Cancer Selectivity of Tetrabranched Neurotensin ... - ACS Publications

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Cancer Selectivity of Tetrabranched Neurotensin Peptides Is Generated by Simultaneous Binding to Sulfated Glycosaminoglycans and Protein Receptors Chiara Falciani,*,†,‡ Jlenia Brunetti,† Barbara Lelli,† Niccolò Ravenni,† Luisa Lozzi,† Lorenzo Depau,† Silvia Scali,† Andrea Bernini,§ Alessandro Pini,† and Luisa Bracci†,‡ †

Department of Medical Biotechnologies, University of Siena, Via Fiorentina 1, 53100 Siena, Italy Istituto Toscano Tumori (ITT), Via Fiorentina 1, 53100 Siena, Italy § Department of Biotechnology, Chemistry, and Pharmacy, University of Siena, Via Fiorentina 1, 53100 Siena, Italy ‡

S Supporting Information *

ABSTRACT: In previous papers we demonstrated that tetrabranched peptides containing the sequence of human neurotensin, NT4, are much more selective than native monomeric analogues for binding to different human cancer cells and tissues. We show here that the much higher binding of NT4 peptides, with respect to native neurotensin, to either cancer cell lines or human cancer surgical samples is generated by a switch in selectivity toward additional membrane receptors, which are specifically expressed by different human cancers. We demonstrate that the branched structure provides NT4 with ability to bind heparin and receptors belonging to the low density lipoprotein receptor (LDLR) family, known to be involved in cancer biology. Systematic modification of neurotensin sequence in NT4 peptides led to identification of a multimeric positively charged motif, which mediates interaction with both heparin and endocytic receptors. Our findings provide the molecular basis for construction of cancer theranostics with high cancer selectivity.

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(CRC)16 and breast cancer.17 Despite the promising features of NT receptors as tumor targets, neurotensin has not yet been successfully developed as a drug. We demonstrated that tetrabranched peptides synthesized on a three-lysine core become extremely resistant to biological degradation by peptidases, while they may maintain biological activity or even increase it because of multimeric binding.18 Because of their biological stability, tetrabranched peptides have a much longer half-life in vivo and are more suitable than monomeric peptides for development as drugs.19−21 In previous research, we synthesized tetrabranched NT peptides (NT4) conjugated to different functional units for selective imaging and killing of cancer cells22 and we demonstrated that unlike monomeric NT peptides,23 NT4 peptides efficiently discriminate between tumor and healthy tissue in human surgical samples of colon and pancreas adenocarcinoma from many patients, with very good statistical significance. Moreover, we proved that NT4 can efficiently and selectively steer functional units24 or liposomes25 for cell imaging or therapy of different human cancer cells. Using NT4 conjugated to methotrexate or 5FdU, we obtained significant reduction of tumor growth in mice.22,23 Since multimeric binding, together with the chemical modification caused by coupling to the branched core, might

INTRODUCTION Selective targeting of tumor cells to enhance the therapeutic index, while limiting possible adverse effects, has long been a goal in oncology. The endogenous peptide neurotensin (NT) has been studied for years as a potential tumor selective targeting agent. NT is a 13 amino acid endogenous neuropeptide, the receptors of which have been reported as overexpressed in different human cancers and cancer cell lines.1 NT has at least three different cell membrane receptors: NTR1 and NTR2, which are both G-protein-coupled receptors, and NTR3/sortilin, which is a type I transmembrane receptor belonging to the Vps10 domain containing (Vps10d) protein family of endocytic receptors.2−4 Numerous studies have reported the high affinity NTR1 as a potential tumor target due to its overexpression in different human cancers, such as colon, pancreas, and prostate carcinomas.5−8 Nonetheless, in the past few years, evidence is accumulating that NTR1 might not be the only relevant NT receptor with a role in cancer cell physiology. An important role for NTR3/sortilin in NTinduced signaling in cancer cells has been repeatedly reported.8−11 Sortilin might be relevant in NT-induced cancer cell proliferation, motility, and invasiveness.12−15 Many different cancer cells, in which NT-induced effects on growth or motility can be measured, do not express NTR1, whereas sortilin was found to be expressed in all cancer cell lines tested.9,12,13 More recently, a determinant role of sortilin in stimulation of cancer cells was reported in colorectal cancer © XXXX American Chemical Society

Received: March 4, 2013

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dx.doi.org/10.1021/jm400329p | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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Figure 1. Ca2+ release induced by branched and monomeric NT peptides in HT29 cells. (A) Endocellular Ca2+ release induced by tetrabranched and monomeric NT peptides in HT29 cells, detected by FLIPR. Right panels: Residual calcium release after the second challenge with native agonist compared to calcium release curves, during first additions of NT (upper panel) or NT4 (lower panel), obtained in experiments reported in (A). First addition curves are reported with the same symbols as in (A). Constants were calculated as mean of three independent experiments. (B) Inhibition of peptide-induced Ca2+ release by the NTR1 antagonist SR48692.

