Targeting surface nucleolin with multivalent HB-19 and related Nucant pseudopeptides results in distinct inhibitory mechanisms depending on the malignant tumor cell type
© Krust et al; licensee BioMed Central Ltd. 2011
Received: 15 March 2011
Accepted: 3 August 2011
Published: 3 August 2011
Nucleolin expressed at the cell surface is a binding protein for a variety of ligands implicated in tumorigenesis and angiogenesis. By using a specific antagonist that binds the C-terminal RGG domain of nucleolin, the HB-19 pseudopeptide, we recently reported that targeting surface nucleolin with HB-19 suppresses progression of established human breast tumor cells in the athymic nude mice, and delays development of spontaneous melanoma in the RET transgenic mice.
By the capacity of HB-19 to bind stably surface nucleolin, we purified and identified nucleolin partners at the cell surface. HB-19 and related multivalent Nucant pseudopeptides, that present pentavalently or hexavalently the tripeptide Lysψ(CH2N)-Pro-Arg, were then used to show that targeting surface nucleolin results in distinct inhibitory mechanisms on breast, prostate, colon carcinoma and leukemia cells.
Surface nucleolin exists in a 500-kDa protein complex including several other proteins, which we identified by microsequencing as two Wnt related proteins, Ku86 autoantigen, signal recognition particle subunits SRP68/72, the receptor for complement component gC1q-R, and ribosomal proteins S4/S6. Interestingly, some of the surface-nucleolin associated proteins are implicated in cell signaling, tumor cell adhesion, migration, invasion, cell death, autoimmunity, and bacterial infections. Surface nucleolin in the 500-kDa complex is highly stable. Surface nucleolin antagonists, HB-19 and related multivalent Nucant pseudopeptides, exert distinct inhibitory mechanisms depending on the malignant tumor cell type. For example, in epithelial tumor cells they inhibit cell adhesion or spreading and induce reversion of the malignant phenotype (BMC cancer 2010, 10:325) while in leukemia cells they trigger a rapid cell death associated with DNA fragmentation. The fact that these pseudopeptides do not cause cell death in epithelial tumor cells indicates that cell death in leukemia cells is triggered by a specific signaling mechanism, rather than nonspecific cellular injury.
Our results suggest that targeting surface nucleolin could change the organization of the 500-kDa complex to interfere with the proper functioning of surface nucleolin and the associated proteins, and thus lead to distinct inhibitory mechanisms. Consequently, HB-19 and related Nucant pseudopeptides provide novel therapeutic opportunities in treatment of a wide variety of cancers and related malignancies.
Keywordsantitumoral action surface nucleolin multivalent pseudopeptides nucleolin antagonist peptide anti-inflammatory action nucleophosmin
Nucleolin is a multifunctional DNA-, RNA- and protein-binding protein ubiquitously expressed in exponentially growing eukaryotic cells. It is involved in fundamental aspects of transcription, cell proliferation and growth [1, 2]. Nucleolin is found at several locations in cells: in the nucleolus it controls many aspects of DNA and RNA metabolism ; in the cytoplasm it shuttles proteins into the nucleus and provides a post-transcriptional regulation of strategic mRNAs [4, 5]; and on the cell surface it serves as an attachment protein for several ligands from growth factors to microorganisms [6–12]. In contrast to nuclear nucleolin, surface nucleolin is glycosylated and is constantly induced in proliferating tumor and endothelial cells [6, 13–15].
Surface nucleolin serves as a low affinity receptor for HIV-1 and various growth factors that interact with its C-terminal domain containing nine repeats of the tripeptide arginine-glycine-glycine, known as the RGG or GAR domain [10, 16–20]. Binding of these ligands results in clustering of cell-surface nucleolin in lipid raft membrane microdomains before endocytosis of the ligand-nucleolin complex [10, 17, 19]. Accordingly, surface nucleolin could shuttle ligands between the cell surface and the nucleus thus act as a mediator for the extracellular regulation of nuclear events [18, 20, 21]. Moreover, ligand binding to surface nucleolin could generate high transitory intracellular Ca2+ membrane fluxes, and thus initiate signal transduction events [13, 22–25]. For an example, the binding of P-selectin to human colon carcinoma cells is shown to induce tyrosine phosphorylation of surface nucleolin and formation of a signaling complex containing nucleolin, phosphatidylinositol 3-kinase (PI3-K) and p38 MAPK .
The importance of cell-surface nucleolin in cancer biology was recently highlighted by studies showing that ligands of nucleolin play critical role in tumorigenesis and angiogenesis [20, 26–36]. Accordingly, we recently reported that both of these events are suppressed by targeting surface nucleolin with the HB-19 pseudopeptide, a potent antagonist that forms an irreversible complex with surface nucleolin [9, 37]. By binding to the RGG domain of nucleolin, HB-19 prevents binding of growth factors to cells, triggers calcium entry into cells, inhibits MAP kinase activation, and down-regulates surface nucleolin without affecting nuclear nucleolin [7, 9, 13, 16, 18, 19, 37]. In nude mice, we showed that HB-19 treatment markedly suppresses the progression of established human breast tumor cell xenografts, and in some cases eliminates measurable tumors . This potent antitumoral effect in vivo is attributed to the direct dual inhibitory action of HB-19 on tumor and endothelial cells . In a more relevant tumor model, we showed that HB-19 treatment for several months delays significantly the onset and frequency of spontaneous melanoma in RET mice, impairs tumor angiogenesis, and reduces metastasis while displaying no toxicity to normal tissue . Other groups have reported that guanosine-rich quadruplex-forming oligodeoxynucleotides (GROs), which interact with surface nucleolin and/or intracellular nucleolin are promising agents for treatment of cancer [39–41]. The aptamer AS1411 is the most recent GRO that is currently being tested in Phase II clinical trials. Finally, we recently reported that treatment of G401 cells derived from a rhabdoid tumor of the kidney and TIII cells derived from a malignant melanoma can affect several criteria implicated in their tumorigenic potential, such as restoration of contact inhibition, reduction of colony formation in soft agar, and impairment of tumorigenicity in mice [38, 42]. Interestingly, these changes are associated with a selective down regulation of genes implicated in tumorigenesis.
