Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2013 Sep 13;8(9):e74548.
doi: 10.1371/journal.pone.0074548. eCollection 2013.

Preclinical development of a novel class of CXCR4 antagonist impairing solid tumors growth and metastases

Luigi Portella  1 , Rosamaria Vitale, Stefania De Luca, Crescenzo D'Alterio, Caterina Ieranò, Maria Napolitano, Anna Riccio, Maria Neve Polimeno, Luca Monfregola, Antonio Barbieri, Antonio Luciano, Andrea Ciarmiello, Claudio Arra, Giuseppe Castello, Pietro Amodeo, Stefania Scala
Affiliations

Preclinical development of a novel class of CXCR4 antagonist impairing solid tumors growth and metastases

Luigi Portella et al. PLoS One. .

Abstract

The CXCR4/CXCL12 axis plays a role in cancer metastases, stem cell mobilization and chemosensitization. Proof of concept for efficient CXCR4 inhibition has been demonstrated in stem cell mobilization prior to autologous transplantation in hematological malignancies. Nevertheless CXCR4 inhibitors suitable for prolonged use as required for anticancer therapy are not available. To develop new CXCR4 antagonists a rational, ligand-based approach was taken, distinct from the more commonly used development strategy. A three amino acid motif (Ar-Ar-X) in CXCL12, also found in the reverse orientation (X-Ar-Ar) in the vMIP-II inhibitory chemokine formed the core of nineteen cyclic peptides evaluated for inhibition of CXCR4-dependent migration, binding, P-ERK1/2-induction and calcium efflux. Peptides R, S and I were chosen for evaluation in in vivo models of lung metastases (B16-CXCR4 and KTM2 murine osteosarcoma cells) and growth of a renal cells xenograft. Peptides R, S, and T significantly reduced the association of the 12G5-CXCR4 antibody to the receptor and inhibited CXCL12-induced calcium efflux. The four peptides efficiently inhibited CXCL12-dependent migration at concentrations as low as 10 nM and delayed CXCL12-mediated wound healing in PES43 human melanoma cells. Intraperitoneal treatment with peptides R, I or S drastically reduced the number of B16-CXCR4-derived lung metastases in C57/BL mice. KTM2 osteosarcoma lung metastases were also reduced in Balb/C mice following CXCR4 inhibition. All three peptides significantly inhibited subcutaneous growth of SN12C-EGFP renal cancer cells. A novel class of CXCR4 inhibitory peptides was discovered. Three peptides, R, I and S inhibited lung metastases and primary tumor growth and will be evaluated as anticancer agents.