Since the high affinity NTR1 receptor is involved in release of endocellular Ca2+, we measured Ca2+ release induced by NT4 or monomeric NT peptides in different cell lines, using a fluorimetric image plate reader (FLIPR). HT29 human colon adenocarcinoma cells expressing NTR1, NTR3/sortilin, and SorLA/LR11 were used. TE671 human rhabdomyosarcoma cells were used as negative controls, since they express NTR3/ sortilin but not NTR1. No expression of NTR2 was detected in either cell line.23 Monomeric native NT induced strong Ca2+ release in HT29 cells. In the same cells, monomeric NT sequence carrying a Cterminal amide (NT-amide) and NT4, which also lacks the free C-terminal COOH, induced much lower Ca2+ release (Figure 1A). As expected from the fact that TE671 cells do not express NTR1 or NTR2, no Ca2+ release was detected in these cells when they were incubated with any NT peptide. We needed to understand whether the lower calcium release induced by NT4, with respect to native NT, was due to lower affinity for NTR1 or partial agonism. Hence, HT29 cells were pulsed with different concentrations of native monomeric NT (Figure 1A, right upper panel) or NT4 (Figure 1A, right lower panel) (first additions) followed by a saturating concentration of native NT (second addition). Endocellular Ca2+ release was monitored during the second addition and compared to that

have affected receptor recognition by the neurotensin sequence, we compared receptor selectivity of branched NT4 peptides with that of native NT with the aim of explaining the much higher cancer selectivity of NT4 than monomeric NT. Our results indicate that the much higher binding of NT4 peptides to cancer cell lines or human cancer surgical samples is generated by a switch in selectivity toward additional membrane receptors, which are very specifically expressed by many different human cancers.

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RESULTS

NT4 and NT Binding to NTR1. The COOH terminal group of the NT sequence and of its C-terminal functional fragment NT(8−13) has been reported to play a crucial role in peptide binding to NTR1 and NTR3/sortilin.26,27 In NT4 tetrabranched peptides, the C-terminal carboxyl group is missing, being engaged in coupling to the three-lysine core. Moreover, tetrabranched peptides constructed by other authors by coupling the NT(8−13) sequence via the N-terminus, rather than via the C-terminus, to a branched core consisting of glutamic acid are reported to have a lower IC50 versus native NT than our tetrabranched NT4 peptides,28 suggesting that the presence of a free C-terminus may modify receptor binding. B



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Figure 2. NT4 binding to sortilin. (A) SPR analysis of NT4 binding to sortilin. Different concentrations of NT4 were injected over recombinant sortilin, which had been covalently immobilized on the sensor chip. (B) Flow cytometry analysis showing binding of ppSortilin-biotin (10 μM) to HT29 (upper left panels) and TE671 (upper right panels) cells and inhibition by NT4 (10 μM) (lower panels).

obtained during the first pulse, at each peptide concentration. Only NTR1 receptors that did not bind the ligand in the first challenge, whether agonist, antagonist, or partial agonist, released Ca2+ during the second addition. NT4 challenge did not impair Ca2+ release by native NT (Figure 1A, right lower panel), meaning that the lower activity was not due to full binding and partial agonism but to lower binding and full agonism. The intersection of the two curves obtained during the first and second pulses made it possible to measure peptide receptor affinity (Figure 1A). These results indicate that NT4 bound the NTR1 receptor with an affinity (3.37 × 10−6) that was 3 logarithmic orders of magnitude lower than that of the native NT (3.35 × 10−9). In order to confirm that peptide-induced endocellular Ca2+ release was caused by the NTR1 receptor, we used specific NTR1 antagonist SR48692,8 which completely inhibited intracellular Ca2+ release induced by any NT peptides. Even the much lower Ca2+ release induced by peptides lacking the free C-terminal COOH was completely inhibited by SR48692, indicating that it was still induced by stimulation of NTR1 (Figure 1B). NT4 Bound Additional Receptors beyond NTR1. We previously found that fluorophore-conjugated NT4 bound membrane receptors on HT29 and TE671 cell lines, though the