Although nucleolin does not possess a hydrophobic transmembrane domain to account for its expression at the cell surface, it behaves as a typical membrane-anchored receptor as demonstrated by its clustering when intact cells are incubated with the anti-nucleolin monoclonal antibody. This clustering occurs at the external side of the plasma membrane and is dependent on its indirect association with the intracellular actin cytoskeleton . An actin based motor protein, the nonmuscle myosin heavy chain 9, could serve as a physical linker between surface nucleolin and actin . Here we report that surface nucleolin exists in a high molecular weight complex, referred to as 500-kDa complex, in association with proteins partners known for their implication in tumorigenesis, inflammation, and bacterial infections. As the 500-kDa complex is highly stable, targeting surface nucleolin could change the organization of this complex and thus interfere with the proper functioning of surface nucleolin and the associated proteins. Indeed, by using HB-19 and related multivalent Nucant pseudopeptides that present pentavalently or hexavalently the tripeptide Lysψ(CH2N)-Pro-Arg (Nucant 3, 6, 6L and 7) [43, 44], we show that such surface nucleolin antagonists exert distinct inhibitory mechanisms depending on the malignant tumor cell type. Accordingly, they inhibit the production of pro-inflammatory cytokines by human blood lymphocytes in response to stimulation with inactivated Staphyloccocus aureus, inhibit adhesion of human breast and prostate carcinoma cells, impair spreading of human colon carcinoma cells, and induce a selective cell killing in leukemia cells. Our results further validate surface nucleolin as a strategic target for an effective cancer drug.
Cells and culture medium
Human breast (MDA-MB-231, MDA-MB-435 ), prostate (LNCaP, from American Type Culture Collection, ATCC, Rockville, MD), and cervical (HeLa ) epithelial cancer cells were grown in DMEM-glutamax medium (Gibco Invitrogen, Cergy-Pontoise, France) containing GlutaMAX™, 4.5 g/l glucose and supplemented with 10% heat-inactivated (56°C for 30 min) fetal bovine serum (FBS; Hyclone, Thermo Fisher Scientific, Inc). Human SW480 cells are derived from a grade 3-4 colon carcinoma, while the highly metastatic counterpart SW620 is established from the lymph node of a 51-year-old Caucasian male. Both SW480 and SW620 cells (obtained from Francis Raul, IRCAD) are highly tumorigenic in nude mice. They were cultured in D-MEM, 10% FCS. The murine melanoma TIII cell line was obtained from Armelle Prévost-Blondel . Murine T29 lymphoma cells, obtained from Philippe Kastner (CNRS, Strasbourg), have been established from IkL/L tumors from mice carrying a hypomorphic mutation (IkL/L) in the Ikaros gene encoding a transcription factor that regulates lymphocyte differentiation, and acts also as a tumor suppressor in T lymphocytes . The T29 cells were cultured in RPMI medium containing 25 mM Hepes pH 7.6, 1 mM sodium pyruvate, and 10% FCS. Human leukemia cell lines, Jurkat (acute T cell leukemia), HuT 78 (cutaneous T lymphocyte), RAJI (Burkitt lymphoma), and HL60 (promyelocytic leukemia) were cultured in RPMI-1640 medium (Gibco) containing GlutaMAX™, and supplemented with 10% heat-inactivated FBS as described before . The wild-type Chinese hamster ovary cell line (CHO K1) was purchased from American Type Culture Collection (ATCC; Rockville, MD). The CHO LRP1-null cell line (CHO-13-5-1) was kindly provided by D. Strickland . They were cultured in Ham's F12K medium. All cells were cultured with 10% (v/v) heat inactivated (56°C, 30 min) fetal bovine serum (FBS) (Roche Molecular Biochemicals, Indianapolis, IN) and 50 international units/mL penicillin-streptomycin (Invitrogen). Cell death was monitored conveniently by uptake of the trypan blue dye due to plasma membrane permeability of dying and/or dead cells.
HB-19 and related Nucant pseudopeptides.
Presents pentavalently the pseudo-tripeptide Lysψ(CH2N)-Pro-Arg coupled to the template: H2NLys-Lys-Lys-Gly-Pro-Lys-Glu-Lys-AhxCONH2.
Nucant 7: N7
Presents hexavalently the pseudo-tripeptide Lysψ(CH2N)-Pro-Arg coupled to a similar template as in HB-19: Ac-Lys-Ala-Lys-Pro-Gly-Lys-Ala-Lys-Pro-Gly-Lys-Ala-Lys-Pro-Gly-CONH2.
Nucant 3: N3
Presents pentavalently the pseudo-tripeptide Lysψ(CH2N)-Pro-Arg coupled to the polypeptide template containing Aib (2-aminoisobutyric acid): Ac-Lys-Aib-Gly-Lys-Aib-Gly-Lys-Aib-Gly-Lys-Aib-Gly-Lys-Aib-Gly-CONH2.
Nucant 6: N6
Presents hexavalently the pseudo-tripeptide Lysψ(CH2N)-Pro-Arg (a mixture in L and D configuration) coupled to the polypeptide template containing Aib: Ac-Lys-Aib-Gly-Lys-Aib-Gly-Lys-Aib-Gly-Lys-Aib-Gly-Lys-Aib-Gly-Lys-Aib-Gly-CONH2.
Nucant 6L: N6L
Presents hexavalently the pseudo-tripeptide Lysψ(CH2N)-Pro-Arg (all bonds in L position) coupled to a polypeptide template as in N6.