PubMed Disclaimer

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Peptides R, I, S, T impair 12G5-CXCR4 binding and CXCL12-induced Ca2+ influx in CCRF–CEM cells.
(A) The amount of peptides or AMD3100 bound was assessed indirectly by flow cytometry using the PE-labelled anti-CXCR4 antibody (clone 12G5). Results are expressed as the percent antibody bound. (B) The Ca2+ influx assay was performed using CCRF-CEM cells and Fluo-3 AM calcium indicator. CCRF-CEM cells were incubated 30 minutes at 37°C with Fluo-3 AM and 15 minutes with AMD3100 (10 µM)/peptides (10 µM) and then treated with CXCL12. Results are expressed as the percent of Fluo-3 AM fluorescence in presence of CXCL12 alone. Each peptide was tested in at least three different experiments. Double tailed T-Test was used for statistical analyses. Differences were considered significant at P<0.05 compared to control. Control (CTRL).
Figure 2
Figure 2. Peptides R, I, S, T inhibit CXCL12 dependent cell migration, wound healing and p-ERK induction.
Experiments were conducted using PES43 cells. (A) Migration was assayed in 24-well Transwell chambers using inserts with 8-µm pore membrane. PES43 cells were placed in the upper chamber (2.5×105 cells/well) in IMDM containing 1% BSA (migration media) in the presence of AMD3100 (10 M) or peptides at several concentrations (10 nM, 100 nM, 1 µM, 10 µM); 100 ng/mL CXCL12 was added to the lower chamber. Migrated cells on the lower surface were fixed, stained with H&E and counted microscopically. The results are expressed as the migration index relative to migration in presence of BSA alone. (B) Delay in wound healing after 6 hours in presence of peptides R, I, S ant T compared with CXCL12. Images were acquired with OKO Time Lapse. (C) Effect of peptides R, I, S, and T on CXCL12 p-ERK induction at 5 minutes. PES43 cells were serum-starved and incubated with CXCL12 (100 ng/ml) alone or in presence of AMD3100/peptides. Each peptide was tested in at least three different experiments. Double tailed T-Test was used for statistical analyses. Differences were considered significant at P<0.05 compared to control.
Figure 3
Figure 3. Peptides R, I and S inhibit murine melanoma lung metastases.
(A) B16-CXCR4 tumor cells pre-treated for 30 minutes with AMD3100 (10 µM), peptide R (10 µM), peptide I (10 µM), or peptide S (10 µM) were inoculated into the tail vein of C57/B female mice and the mice were further treated intraperitoneally for 10 days with 1.25 mg/kg AMD3100, or 2 mg/kg peptide R, peptide I or peptide S. Gross examination of representative lungs; (B) Graphical representation of the number of lung metastases in treated mice. Double tailed T-Test was used for statistical analyses. The experiments were repeated three times.
Figure 4
Figure 4. Peptides R, I and S inhibit murine osteosarcoma lung metastases.
(A) Twenty-five 6-8-week-old female Balb/c mice were injected via tail vein with 2.5×105 K7M2 cells pre-treated for 30 minutes with AMD3100 (10 µM), peptide R (10 µM), or peptide I (10 µM) or peptide S (10 µM). The animals were then further treated intraperitoneally for 15 days with 2.5 mg/kg AMD3100 or 10 mg/kg peptide R, peptide I or peptide S. (B) Graphical representation of the number of lung metastases in treated mice. Double tailed T-Test was used for statistical analyses. The experiments were repeated three times.
Figure 5
Figure 5. Peptides R, I, and S inhibit the growth of primary human SN12C-pEGFP tumors.
6 to 8-week-old female CD1 nude mice were injected subcutaneously with 2×106 pEGFP-SN12C cells pre-treated with 10 µM AMD3100 or 10 µM of each peptide. Beginning the following day mice were further treated intraperitoneally for 10 days with 1.25 mg/kg AMD3100 or 2 mg/kg of peptides R, I or S. A week later mice were euthanized and tumor volume evaluated directly from the flank of mice using a Leica MacroFluo fluorescence stereomicroscope (Leica microsystem, Houston, TX) (A). The experiments were repeated three times. B. SN12C-PEGFP cells migration toward CXCL12 in the presence of AMD3100 or Peptide R, S and I.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Baggiolini M (2001) Chemokines in pathology and medicine. J Intern Med 250: 91–104. - PubMed
    1. Allen SJ, Crown SE, Handel TM (2007) Chemokine: receptor structure, interactions, and antagonism. Annu Rev Immunol 25: 787–820. - PubMed
    1. Wu X, Lee VC, Chevalier E, Hwang ST (2009) Chemokine receptors as targets for cancer therapy. Curr Pharm Des 2009 15: 742–57. - PubMed
    1. Teicher BA, Fricker SP (2010) CXCL12 (SDF-1)/CXCR4 pathway in cancer. Clin Cancer Res 16: 2927–31. - PubMed
    1. Muller A, Homey B, Soto H, Ge N, Catron D, et al. (2001) Involvement of chemokine receptors in breast cancer metastasis. Nature 410: 50–6. - PubMed

Publication types

MeSH terms

Grants and funding

This work was supported by Alleanza Contro il Cancro (ACC9), Associazione Italiana per la Ricerca sul Cancro IG 13192, and FIRB RBAP11884M-008. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.