latter does not express NTR1.25 Confocal microscopy and flow cytometry experiments confirmed binding and cell internalization of NT4 peptides in both cell lines (Supporting Information Figure 1). Taken together, confocal microscopy, flow cytometry, and Ca2+ release experiments indicated that NT4 binds to additional receptors, beyond NTR1. Binding of NT4 to NTR3/sortilin was checked by SPR on the recombinant protein, which had previously been immobilized covalently on the surface of a Biacore sensor chip. As shown in Figure 2A, NT4 clearly bound to recombinant sortilin. Sortilin, belonging to the Vps10d family, is synthesized as a precursor and converted into the mature receptor by intracellular cleavage and removal of a propeptide.2 The propeptide generated by protein cleavage specifically binds the mature receptor and may inhibit binding of other ligands.3,4 We synthesized sortilin propeptide (ppSortilin) and tested inhibition of biotinylated ppSortilin binding to HT29 or TE671 cells by NT4, by flow cytometry (Figure 2B). NT4 inhibited binding of ppSortilin to HT29 and TE671 cells, confirming that NT4 maintains the ability of native neurotensin to bind sortilin. NT4 Bound Heparin and Heparan Sulfate. Membrane receptors belonging to the Vps10d family2 or the contiguous family of low density lipoprotein receptors (LDLR)29 are C



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endocytic receptors that share multiple ligands.4,30−33 Most ligands of LDLR, such as ApoE, are heparin-binding proteins, which bind LDLR and heparan sulfate proteoglycans (HSPGs) by means of repeated pairs of positively charged residues, establishing multimeric ionic interactions with highly repeated negatively charged residues on LDLR or HSPG.34−37 Since the neurotensin sequence has one lysine at position 6 and two contiguous arginines at positions 8 and 9, repeated four times in the branched structure of NT4, it seemed reasonable to test whether NT4 bound heparin and other glycosaminoglycans (GAGs) . We tested binding of heparin, heparan sulfate (HS), and hyaluronic acid (HA) to NT4 by SPR. Biotin-conjugated NT4 was captured on a streptavidin-coated sensor chip, and GAGs were injected as analytes. Heparin and HS bound to NT4, and binding of HA was much lower (Figure 3A).

and not HA, completely inhibited binding of NT4 to cancer cells (Figure 4A). IC50 was measured (6.5 × 10−8) as reported in Supporting Information Figure 3. Inhibition of NT4 binding to cancer cells by heparin was confirmed by confocal microscopy on TE671 (Figure 4B) and HT29 (Supporting Information Figure 2) cell lines. We previously demonstrated that NT4 peptides can efficiently discriminate between tumor and healthy tissue with very good statistical significance in a large percentage of patients undergoing surgical resection for either CRC or pancreas ductal adenocarcinoma.23 In order to merge results from cell models and tissue samples, we tested whether heparin interfered with binding of NT4 on human tissue samples. Surgical samples of cancer and healthy tissue from three patients with CRC and three patients with pancreas adenocarcinoma were analyzed by confocal microscopy for binding of NT4 (Figure 4C). Images were analyzed as already described23 for pixel distribution in the green color range, thus translating the immunofluorescence signals into numbers representing the median value of green staining, in the range of the RGB system. Results confirmed the already reported selectivity of NT4 peptides for cancer versus healthy tissue in all samples analyzed, whereas binding of monomeric NT was not detected. The effect of heparin on binding of NT4 was then tested on surgical samples from cancer and healthy tissues. The presence of heparin produced a reduction of NT4 binding to cancer tissues (Figure 4C), which brought staining to the cutoff level (Figure 4D), thus abolishing NT4 selectivity for cancer tissues. Binding of NT4 to Cancer Cells Was Inhibited by Heparin Binding Proteins. Binding of NT4 to heparin and HS indicates that NT4 might mimic heparin-binding sites of proteins, such as apolipoprotein E (ApoE) and the growth factor midkine, which also bind members of Vps10d, LDLR, or the low density lipoprotein receptor-related (LRP) protein family through repeated groups of proximal positively charged amino acids. ApoE is known to bind heparin as well as different members of LDLR, LRP, and Vps10d protein families.30−37 Midkine is a 13 kDa growth factor largely expressed during embryogenesis and with limited expression in adult tissues but overexpressed in many different tumors. It plays important roles in cell proliferation, differentiation, and migration.38 Midkine has been reported to bind different cell membrane receptors, including LRP1, LRP6, integrins, and sulfated proteoglycans, by means of clusters of positively charged residues.38−41 ApoE and midkine were analyzed by flow cytometry for their ability to inhibit NT4 binding to TE671 cancer cells. Both heparin-binding proteins very clearly inhibited binding of NT4 to cancer cells (Figure 5). Structural Similarity between NT4 and Midkine. To compare NT4 with heparin-binding proteins from a structural point of view, the branched peptide was modeled as a threedimensional structure and compared to the midkine structure, which was built from the N- and C-terminal domains available in Protein Data Bank (PDB codes 1MKN and 1MKC, respectively). As illustrated in Figure 6, a very similar spatial distribution of clusters of positively charged residues is obtained in the two structures. K6, R8, and R9 of neurotensin peptides overlap with analogous consensus clusters of positively charged residues in midkine. Interestingly, these clusters, which involve K86, K87, and R89 in the midkine C-terminal domain and K2,

Figure 3. NT4 binding to heparin and other glycosaminoglycans: (A) SPR analysis of GAGs (50 μg/mL) binding to surface immobilized NT4; (B) branched NT4 (250 nM) or native monomeric NT (NT1− 13) and NT-amide (1 μM) binding to surface-immobilized heparin.