Analysis of nucleolin by immunoblotting
Nucleolin was analyzed by immunoblotting using either the monoclonal antibody (mAb) D3 against nucleolin (kindly provided by Dr. J. S. Deng) , or rabbit polyclonal antibodies directed against a synthetic peptide corresponding to the first 26 (MVKLAKAGKNQGDPKKMAPPPKEVEE) and the last 16 (GGGGDHKPQGKKTKFE) amino acid residues of human nucleolin as described before . Nucleophosmin was revealed using rabbit monoclonal antibody EP1848Y (abcam). The murine mAb D3 and rabbit antibodies were revealed with horseradish peroxidase-conjugated sheep anti-mouse and goat anti-rabbit immunoglobulin (Jackson ImunoResearch), respectively. The reacting bands were visualized with an enhanced chemiluminescence (ECL) reagent and by exposure to autoradiography film (Amersham Biosciences). In some experiments, the presence of actin was monitored as a control with mAb anti-actin A-4700 (Sigma).
Preparation of cytoplasmic and nuclear extracts
Cells washed in phosphate-buffered saline (PBS) were lysed in buffer E (20 mM Tris-HCl, pH 7.6, 150 mM NaCl, 5 mM MgCl2, 5 mM β-mercaptoethanol, protease inhibitor cocktail (Sigma) and 0.5% Triton X-100) and the nuclei were pelleted by centrifugation (1000 g for 5 min). For the preparation of nuclear extracts, the nuclear pellet was disrupted in buffer I (20 mM Tris-HCl, pH 7.6, 50 mM KCl, 400 mM NaCl, 1 mM EDTA, 5 mM β-mercaptoethanol, protease inhibitor cocktail, 1% Triton X-100, and 20% glycerol). Cytoplasmic and nuclear extracts were then centrifuged at 12,000 g for 10 min, and the supernatants (nucleoplasm) were stored at -20°C. Aliquots of crude cell extracts were diluted in 2 fold concentrated electrophoresis sample buffer containing SDS, and processed for immunoblotting analysis [9, 10]. To monitor the profile of proteins in crude cells extracts, gels were stained with Brilliant Blue G-Colloidal Concentrate from Sigma .
Three days after passage of HeLa P4 cells (10 × 150 cm2 flasks), subconfluent cell monolayers were washed twice in PBS containing 1 mM EDTA before preparation of cytoplasmic extracts using 10 × 200 μl buffer E. For the gel filtration of surface nucleolin bound to the biotinylated HB-19 (HB-19/Btn), HeLa cells were first incubated with 5 μM of HB-19/Btn (45 min at room temperature) before preparation of extracts. Cytoplasmic extracts were diluted in PBS containing 1 mM EDTA then centrifuged at 12,000 g for 10 min, and filtered using Costar® Spin-X® centrifuge tubes filters (0.45 μm membrane pores size). Gel-filtration chromatography was carried out as previously described  on a GE Pharmacia fast protein liquid chromatography (FPLC) system. Briefly, a Superose™ 6 column (1.6 cm × 50 cm) was equilibrated in buffer GF containing 20 mM Tris/HCI, pH 7.6, 50 mM NaCl and 0.1% Triton X-100 at 0.5 ml/min. The column (bed volume 100 ml) was calibrated using cell extracts supplemented with gel filtration standard proteins from GE Healthcare Life Sciences: thyroglobulin (669-kDa), catalase (232-kDa) and BSA (67-kDa). Elution was in buffer GF with collection of 1-ml fractions/2 min, with a void volume (Vo) and total elution volume (Vc) at 30 ml and 114 ml, respectively. The void volume (Vo) was determined from the elution profile of Blue Dextran 2000 and total elution volume from the elution profile of Bromophenol blue. The sample (1 ml) was loaded on the column, and the eluate was monitored at 280 nm. Aliquots from each fraction were assayed for DPP IV activity by the cleavage of Gly-Pro-NH-Np (Sigma) in order to determine the peak of the 110-kDa DPP IV. The peaks of thyroglobulin, catalase and BSA were determined by polyacrylamide gel electrophoresis. The presence of nucleolin in various fractions was revealed by immunoblotting.
Purification of the cell surface expressed 500-KDa complex containing surface nucleolin for microsequencing of nucleolin-associated proteins
Twenty-four hours after passage, CEM cells (109 cells) were washed extensively with PBS before incubation in culture medium (RPMI, 10% FCS) at room temperature for 30 min with 5 μM HB-19/Btn. After washing extensively in PBS containing 1 mM EDTA (PBS/EDTA), cytoplasmic extracts were prepared in lysis buffer E. The complex formed between cell-surface-expressed nucleolin and the HB-19/Btn was isolated by purification of extracts using avidin-agarose (Simon-Pure Immobilized Avidin from Pierce) in PBS/EDTA. After 2 h of incubation at 4°C, the samples were washed extensively with PBS/EDTA. The purified proteins were denatured by heating in the electrophoresis sample buffer containing SDS and analyzed by SDS-PAGE (7.5%). The proteins were transferred to a PVDF membrane before microsequencing the NH2-terminal ends (performed by the Protein-Sequencing Laboratory at Institut Pasteur, Paris).
Triggering the production of pro-inflammatory cytokines by peripheral blood lymphocytes
Peripheral blood mononuclear cells (PBMC) from healthy donors were prepared by Ficoll-Hypaque density gradient centrifugation  and suspended in RPMI 1640 medium containing 1% (v/v) of human serum AB (from Invitrogen). PBMC (106 cells/0.5 ml) in the absence or presence of 10 μM of each of HB-19, N3, N6, and N7, or 1 μg/ml of dexamethasone (Sigma) were stimulated with 108 particles/ml of Heat-killed Staphylococcus aureus (HKSA; InvivoGen, San Diego, USA). The cultures were incubated at 37°C in a 5% CO2 incubator, and the level of TNF-α and IL-6 was monitored in the culture supernatants by ELISA (R & D Systems) at 20 hours post-stimulation.
