complexed curcumin exhibits anti-inflammatory and antiproliferative activities superior to those of curcumin through higher cellular uptake. Biochem. Pharmacol. 2010, 80, 1021−1032. (11) Anand, P.; Nair, H. B.; Sung, B.; Kunnumakkara, A. B.; Yadav, V. R.; Tekmal, R. R.; Aggarwal, B. B. Design of curcumin-loaded PLGA nanoparticles formulation with enhanced cellular uptake, and increased bioactivity in vitro and superior bioavailability in vivo. Biochem. Pharmacol. 2010, 79, 330−338. (12) Miyamoto, S.; Yagi, H.; Yotsumoto, F.; Kawarabayashi, T.; Mekada, E. Heparin-binding epidermal growth factor-like growth factor as a novel targeting molecule for cancer therapy. Cancer Sci. 2006, 97, 341−347. (13) Goishi, K.; Higashiyama, S.; Klagsbrun, M.; Nakano, N.; Umata, T.; Ishikawa, M.; Mekada, E.; Taniguchi, N. Phorbol ester induces the rapid processing of cell surface heparin-binding EGF-like growth factor: conversion from juxtacrine to paracrine growth factor activity. Mol. Biol. Cell 1995, 6, 967−980. (14) Higashiyama, S.; Iwamoto, R.; Goishi, K.; Raab, G.; Taniguchi, N.; Klagsbrun, M.; Mekada, E. The membrane protein CD9/DRAP 27
ASSOCIATED CONTENT
* Supporting Information S
Supplementary figures: Characterization of recombinant receptor binding domain of DT (RDT) expressed in E. coli, HB-EGF expression on the cell surface of U-87 MG cells, interactions of different residues of RDT with curcumin, fluorescence emission spectra of curcumin, RDT, and BSA in PBS, fluorescence emission spectra of curcumin−RDT complexes with different molar ratios of curcumin and RDT, and solid-phase ELISA showing that the curcumin−RDT 216
dx.doi.org/10.1021/mp400378x | Mol. Pharmaceutics 2014, 11, 208−217
Molecular Pharmaceutics
Article
factor as a novel targeting molecule. Anticancer Res. 2007, 27, 3713− 3721. (35) Yagi, H.; Yotsumoto, F.; Sonoda, K.; Kuroki, M.; Mekada, E.; Miyamoto, S. Synergistic anti-tumor effect of paclitaxel with CRM197, an inhibitor of HB-EGF, in ovarian cancer. Int. J. Cancer 2009, 124, 1429−1439. (36) Gaillard, P. J.; Brink, A.; De Boer, A. G. Diphtheria toxin receptor-targeted brain drug delivery. Int. Congress Series 2005, 1277, 185−198. (37) Vugmeyster, Y.; Xu, X.; Theil, F. P.; Khawli, L. A.; Leach, M. W. Pharmacokinetics and toxicology of therapeutic proteins: Advances and challenges. World. J. Biol. Chem. 2012, 3, 73−92. (38) Tizard, I. R. Immunology: An Introduction, 4th ed.; Thomson Asia Pte. Ltd: Singapore, 2004. (39) Fasano, M.; Curry, S.; Terreno, E.; Galliano, M.; Fanali, G.; Narciso, P.; Notari, S.; Ascenzi, P. The extraordinary ligand binding properties of human serum albumin. IUBMB Life 2005, 57, 787−796. (40) Sneharani, A. H.; Karakkat, J. V.; Singh, S. A.; Rao, A. G. Interaction of Curcumin with β-LactoglobulinStability, Spectroscopic Analysis, and Molecular Modeling of the Complex. J. Agric. Food Chem. 2010, 58, 11130−11139. (41) Mishima, K.; Higashiyama, S.; Asai, A.; Yamaoka, K.; Nagashima, Y.; Taniguchi, N.; Kitanaka, C.; Kirino, T.; Kuchino, Y. Heparin-binding epidermal growth factor-like growth factor stimulates mitogenic signaling and is highly expressed in human malignant gliomas. Acta Neuropathol. 1998, 96, 322−328. (42) Wang, P.; Liu, Y.; Shang, X.; Xue, Y. CRM197-induced bloodbrain barrier permeability increase is mediated by upregulation of caveolin-1 protein. J. Mol. Neurosci. 2011, 43, 485−492. (43) Shehzad, A.; Wahid, F.; Lee, Y. S. Curcumin in Cancer Chemoprevention: Molecular Targets, Pharmacokinetics, Bioavailability, and Clinical Trials. Arch. Pharm. Chem. Life Sci. 2010, 9, 489− 499. (44) Miyazono, K. Ectodomain shedding of HB-EGF: a potential target for cancer therapy. J. Biochem. 2012, 151, 1−3.