In order to analyze the possible contribution of multimeric interactions, NT4 binding to surface immobilized heparin was compared with that of monomeric NT and NT-amide. Biotinylated heparin was immobilized on a streptavidin-coated sensor chip, and NT peptides were injected as analytes. NT4 very clearly bound to surface-immobilized heparin, whereas no binding was detected for monomeric NT, with or without a free terminal carboxyl group (Figure 3B), indicating that multimericity rather than the terminal carboxyl group is determinant for heparin binding. Heparin Inhibited Binding of NT4 to Cancer Cells and Tissues. We proved by flow cytometry that heparin and HS, D



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Figure 4. Heparin inhibited NT4 binding to human cancer cells and tissues. (A) Inhibition of NT4-biotin (250 nM) binding to TE671 cells by GAGs (100 μg/mL) as analyzed by flow cytometry. (B) TE671 cells stained with NT4-biotin (1 μM), followed by 0.5 μg/mL streptavidin-FITC (green), with and without heparin (10 μM). (C) NT4-biotin binding (1 μM) (green) to colon adenocarcinoma (K) and healthy human colon tissue (H) without (upper panels) and with (lower panels) addition of heparin (10 μM). Nuclei were stained with DAPI (blue). (D) Mean fluorescent value of NT4 binding to cancer and healthy human tissues from three patients with colon and three patients with pancreas adenocarcinoma, with or without heparin. The dotted line represents the cutoff, corresponding to the mean fluorescent value of NT4 binding on 29 colon and 24 pancreas samples of healthy tissue.

Figure 5. Heparin-binding proteins inhibited NT4 binding to cancer cells. Inhibition of NT4-biotin (250 nM) binding to TE671 cells by 1 μM ApoE (A) or midkine (B), analyzed by flow cytometry.

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Figure 6. NT4 and midkine models. Structures of NT4 modeled as extended conformation (left) and midkine (right) compared on the same scale. Lysine residues from the scaffold of NT4 are colored in yellow, orange, and cyan, while neurotensin peptides are colored in green. Selected clusters of positively charged residues are shown as spheres (white = carbon, blue = nitrogen) on both structures. Similarity in spatial distribution of such groups is visible.

Figure 7. NT4 binding to LRP1, LRP6, and sortilin was inhibited by heparin. SPR analysis of monomeric (100 μM, dotted line) and tetrabranched (250 nM, solid line) NT peptides binding to immobilized sortilin (A), LRP6 (B), and LRP1 (C). (D) Inhibition of NT4 binding to sortilin, LRP6, and LRP1 analyzed by SPR with increasing concentrations of heparin.

K3, and K5 in its N-terminal domain, are reported to potentially interact with heparin.39 NT4 Bound LRP1 and LRP6 and Competed with Heparin for Binding. To determine whether NT4 also binds members of the LDLR protein family by mimicking ApoE and midkine heparin binding sites, we tested NT4 binding on two different LRP receptors: LRP6, described as a receptor for midkine and known to be overexpressed in several cancers, where it functions as receptor for the morphogenic ligand Wnt,42 and LRP1, a receptor for ApoE and midkine and described to be overexpressed in cancer.29,33−37,43

We found that NT4 bound recombinant LRP6 and LRP1 in Biacore and that binding was inhibited by heparin. Notably, binding of NT4 and inhibition by heparin were very similar on immobilized recombinant sortilin, LRP6, and LRP1, suggesting similar interaction of NT4 with the three receptors (Figure 7). Binding of monomeric NT, with or without a free terminal carboxyl group, was not detected on any protein receptor nor was it detected on heparin, confirming that the branched structure was even essential for binding to protein receptors. Decoding NT4 Binding to LRP1, LRP6, Sortilin, and Heparin. In order to verify whether binding of NT4 to protein receptors and heparin was mediated by the same residues, we F