Inhibition of surface nucleolin function as a cell surface receptor for HIV-1 entry into HeLa CD4+ cells
Surface nucleolin serves as a low affinity receptor implicated in the binding and entry of HIV-1 particles into permissive cells. Consequently, HB-19 treatment of cells prevents HIV binding to cells and thus HIV entry and infection [9, 16]. Here HIV-1 LAI entry was monitored in HeLa-CD4-LTR-LacZ cells containing the bacterial lacZ gene under the control of HIV-1 LTR. Virus entry and replication result in trans-activation of HIV-1 LTR by the viral Tat protein, leading to the expression of β-galactosidase. At 48 h post-infection, cell monolayers are lysed in a phosphate buffer containing Nonidet P-40 (Sigma) (1%; v ⁄ v) and assayed for β-galactosidase activity by measuring optical density at 570 nm .
Analysis of the cell-surface-expressed nucleolin
Two days after seeding, subconfluent cells (about 5 × 106 cells/75 cm2 flask) were incubated (45 min, 20°C) with 5 μM of HB-19/Btn. After washing extensively in PBS containing 1 mM EDTA (PBS-EDTA), nucleus-free cell extracts were prepared in lysis buffer E. The complex formed between cell-surface expressed nucleolin and HB-19/Btn was isolated by purification of the extracts using NeutrAvidin agarose (100 μl; Pierce Biotechnology) in PBS-EDTA. After 3 hours at 6°C, the avidin-agarose samples were washed extensively with PBS-EDTA. The purified surface nucleolin was eluted in the electrophoresis sample buffer containing SDS and analyzed by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The presence of nucleolin was then revealed by immunoblotting using mAb D3 as described before [10, 16]. All cells investigated in this study (MDA-MB-231, MDA-MB-435, LNCaP, HeLa, SW480, SW620, and T29) expressed the cell surface nucleolin assayed by this procedure.
Cells were plated 24 hours before the experiment in eight-well glass slides (Lab-Tek Brand; Nalge Nunc International, Naperville, IL). Cells were fixed with PFA/Triton X-100 solution (PFA/Triton) for staining intracellular biotinylated pseudopeptides (HB-19, N3, and N7) and nucleolar nucleolin [6, 16]. Polylysine (0.01%) coated glass slides were used for adhesion of cells that proliferate in suspension. SW480 and SW620 cells plated in glass slides were cultured in the absence (control) or presence of 10 μM N6L for several days. Cells were either photographed as such or washed with PBS before fixation with PFA/Triton solution and processed for the detection of nuclear nucleolin using mAb D3. The secondary antibodies were the following: FITC-conjugated goat anti-mouse IgG (Sigma) and rabbit anti-biotin concentrate (IgG fraction; Enzo Dioagnostics, Inc., New York). The nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI). It should be noted that artifactual results are observed when cells are fixed with methanol/acetate (3/1) to reveal intracellular localization of HB-19 or Nucant pseudopeptides, as it is the case for intracellular nucleolin [6, 15].
Analysis of DNA fragmentation in response to Nucant treatment
Extraction of nuclei in buffer I (prepared as above) from viable cells results in the recovery of chromatin-free nucleoplasm. However, during cell death when DNA fragmentation occurs, the low molecular weight DNA fragments are recovered in the nucleoplasm. Consequently, supernatant of nuclear extracts from cells undergoing programmed cell death contain low molecular weight DNA fragments, whereas the pellet of the nuclear extracts contain the high molecular weight chromatin. By this experimental approach, DNA fragmentation could be analyzed without interference of the bulky DNA . For the preparation of the low molecular weight DNA fragments, nucleoplasm was incubated with 1 mg/ml RNase for 1 hour at 50°C, then 0.5 mg/ml proteinase K for 1 hour at 50°C, followed by extraction with phenol/chloroform/isoamyl alcohol (25:24:1) and precipitation with ethanol. The DNA pellet was resuspended in electrophoresis buffer (10 mM Tris-HCI, pH 8.0, 1 mM EDTA) and analyzed by electrophoresis on 1.5% agarose gels containing 0.5 pg/ml ethydium bromide.
mRNA expression monitored by reverse transcription-polymerase chain reaction (RT-PCR)
SW620 cells were cultured in the absence or presence of N7 before extraction for total RNA using RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. RT was carried out with oligo(dT) and 2-4 μg of total RNA using Superscript II Reverse Transcriptase (Invitrogen). PCR was performed in a RoboCycler 96 (Stratagene, La Jolla, CA, USA) with specific primers for human nucleolin (referred to as NCL) 5'-TTGAATTCATCATGGTGAAGCTCGCGAAGGC-3' and 5'-TAGGGCCCAGGCTCTTCCTCCTC-3' (835 bp); glyceraldehyde-3-phosphate dehydrogenase (GAPDH) 5'-TGAAGG-TCGGAGTCAACGGATTTGGT-3' and 5'-CATGTGGGCCATGAGGTCCA-CCAC-3' (983 bp); nucleophosmin (NPM or B23) 5'-TGGTTCTCTTCCCAAAGTGG-3' and 5'-TAAAACCAAGCAAAGGGTGG-3' (320 bp); matrix metalloproteinase-2 (MMP-2) 5'-GTGCTGAAGGACACACTAAAGAAGA-3' and 5'-TTGCCATCCTTCTCAAAGTTGTAGG-3' (580 bp); matrix metalloproteinase-9 (MMP-9) 5'-CACTGTCCACCCC TCAGAGC-3' and 5'-GCCACTTGTCGGCGATAAGG-3' (243 bp); tissue inhibitor of metalloproteinase 1 (TIMP-1) 5'-ATCCTGTTGTTGCTGTGGCTGATAG-3' and 5'-TGCTGGGTGGTAACTCTTTATTTCA-3' (667 bp); tissue inhibitor of metalloproteinase 2 (TIMP-2) 5'-AAACGACATTTATGGCAACCCTATC-3' and 5'-ACAGGAGCCGTCACTTCTCTTGATG-3' (405 bp). PCR amplification conditions were: 95°C for 5 min, 30 cycles at 95°C for 30 sec, 60°C for 30 sec and 72°C for 45 sec, and 72°C for 5 min (for NCL and GAPDH), 95°C for 5 min, 30 cycles at 95°C for 30 sec, 53°C for 30 sec and 72°C for 30 sec, and 72°C for 5 min (for NPM); 95°C for 5 min, 35 cycles at 95°C for 45 sec, 56°C for 45 sec and 72°C for 45 sec, and 72°C for 5 min (for MMP2 and MMP9); 95°C for 5 min, 30 cycles at 95°C for 45 sec, 59°C for 1 min and 72°C for 1 min 30 sec, and 72°C for 5 min (for TIMP1 and TIMP2).