potentiates the juxtacrine growth factor activity of the membraneanchored heparin-binding EGF-like growth factor. J. Cell Biol. 1995, 128, 929−938. (15) Collier, R. J. Understanding the mode of action of diphtheria toxin: a perspective on progress during the 20th century. Toxicon 2001, 39, 1793−1803. (16) Grosdidier, A.; Zoete, V.; Michielin, O. SwissDock, a proteinsmall molecule docking web service based on EADock DSS. Nucleic Acids Res. 2011, 39, W270−W277. (17) Irwin, J. J.; Shoichet, B. K. ZINC–a free database of commercially available compounds for virtual screening. J. Chem. Inf. Model. 2005, 45, 177−182. (18) Pettersen, E. F.; Goddard, T. D.; Huang, C. C.; Couch, G. S.; Greenblatt, D. M.; Meng, E. C.; Ferrin, T. E. UCSF Chimera–a visualization system for exploratory research and analysis. J. Comput. Chem. 2004, 25, 1605−1612. (19) Kunwar, A.; Barik, A.; Pandey, R.; Priyadarsini, K. I. Transport of liposomal and albumin loaded curcumin to living cells: an absorption and fluorescence spectroscopic study. Biochim. Biophys. Acta 2006, 1760, 1513−1520. (20) Wang, Y. J.; Pan, M. H.; Cheng, A. L.; Lin, L. I.; Ho, Y. S.; Hsieh, C. Y.; Lin, J. K. Stability of curcumin in buffer solutions and characterization of its degradation products. J. Pharm. Biomed. Anal. 1997, 15, 1867−1876. (21) Mosmann, T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 1983, 65, 55−63. (22) Riccardi, C.; Nicoletti, I. Analysis of apoptosis by propidium iodide staining and flow cytometry. Nat. Protocols 2006, 1, 1458−1461. (23) Brooke, J. S.; Cha, J. H.; Eidels, L. Diphtheria toxin:receptor interaction: association, dissociation, and effect of pH. Biochem. Biophys. Res. Commun. 1998, 248, 297−302. (24) Zhang, Y.; Xiang, J.; Liu, Y.; Zhang, X.; Tang, Y. Constructing transferrin receptor targeted drug delivery system by using doxorubicin hydrochloride and vanadocene dichloride. Bioorg. Med. Chem. Lett. 2011, 21, 5982−5986. (25) Bachar, M.; Mandelbaum, A.; Portnaya, I.; Perlstein, H.; EvenChen, S.; Barenholz, Y.; Danino, D. Development and characterization of a novel drug nanocarrier for oral delivery, based on self-assembled beta-casein micelles. J. Controlled Release 2012, 160, 164−171. (26) Priyadarsini, K. I. Photophysics, photochemistry and photobiology of curcumin: Studies from organic solutions, bio-mimetics and living cells. J. Photochem. Photobiol., C 2009, 81−95. (27) Leung, M. H.; Kee, T. W. Effective stabilization of curcumin by association to plasma proteins: human serum albumin and fibrinogen. Langmuir 2009, 25, 5773−5777. (28) Barik, A.; Priyadarsini, K. I.; Mohan, H. Photophysical studies on binding of curcumin to bovine serum albumins. Photochem. Photobiol. 2003, 77, 597−603. (29) Gupta, S. C.; Prasad, S.; Kim, J. H.; Patchva, S.; Webb, L. J.; Priyadarsini, I. K.; Aggarwal, B. B. Multitargeting by curcumin as revealed by molecular interaction studies. Nat. Prod. Rep. 2011, 28, 1937−1955. (30) Yan, Y.; Marriott, G. Analysis of protein interactions using fluorescence technologies. Curr. Opin. Chem. Biol. 2003, 7, 635−640. (31) Bong, P. H. Spectral and Photophysical Behaviors of Curcumin and Curcuminoids. Bull. Korean Chem. Soc. 2000, 21, 81−86. (32) Aoki, H.; Takada, Y.; Kondo, S.; Sawaya, R.; Aggarwal, B. B.; Kondo, Y. Evidence that curcumin suppresses the growth of malignant gliomas in vitro and in vivo through induction of autophagy: role of Akt and extracellular signal-regulated kinase signaling pathways. Mol. Pharmacol. 2007, 72, 29−39. (33) Karmakar, S.; Banik, N. L.; Ray, S. K. Curcumin suppressed antiapoptotic signals and activated cysteine proteases for apoptosis in human malignant glioblastoma U87MG cells. Neurochem. Res. 2007, 32, 2103−2113. (34) Miyamoto, S.; Yagi, H.; Yotsumoto, F.; Horiuchi, S.; Yoshizato, T.; Kawarabayashi, T.; Kuroki, M.; Mekada, E. New approach to cancer therapy: heparin binding-epidermal growth factor-like growth 217
dx.doi.org/10.1021/mp400378x | Mol. Pharmaceutics 2014, 11, 208−217