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switch of receptor selectivity by decreasing affinity to highaffinity NTR1 and simultaneously allowing binding to additional receptors. We demonstrated here that NT4 can bind sortilin. Sortilin shares ligands, such as apolipoprotein E,2−4,30−37 with the related protein family of low density lipoprotein receptors. A hallmark of most LDLR ligands is the ability to bind heparin and sulfated proteoglycans by means of a common module consisting of repeated positively charged residues that interact with multiple negatively charged groups on HSPG. It was proposed that recognition of LDLR by its heparin-binding ligands is mediated by the same positively charged modules, which interact with aspartic acid motives, present in the repeated complement-like domains of LDLR.35−37 A negatively charged repeated motif, called Aspbox, is also present in the β-propeller, i.e., binding site, of Vps10d proteins.45 Indeed, members of the Vps10d and LDLR families have common structural features, including βpropellers, flanked by hydrophobic modules.2,29 Considering these structural and functional similarities between the Vps10d receptors and LDLR and that the neurotensin sequence has one lysine and two adjacent arginine residues that are repeated four times in the branched molecules, the hypothesis that NT4 acts as a heparin-binding protein is verified. We report here that heparin and heparan sulfate both bind NT4 and inhibit NT4 binding to different cancer cell lines and even to human surgical samples, in which heparin cancels NT4 selectivity for cancer versus healthy tissue. We also found that NT4 binds LRP1 and LRP6 and its binding is inhibited by heparin. Moreover, known ligands of LRP1 and LRP6 receptors, like ApoE and midkine, can inhibit binding of NT4 to cancer cells. By systematic substitutions of residues in the NT sequence of NT4, we proved that binding of NT4 to the protein receptors sortilin, LRP6, and LRP1 is mediated by the same residues, which are also responsible for heparin binding. The determinant role of the cluster of positively charged residues of the NT sequence for NT4 binding to protein receptors and heparin was clearly confirmed. In conclusion, we believe that the multimericity of our tetrabranched peptides, associated with the loss of the NT Cterminal carboxyl group, produced a switch in selectivity toward receptors that are more selectively expressed in cancer tissues than NTR1, like LRP1 and LRP6, which have both already been described as important tumor markers.37,42,43 Moreover, our results confirm that GAGs can mimic the ligand binding site of different LDLR and other endocytic receptors, including member of the Vps10d protein family, and suggest that by reproduction of the multimeric and positively charged heparin-binding site of LDLR ligands, it is possible to target multiple endocytic receptors and sulfated proteoglycans at the same time, obtaining extremely high selectivity toward many different human cancers. It is becoming clear that HSPGs are important in many aspects of cancer progression.46,47 The extremely high cancer selectivity that can be achieved by simultaneous targeting sulfated glycosaminoglycans and endocytic receptors confirms the determinant role of HSPGs as tumor markers and reinforces the hypothesis of their synergic action with LDLR in cancer cell development. An attractive hypothesis, which seems to be reinforced by our data, is that negatively charged HSPGs may function as a sort of spider web for protein growth factors containing multimeric positively charged binding motives. Multimericity of both ligand and receptor binding motives may enable formation of trimetric complexes in which HSPGs attract ligands, trapping

synthesized modified tetrabranched NT4 in which each residue of the NT sequence was substituted with alanine. Binding of modified NT4 peptides was then tested by SPR on recombinant sortilin, LRP6, and LRP1 and on heparin and compared with that of unmodified NT4 at identical concentrations. A single modification of the C-terminal 8−13 sequence proved to be more critical than modifications of the N-terminal sequence. Substitution of the arginines at positions 8 and 9 completely abolished binding on protein receptors and on heparin (Figure 8). Moreover, substitution of any residue in

Figure 8. Decoding NT4 binding to sortilin, LRP6, LRP1, and heparin. Binding of NT4 Ala-modified peptides (250 nM) to heparin and cell membrane receptors analyzed by SPR on immobilized molecules. Results are expressed as a percentage of unmodified NT4 peptide binding on the same molecule under identical conditions.

the C-terminal hydrophobic region (YIL) also resulted in complete loss of binding on all protein receptors and on heparin as well. In the 8−13 sequence, only substitution of proline at position 10 did not modify binding on protein receptors or heparin. Substitution of lysine at position 6 and tyrosine at position 3 produced a clear reduction of binding. These results indicate that binding of NT4 to sortilin, LRP6, and LRP1 is mediated by the same residues, which are also responsible for heparin binding, with a determinant role of the cluster of positively charged residues.