The significance of variability between the results of each group and its corresponding control was determined by unpaired t-test and Mann-Witney Anova. All results are expressed as mean ± standard errors of the means from at least two independent experiments.
Results and Discussion
The cell-surface expressed nucleolin exists in a complex of high molecular weight
In order to differentiate between nucleolin expressed on the cell surface from that found in the cytoplasm, we carried out a similar gel filtration experiment but using extracts from cells that were preincubated at room temperature with biotinylated HB-19 (HB-19/Btn). At reduced temperatures, HB-19 binds surface nucleolin and forms a stable complex but it is not internalized, thus allow the differentiation between surface and cytoplasmic nucleolin. After gel filtration, nucleolin was recovered in two main peaks located in fractions 3-9 and 19-23 corresponding to apparent molecular weight of 500- and 100-kDa, respectively, whereas the peak at the molecular weight of 200-kDa was greatly reduced (Figure 1B). For the recovery of nucleolin complexed to HB-19/Btn, fractions 1-7 and 17-24 were purified by affinity chromatography using avidin-agarose Figure 1C and 1D). The results indicate that the peak of nucleolin eluting at an apparent molecular weight of 500-kDa represents surface nucleolin, whereas the 100-kDa peak corresponds to nucleolin present in the cytosol. The marked reduction of the 200-kDa nucleolin peak might reflect the shift of this form of nucleolin towards the cell surface during the incubation period of cells with HB-19/Btn. Taken together, these results indicate that the existence of surface nucleolin in a high molecular weight complex is independent of ligand binding to surface nucleolin.
Identification of proteins associated with the surface expressed nucleolin in the 500-kDa complex
Identification of nucleolin associated proteins in the 500-kDa complex.
Protein Band (MW)
Obtained Sequence 1 /Medline Sequence 2
Band 5 (90-kDa)
1-MHRNFRK 2 = Wnt related protein A 3
Band 6 (85-kDa)
1-MRPMTFIVGLK 2 = Wnt related protein B 4
Band 7 (80-kDa)
1-MVRSGNKAAVVLCMDVGFTMS = Ku80 5
Band 8 (72-kDa)
1-MASGGSGGVSVPA 2 = SRP72 6
Band 9 (68-kDa)
1-MAAEKQVPGGGGGGGS 2 = SRP68 6
Band 11 (32-kDa)
74-LHTDGDKAFVDFLSD 2 = p32/p33/HABP1/gC1q-R 7
Bands 12 (25-kDa):
1-MARGPKKHLK 2 = S4 8
1-MKLNISFPAL 2 = S6 8
Characteristics of proteins associated with the cell surface expressed nucleolin.
Wnt related protein A and B. Protein A shares homology with human Wnt-7b , while protein B shares homology with Wnt-1 of the Mexican axolotl Ambystoma mexicanum . The Wnt proteins are a family of secretory glycoproteins mostly associated with cell membranes and the extracellular matrix. They are implicated in proliferation and differentiation of both normal and malignant cells. Many members of the WNT gene family, including WNT-7, are up-regulated in bladder and breast carcinoma and as well as in chronic lymphocytic leukemia, suggesting involvement of Wnt signaling pathways in tumorigenesis [92–94].
80-kDa subunit of Ku. The Ku autoantigen is a heterodimeric protein made up of 70- and 80-kDa subunits. Besides its central importance to DNA repair, Ku has a key role in a number of other fundamental cellular processes such as telomere maintenance, transcription and cell death . The cell surface expressed Ku80 is detected in a variety of tumor cells, including leukemia and solid tumor cells . Surface Ku contributes to adhesion and invasion of tumor cells thus potentiating tumor metastasis .
SRP68 and SRP72. These are the 68- and 72-kDa subunit in the signal recognition particle, a ribonucleoprotein complex composed of 7S RNA and 6 proteins of 9-, 14-, 19-, 54-, 68-, 72-kDa. SRP comprise the major cellular machinery that mediates the cotranslational targeting of proteins to cellular membranes [60, 61]. SRP68 and SRP72 are functionally linked. Experimental evidence has demonstrated that SRP68 binds SRP72 and forms a highly stable heterodimer .
32-kDa protein referred to as p32/p33, HABP1, or gC1q-R. A multifunctional and muticompartmental cellular protein that was originally isolated based on its copurifiation with the nuclear splicing factor SF2 (p32/p33). It has also been described on the cell surface as the hyaluronan binding protein 1 (HABP1) and the receptor for complement component C1q (gC1q-R) . Hyaluronan is a glycosaminoglycan that with its surface receptors regulate tumor cell adhesion, migration and invasion . Preferentially over expressed in adenocarcinoma cells, gC1q-R is a molecular target in tumor cells and tumor stroma [90, 99]. In addition to cancer, gC1q-R is considered to play an important role in bacterial infections and inflammation [54, 55].
Ribosomal proteins S4 and S6. S4 and S6 are respectively components of the 40S and 60S ribosomal subunits, which generate ribosomal 80S subunit implicated in the cellular process of translation. Ribosomal proteins with nucleolin are also implicated in the processing and assembly of pre-ribosomal particles in the nucleolus. Like HB-19, several ribosomal proteins bind nucleolin via its RGG domain [9, 91]. The role, if any, of the surface expressed S4 and S6 remains to be investigated. It is worthwhile to note that several ribosomal protein genes have been reported to act as cancer genes in zebrafish .
Interestingly, most of surface-nucleolin associated proteins are implicated in cell signaling, tumor cell adhesion, migration, invasion, cell death, and inflammation (Table 3). In addition, Ku and nucleolin are important autoantigens in patients with systemic lupus erythematosus and other systemic autoimmune disorders [57, 58], thus suggesting the potential implication of the 500-kDa complex in autoimmune diseases. The mechanism by which Ku is translocated to the cell surface is not known. However, it is of interest to note that in the cytoplasm both Ku and nucleolin are packaged within small vesicles, and are translocated to the cell surface by a mechanism independent of the conventional endoplasmic reticulum/Golgi secretory pathway [6, 59]. In view of the observation that SRP68 and SRP72 are associated with the high molecular weight nucleolin complex (Table 2), and the implication of SRP in the cotranslational delivery of nascent secretory and membrane proteins [60, 61], it is tempting to speculate that SRP could coordinate active translocation of Ku and nucleolin towards the plasma membrane.
None of the isolated proteins in the 500-kDa complex including nucleolin possess a hydrophobic transmembrane domain to account for anchorage in the plasma membrane. In spite of this, the 500-kDa complex is tightly associated with the plasma membrane because extensive washing of cells with high concentrations of EDTA, EGTA, or NaCl have no effect. On the other hand, the 500-kDa complex is readily recovered by solubilization of the plasma membrane with non-ionic detergents, Triton X-100 or NP-40, thus indicating that its association with the cell surface is through non-covalent interactions. The surface nucleolin in the 500-kDa complex should be associated, directly or indirectly, to an integral membrane protein partner that holds this complex to coordinate clustering of surface nucleolin along with active endocytosis of various nucleolin-binding ligands via lipid rafts [10, 17, 19]. A potential candidate for a transmembrane protein partner of the 500-kDa complex is the low-density lipoprotein (LDL) receptor related protein (LRP1), which is a large scavenger receptor mediating endocytosis of various biological components and is largely implicated in cytoskeleton organization [7, 62, 63]. The link between the 500-kDa complex and LRP1 could be apoplipoprotein E-enriched LDL that in addition to LRP1 binds also surface expressed nucleolin. Accordingly, anti-nucleolin antibody has the capacity to inhibit significantly the binding of LDL to the cell surface . Interestingly, active internalization of specific surface nucleolin ligands (midkine, pleiotrophin, lactoferrin, HB-19) is dependent on the expression of LRP-1 [7, 15, 17–19, 21, 65]. Moreover, the expression of surface nucleolin appears to be dependent on the expression of LRP1. For example, in Chinese hamster ovary CHO LRP1-null cells, although nucleolin is present abundantly in the nucleus and in the cytoplasm, it remains undetectable at the cell surface (Additional file 3, Figure S3). Consequently, in the absence of surface nucleolin in such LRP1-null cells, ligands of nucleolin become internalized by a receptor-independent passive process (Additional file 3, Figure S4). These reports and observations suggest that LRP1 might be one of the potential transmembrane anchored partners that allow surface expression of nucleolin in the 500-kDa complex. The mechanism by which LRP1 expression could coordinate the expression of nucleolin at the cell surface remains to be elucidated.
HB-19 and related Nucant pseudopeptides exert distinct inhibitory effects on different types of tumor cells in culture
Finally, treatment of cells with HB-19 and Nucant constructs results in a drastic down regulation of surface/cytoplasmic nucleolin without affecting nuclear nucleolin (Figure 2B). Interestingly, the reduction of surface/cytoplasmic nucleolin is greater in cells treated with N6 and N7 compared to cells treated with HB-19 and N3. In fact, the level of surface/cytoplasmic nucleolin is almost completely abolished in cells treated with either N6 or N7. This is due to a selective reduction of surface/cytoplasmic nucleolin, since the profile of cytoplasmic proteins assayed by Brilliant Blue G-Colloidal Concentrate staining is comparable in the untreated control and HB-19 or Nucant treated cells (Figure 2B). Consistent with its higher activity as an antagonist of surface nucleolin (Figure 2C), N6L treatment causes a drastic down regulation of surface but not nuclear nucleolin in a dose dependent manner (Figure 2D). At 1 μM of N6L surface nucleolin level is reduced by more than 80%, whereas at 2 μM concentration surface nucleolin is no longer detectable. Taken together, these results indicate that selective reduction of surface/cytoplasmic nucleolin occurs independently of nuclear nucleolin, which further illustrates that HB-19 and Nucant pseudopeptides exert their inhibitory effects without toxicity. We have recently reported that in spite of reduction of surface/cytoplasmic nucleolin protein, nucleolin mRNA is continuously induced but it is not translated . The molecular mechanism of such a specific translational block on nucleolin mRNA in HB-19 and Nucant treated cells remains to be investigated. However, it is tempting to speculate that nucleolin mRNA might require the nucleolin protein for its translation as it is the case for metalloproteinase-9 (MMP-9) and bcl2 oncogene mRNA [4, 5].
Inhibitory activity of hexavalent Nucant pseudopeptides N6, N7, and N6L in tumor cell lines of different origins.