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DISCUSSION AND CONCLUSIONS In previous papers we reported much higher selectivity of tetrabranched NT4 peptides than native monomeric NT peptides toward cancer cells and tissues. We also demonstrated that NT4 can be coupled to many different functional units for selective cancer cell tracing or therapy and can induce tumor growth reduction in animal models.22−25 We then proposed NT4 as promising cancer selective theranostics for different human cancers, including CRC, pancreas adenocarcinoma, and urinary bladder cancer.44 Nonetheless, we were aware that multimeric binding of tetrabranched peptides, together with the chemical modification produced by coupling to the branched core, might modify receptor selectivity of NT4 with respect to native monomeric NT and we had no conclusive indication about which receptor our branched NT4 peptides were binding to. The much higher cancer selectivity of NT4 with respect to native NT deserved investigation on the specific receptors of branched NT4 in cancer cells. The results of the present paper demonstrate that synthesis of the neurotensin sequence in a tetrabranched form induces a G



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serum, 200 mg/mL glutamine, 100 mg/mL streptomycin, and 60 mg/ mL penicillin. TE671 human rhabdomyosarcoma cells were grown in DMEM (Dulbecco’s modified Eagle medium) supplemented as above. Intracellular Ca2+ Release. HT29 and TE671 cells were plated at a density of 5 × 104 cells/well in 96-well plates and incubated at 37 °C for 24 h. Cells were loaded with Fluo-4 AM (Invitrogen) for 30 min at 37 °C. After washing, intracellular calcium fluxes in the presence of various concentrations of peptides (from 10 μM to 100 pM) were measured on a fluorimetric image plate reader (FLIPR, Molecular Devices). The second addition was carried out using 500 nM NT, corresponding to the EC100 concentration. For the inhibition of Ca2+ release by the NTR1 antagonist SR48692, 5 × 104 cells/well were incubated with 10 μM NT4 or with 5 nM NT in the presence of SR48692 (from 10 μM to 100 pM). EC50 values were determined by nonlinear regression analysis using the GraphPad Prism 5.03 software and were calculated on three independent experiments. Flow Cytometry. All experiments were performed using 1 × 105 HT29 or TE671 cells in 96-well U-bottom plates. A total of 10 000 events in a BD FACS Canto II or in a BD FACS Calibur (Becton Dickinson, NJ, U.S.) were analyzed. Results were analyzed using FCS Express 4 Plus software. For peptide binding, cells were incubated with 1 μM NT4-biotin for 45 min at room temperature. Inhibition of propeptide (ppSortilin) binding by NT4 was measured incubating cells with 10 μM ppSortilinbiotin and 10 μM NT4 for 30 min at room temperature. Inhibition of NT4 binding by GAGs was carried out incubating cells with 250 nM biotinylated NT4 and 100 μg/mL GAGs. Inhibition of NT4 binding by heparin-binding proteins was performed incubating cells with 250 nM NT4-biotin and 1 μM apolipoprotein E4 or midkine. Cells were finally incubated with 1 μg/mL streptavidin-FITC. All dilutions were performed in PBS, containing 5 mM EDTA and 0.5% BSA. Surface Plasmon Resonance. All experiments were performed on a BIA T100 (GE Healthcare). Binding of glycosaminoglycans was performed on a sensor chip previously coated with NT4. Briefly, an amount of 3000 RU of streptavidin was immobilized via standard procedures on the dextran matrix of a CM5 sensor chip and then 10 μg/mL biotinylated NT4 in HBS-EP+ (10 mM Hepes, 150 mM NaCl, 3.4 M MEDTA, 0.05% polysorbate 20, pH 7.4) was injected for 90 s at the flow rate of 10 μL/min on the flow cell, obtaining 1500 RU. Heparin, heparan sulfate, and hyaluronic acid (50 μg/mL, in HBS-EP +) were injected for 120 s at a flow rate of 20 μL/min on immobilized NT4. A flow cell coated only with streptavidin was used as reference. Regeneration of the matrix was achieved with a short pulse of 1 M NaCl−10 mM NaOH. For NT4 binding to recombinant protein receptors, sortilin, LRP6, and LRP1 were immobilized via standard amine coupling on the dextran matrix of a CM4 sensor chip, obtaining 2400, 1600, and 1100 RU, respectively. For binding of NT4 to immobilized heparin, biotinylated heparin diluted in HBS-EP+ at 100 μg/mL was injected for 90 s at the flow rate of 10 μL/min on the surface of a SA sensor chip obtaining 50 RU. NT4 (250 nM in HBS-EP+) or monomeric NT peptides (100 μM) were injected for 120 s at the flow rate of 10 μL/ min on immobilized sortilin, LRP6, LRP1, and heparin. Competition experiments were carried out injecting 250 nM NT4 with various concentrations (500, 100, 50, and 10 ng/mL) of heparin. Experiments were repeated at least three times. Regeneration of the matrix was achieved by a short pulse of 1 M NaCl. Constants were calculated from a single significative experiment. Binding of alanine-modified NT4 peptides was analyzed injecting 250 nM peptides for 120 s at a flow rate of 10 μL/min on immobilized sortilin, LRP6, LRP1, and heparin. Modeling. NT4 was modeled as extended conformation structure with PyMol (The PyMOL Molecular Graphics System, version 1.4, Schrödinger, LLC) and refined by energy minimization with the Gromacs package48 and Amber force field.49 A new force field entry was created for lysine forming the scaffold by reparameterization of the standard lysine residue from the Amber library to take covalent bonding of the side chain amine into account. Neurotensin peptides were linked to the available amines of the scaffold. Midkine structure

and concentrating them near their membrane receptors. In this view, multimeric positively charged ligands, which allow simultaneous targeting of HSPGs and protein receptors, may provide very selective targeting agents for many different cancers.