Tumor cell origin
% Growth inhibition
% Cell death
After 3 days
(N6/N7 - N6L)
After 24 hours
(N6/N7 - N6L)
Hu breast cancer
92 - 95%
Hu breast cancer
58 - 66%
Hu prostate cancer
88 - 90%
Hu cervical cancer
46 - 60%
Hu colon carcinoma
25 - 35%
Hu colon carcinoma
45 - 55%
Mu melanoma cells
55 - 83%
Hu cutaneous T cell leukemia
62 - 83%
44 - 55%
Hu T-cell leukemia
80 - 85%
45 - 60%
Hu Burkitt lymphoma
75 - 95%
42 - 71%
Hu acute promyelocytic leukemia
65 - 80%
35 - 48%
Mu T-cell lymphoma
85 - 95%
45 - 65%
The inhibitory action of HB-19 and related Nucant pseudopeptides on the production of pro-inflammatory cytokines by human blood lymphocytes in response to stimulation by heat inactivated Staphylococcus aureus
The inhibitory activity on the production of inflammatory cytokines provide an important contribution to the overall anti-tumorigenic action of HB-19 and related Nucant pseudopeptides, since inflammation could constitute a risk factor for a variety of epithelial cancers by generation of free radicals, stimulation of cytokines, chemokines, and growth and angiogenic factors . It is of interest to note that surface nucleolin is an eukaryotic receptor for the adhesin intimin-gamma of enterohemorrhagic Escherichia coli [11, 67]. In view of this and the capacity of nucleolin antagonist pseudopeptides to block functioning of gC1q-R, we suggest that HB-19 and related Nucant pseudopeptides could also provide efficient inhibitors of pathogenic bacteria.
Nucant pseudopeptides inhibit cell adhesion and spreading of human carcinoma cells
Inhibitory activity of N3, N6, and N6L on melanoma TIII cell adhesion and proliferation.
% Inhibition of cell
N3: 5 μM
N3: 10 μM
N3: 20 μM
N6: 2.5 μM
N6: 5 μM
N6: 10 μM
N6: 20 μM
N6L: 2.5 μM
N6L: 5 μM
N6L: 10 μM
N6L: 20 μM
The inhibitory effect of Nucant on the spreading of human colon carcinoma cells is associated with down regulation of MMP-9 and nucleolin transcripts
Nucant pseudopeptides inhibit proliferation of human colon carcinoma cells.
Number of cells
N7: 10 μM
N7: 20 μM
N6L: 10 μM
N7: 10 μM
N7: 20 μM
N6L: 10 μM
Multiplication of T29 lymphoma cells is inhibited by the multivalent Nucant pseudopeptides due to a selective mechanism of cell death
In epithelial type tumor cell cultures, HB-19 and Nucant pseudopeptides could affect cell adhesion (Figure 4), migration (Additional file 5, Figure S7), spreading (Figure 5), and restore contact inhibition [38, 42](Additional file 5, Figure S6), but have no significant effect on cell viability as demonstrated by the lack of trypan blue uptake in cells at 24 hours after treatment (Table 4, Figure 4B). In contrast to epithelial cells however, these pseudopeptides induce cell death in several leukemia cells (Table 4), in which nucleolin is highly expressed at the cell surface . The binding and internalization of pseudopeptides in Leukemia cells result in a selective down regulation of surface but not nuclear nucleolin in a dose dependent manner (data not shown), as it is the case in epithelial cells (Figure 2B, D)[13, 37].
IC50 values for the induction of cell death and reduction of cell number in T29 cells treated with HB-19 and related multivalent pseudopeptides.
Cell death: IC50 24 hours post-passage
Cell number: IC50 72 hours post-passage
> 20 μM
> 20 μM
Like in other cell types , surface/cytoplasmic nucleolin in T29 cells is degraded selectively upon treatment of cells with the Nucant pseudopeptides, since degradation of surface/cytoplasmic nucleolin occurs without any apparent effect on the level of nuclear nucleolin (Figure 7C). Interestingly, rabbit polyclonal antibodies directed against the last 16 but not the first 26 amino acid residues of nucleolin reveal the presence of partial cleavage products of 70- and 60-kDa (referred to as p70 and p60), thus indicating that such cleavage fragments are derived from the COOH-terminal end of nucleolin. Finally, we investigated whether Nucant-mediated cell death in leukemia cells is associated with internucleosomal DNA fragmentation (Figure 7D). No low molecular weight DNA fragments are observed in the nucleoplasm of control cells, whereas there is a Nucant-dose dependent increase of DNA fragments with a characteristic internucleosomal DNA cleavage ladder-pattern, generally observed in cells undergoing programmed cell death (PCD). A similar profile of DNA fragments was observed in cells treated with the anti-cancer drug bisphosphonate or the protein synthesis inhibitor cycloheximide , which are known to induce apoptosis.
The fact that Nucant does not affect cell viability in epithelial tumor cells indicates that the capacity of Nucant to induce cell death in leukemia cells is by a selective mechanism rather than due to non-specific cellular injury. The pathway by which Nucant induces cell death and the type of PCD remains to be investigated. The early loss of plasma membrane integrity in different types of leukemia cells in response to Nucant treatment favors necrotic type of PCD, since plasma membrane integrity is preserved during PCD by apoptosis in spite of internucleosomal DNA fragmentation [51, 75]. Intriguingly, Nucant induced early loss of plasma membrane integrity is associated with internucleosomal cleavage of cellular DNA with the characteristic ladder pattern, which is generally observed in cells undergoing apoptosis. Previously, internucleosomal DNA cleavage, visualized as ladders, has been reported during necrosis in various types of cells, such as hepatocytes, thymocytes, Jurkat and MDCK cells .
Nucant pseudopeptides bind surface nucleolin at a higher affinity compared to binding to nucleophosmin
The capacity of HB-19 and related multivalent Nucant pseudopeptides to bind ANP32A1, SET1, and nucleophosmin2 expressed on the cell surface of cells in addition to nucleolin.
ANP32A: The acidic nuclear phosphoprotein 32 family, member A. In the literature it has also been referred to as LANP, MAPM, PP32, PHAP I, I1PP2A, C15orf1, MGC119787, and MGC150373. ANP32 is a 30-kDa phosphoprotein that is mainly described in the nucleus. It is characterized by an N-terminal tandem arrays of a leucine rich repeat and an acidic carboxyl half. ANP32A is implicated in a number of cellular processes, including modulation of cell signaling and transduction of gene expression to regulate the morphology and dynamics of the cytoskeleton, cell adhesion and differentiation, and caspase-dependent and caspase-independent apoptosis .