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EXPERIMENTAL SECTION

Recombinant Proteins. Recombinant human LRP-1 cluster IV Fc chimera (code 5395-L4), recombinant human sortilin (code 3154-ST), and recombinant human LRP-6 Fc chimera (code 1505-LR) were from R&D Systems. Recombinant human midkine (code SRT3114), recombinant human apolipoprotein E4 (code A3234), and α2macroglobulin (code M6159) were from Sigma Aldrich. Glycosaminoglycans. Heparin (code H3149), heparin-biotin (code B9806), hyaluronic acid (code H7630), and heparan sulfate (code H7640) were from Sigma Aldrich. All proteins and glycosaminoglycans tested had a purity of at least 95% as stated by the producer. Peptide Synthesis. Peptides were synthesized on an automated multiple synthesizer (MultiSynTech, Germany) by standard Fmoc chemistry. Protected L-amino acids, TentaGel resin, and Fmoc4-Lys2Lys-β-Ala-Wang resin were purchased from Iris Biotech, Germany. The coupling reagent HBTU (O-benzotriazole-N,N,N′,N′-tetramethyluronium hexafluorophosphate) was from MultiSynTech, and DIPEA (N,N-diisopropylethylamine) was from Merck as base. Tetrabranched peptides were synthesized on Fmoc4-Lys2-Lys-beta-Ala-Wang resin or built using two consecutive Fmoc-Lys(Fmoc)-OH coupling steps to form the branched core on TentaGel resin. NT4-biotin was synthesized on TentaGel resin with Fmoc-Lys(biotin)-OH as the first coupling step and Fmoc-PEG12-OH as the second. FmocLys(Fmoc)-OH was then used to build the tetrameric core. Pyro-GluO-pentachlorophenyl ester (Bachem, Switzerland) was used for the last coupling step, since pyro-Glu is the N-terminal acid of the neurotensin sequence. Peptides were then cleaved from the resin, deprotected, and lyophilized. Monomeric peptides were synthesized on TentaGel resin as amides. NT conjugated to biotin at the C-terminal was obtained using FmocLys(biotin)-OH as the first and Fmoc-PEG12-OH as the second coupling step. NT conjugated to biotin at the N-terminal was obtained on Fmoc-Leu-Wang resin using Fmoc-Lys(biotin)-OH in the last coupling step and glutamine in place of pyro-glutamic acid. The biotin carrying lysine was acetylated at the N-alpha before cleavage and deprotection. HPLC purification was performed on a C18 Jupiter column (Phenomenex). Water with 0.1% TFA (A) and methanol (B) were used as eluents. Linear gradients of B in 30 min were run at flow rates of 0.8 and 4 mL/min for analytical and preparatory procedures, respectively. All compounds were also characterized on a Bruker Ultraflex MALDI TOF/TOF mass spectrometer. NT (pyELYENKPRRPYIL-OH) MS, m/z calculated for C78H121N21O20 [M + H]+: 1672.92. Found 1672.89. tR (from 80% A to 20% A), 22.23 min. NTamide (pyELYENKPRRPYIL-NH 2 ) MS, m/z calculated for C78H122N22O19 [M + H]+: 1671.93. Found 1671.79. tR (from 80% A to 5% A), 21.03 min. NT-biotin (pyELYENKPRRPYIL-Peg12K(biotin)-NH2) MS, m/z calculated for C121H201N27O35S[M + H]+: 2626.11. Found 2625.83. tR (from 80% A to 1% A) 22.07 min. BiotinSort (biotin LC-QDRLDAPPPPAAPLPRWSGPIGVSWGLRAAAAGGAFPRGGRW--RR) MS, m/z calculated for C225H1346N72O55S [M + H]+: 4985.69. Found 4987.08. tR (from 80% A to 5% A), 25.33 min. NT4 (pyELYENKPRRPYIL)4K2K-β-Ala MS, m/z calculated for C333H519N91O81 [M + H]+: 7094.24. Found 7095.15. tR (from 80% A to 20% A), 26.63 min. Biotinylated NT4 (pyELYENKPRRPYIL)4K2K-PEG12-K(biotin) MS, m/z calculated for C373H594N96O95S [M + H]+: 7976.35. Found 7978.72. tR (from 80% A to 20% A), 26.99 min. All peptides tested had a purity of at least 95% as established by HPLC. Cell Lines. Cell lines were purchased from Istituto Zooprofilattico Sperimentale (Brescia, Italy). HT29 human colon adenocarcinoma cells were grown in McCoy’s 5A, supplemented with 10% fetal calf H