SET nuclear oncogene. In the literature it has also been referred to as 2PP2A, IGAAD, TAF-I, I2PP2A, IPP2A2, PHAP II, and TAF-IBETA. SET is a 39-kDa phosphoprotein with a highly acidic carboxyl-terminus. It is a multifunctional protein widely expressed in various tissues and localizes predominantly in the nucleus. It is involved in apoptosis, transcription, nucleosome assembly and histone binding .
Nucleophosmin. Also referred to as B23, nucleophosmin is a 37-kDa protein ubiquitously expressed chaperone that shuttles rapidly between the nucleus and cytoplasm, but predominantly resides in the nucleolus . It is implicated in several cellular processes, including ribosome biogenesis, centrosome duplication, cell cycle progression, and apoptosis . Somatic mutations in the exon 12 of the nucleophosmin gene (NPM1) are the most frequent genetic abnormality in adult acute myeloid leukemia leading to aberrant localization of nucleophosmin into the cytoplasm [77, 101], which might be a critical event for leukogenesis .
Like nucleolin, nucleophosmin is a nucleo-cytoplasmic shuttling multifunctional protein with prominent nuclear localization. It is involved in many cellular processes, including the transport of pre-ribosomal particles and ribosome biogenesis, the response to stress stimuli, the maintenance of genomic stability, regulation of DNA transcription, and regulation of crucial tumor suppressors such as p53 and ARF . The expression of nucleophosmin at the cell surface, and its implication in the overall mechanism of action of Nucant pseudopeptides against various tumor cell types remain to be characterized. Nevertheless, nucleophosmin and nucleolin expressed at the cell surface interact with K-Ras, and play a critical role in signal transduction via the MAPK pathway . In general nucleophosmin appears to be relatively stable compared to the expression of surface nucleolin, which is constantly induced in association with the proliferative state of tumor cells .
HB-19 and related multivalent Nucant pseudopeptides are potent antitumoral agents by their capacity to exert multiple and distinct inhibitory effects
By their capacity to bind surface nucleolin in the 500-kDa protein complex, we show that HB-19 and related multivalent Nucant pseudopeptides exert multiple and distinct inhibitory effects on cell proliferation, adhesion, spreading, inflammation, and cell death. This is a unique property of HB-19 and related Nucant pseudopeptides, since other antitumoral agents do not exert a differential mode of action depending on a given tumor cell type. In addition to surface nucleolin, HB-19 and related Nucant pseudopeptides at high concentrations can bind directly other proteins expressed on the cell-surface, such as nucleophosmin  and the putative HLA class II-associated protein PHAP I and PHAP II (Table 8).
The official name of PHAP I and PHAP II is ANP32A for the acidic nuclear phosphoprotein 32 family member A, and the SET nuclear oncogene, respectively. Both ANP32A and SET have been described by several groups who have named them according to a specific function (Table 8). Like nucleolin, ANP32A and SET are nucleo-cytoplasmic shuttling phosphoproteins with various functions in cell metabolism. The common features between ANP32A and SET are the presence of an acidic carboxyl-terminal tail, association with HLA class II molecules, protein phosphatase 2 inhibitory activity, histone acetyltransferase inhibitory activity, and implication in mechanisms initiating cell death [78–81]. Consequently, Nucant-mediated occurrence of cell death in leukemia cells might be associated, at least in part, with the functioning of ANP32A and SET. In this respect, it is worthwhile to note that cross-linking of HLA class II has been reported to induce caspase independent cell death in lymphocytes . Moreover, engagement of class II molecules mediates the transduction of signals leading to cell death, which is associated with the enhanced expression of IL-1β and TNF-α mRNA [83, 84].
Several reports have now provided evidence that surface nucleolin is a promising target for cancer therapy [37, 38, 40, 85, 86]. Chemotherapy by targeting surface nucleolin could be less toxic compared to conventional cancer drugs, since nucleolin is continuously and abundantly expressed in tumor compared to normal cells, thus making tumor cells the preferential targets of inhibitors of surface nucleolin . Another parameter that could contribute to the lack of toxicity is the capacity of HB-19 and related Nucant pseudopeptides to block the functioning of surface nucleolin without affecting nuclear nucleolin, which controls many aspects of cellular metabolism [1, 3, 15, 37, 38]. Indeed, after specifically binding to surface nucleolin, HB-19 and related Nucant pseudopeptides enter cells and accumulate in the cytoplasm without crossing the nuclear membrane. Consequently, the effect of these nucleolin antagonists is exerted differentially via the cell surface expressed nucleolin without affecting nuclear nucleolin.
The fact that nucleolin has several protein partners at the cell-surface, and the capacity of HB-19 and related Nucant pseudopeptides to bind additional cell surface proteins besides nucleolin, suggest that the response of tumor cells to these multivalent pseudopeptides should be associated with the expression and/or the level of surface nucleolin and the different nucleolin-partners in tumor cells. Consequently, these surface nucleolin antagonist pseudopeptides exert distinct inhibitory mechanisms depending on a given tumor cell type. Taken together, our results indicate that HB-19 and related Nucant pseudopeptides represent a unique multi-action drug, which provides novel therapeutic opportunities in treatment of a wide variety of cancers and related malignancies.
the surface-nucleolin antagonist-pseudopeptide that presents pentavalently the tripeptide Kψ(CH2N)PR
nucleolin antagonist pseudopeptide that presents the tripeptide Kψ(CH2N)PR either pentavalently or hexavalently
HB-19 related pseudopeptide that presents pentavalently the tripeptide Kψ(CH2N)PR
HB-19 related pseudopeptides that present hexavalently the tripeptide Kψ(CH2N)PR
tissue inhibitor of metalloproteinase 1
tissue inhibitor of metalloproteinase 2
programmed cell death
putative HLA class II-associated protein
acidic nuclear phosphoprotein 32 family member A.
This work was supported by CNRS-France (Centre National de la Recherche Scientifique; FRE 3235, Director: Philippe Djian).
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