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was built from the N- and C-terminal domains available in Protein Data Bank (PDB codes 1MKN and 1MKC, respectively). Confocal Microscopy. HT29 and TE671 cells were plated at a density of 3 × 104 cells/well in 24-well plates with cover glass slides. Samples were fixed through incubation with a phosphate buffered saline (137 mM NaCl, 2 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4, PBS)−4% paraformaldehyde (PFA) solution for 15 min, saturated for 30 min at 37 °C with PBS−1% bovine serum albumin (BSA), and incubated with 1 μM biotinylated peptides for 30 min at room temperature, then with 0.5 μg/mL streptavidin-FITC for 15 min at room temperature. For peptide internalization, HT29 and TE671 cells plated and saturated as above were incubated with 1 μM biotinylated peptides for 30 min at room temperature to enable peptide binding followed by 0.3 μg/mL streptavidin-Atto 550 for 15 min at room temperature. Cells were incubated at 37 °C with prewarmed culture medium for different times (0, 1, 2, 4 h) and then fixed with PBS−4% PFA for 10 min at room temperature. Cell membranes were stained with 0.3 μg/mL lectin-FITC for 10 min at room temperature. Samples were mounted using Fluoroshield with DAPI (Sigma Aldrich). Peptide binding and cell localization were analyzed by confocal laser microscope (Leica TCS SP5) with 364/488/555 nm excitation and 458/519/565 nm emission filters for DAPI, FITC and Atto 550, respectively. All images were processed using ImageJ software (NIH). Human Specimens. Samples of colon or pancreas adenocarcinoma and corresponding healthy tissue were collected from patients who had undergone surgical resections at the Third Division of General and Oncologic Surgery, Careggi Regional and University Hospital, Florence, Italy. Healthy tissues were obtained from the normal mucosa 10 cm from the tumor edge. All patients were given an informed written consent module to read and sign before surgery, including detailed explanations of tissue sample collection and sensitive data management. This study was carried out with approval of the Local Ethics Committee. Human Tissue Analysis. Samples were embedded in Tissue-Tek (Sakura Fine Technical) immediately after surgery and stored in liquid nitrogen. The 10 μm thick sections, obtained with a Leica CM1850 UV cryostat, were dried at 37 °C for 10 min, fixed with PBS−4% PFA for 15 min at room temperature, and incubated in 0.1 M glycine overnight at 4 °C. Then blocking with FBS for 30 min at 37 °C was performed, followed by incubation with biotinylated peptides (1 μM in PBS−1% BSA) for 30 min at room temperature and incubation with streptavidin-FITC (0.5 μg/mL in PBS−1% BSA) for 15 min at room temperature. Samples were mounted using Fluoroshield with DAPI.

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ABBREVIATIONS USED ApoE, apolipoprotein; CRC, colorectal cancer; DAPI, 4′,6diamidino-2-phenylindole; DMEM, Dulbecco’s modified Eagle medium; 5FdU, 2-deoxy-5-fluorouridine; FITC, fluorescein isothiocyanate; FLIPR, fluorimetric image plate reader; GAG, glycosaminoglycan; HA, hyaluronic acid; HS, heparan sulfate; HSPG, heparan sulfate proteoglycan; LDLR, low density lipoprotein receptor; LRP, low density lipoprotein; LRP1, low density lipoprotein receptor-related protein 1; LRP6, low density lipoprotein receptor-related protein 6; NT, neurotensin; NTR1, neurotensin receptor 1; NTR2, neurotensin receptor 2; NTR3/sortilin, neurotensin receptor 3/sortilin; ppSortilin, sortilin propeptide; RGB, red green blue; SPR, surface plasmon resonance; Vps10, vacuolar protein sorting 10

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

Binding and internalization of NT4 in different cancer cell lines and inhibition of NT4 binding to cancer cells by heparin. This material is available free of charge via the Internet at http:// pubs.acs.org.

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Article

AUTHOR INFORMATION

Corresponding Author

*Phone: +390577 234928. E-mail: [email protected]. Notes

The authors declare the following competing financial interest(s): L.B., C.F., and A.P. are founders of SetLance srl, a company holding IPRs on NT4 peptides..

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ACKNOWLEDGMENTS The authors thank Stefano Bindi for skillful technical assistance and Prof. Neri Niccolai for helpful discussions. This work was supported by Associazione Italiana per la Ricerca sul Cancro AIRC [IG2009] and by Istituto Toscano Tumori-ITT. I



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