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The synthetic peptide P111-136 derived from the C-terminal domain of heparin affin regulatory peptide inhibits tumour growth of prostate cancer PC-3 cells.

Hamma-Kourbali Y, Bermek O, Bernard-Pierrot I, Karaky R, Martel-Renoir D, Frechault S, Courty J, Delbé J - BMC Cancer (2011)

Bottom Line: In vivo anti-angiogenic effect was confirmed using a mouse Matrigel™ plug assay.The angiostatic effect of P111-136 on HARP was also confirmed using an in vivo Matrigel™ plug assay in mice Our results demonstrate that P111-136 strongly inhibits the mitogenic effect of HARP on in vitro and in vivo growth of PC-3 cells.In vivo, the P111-136 treatment significantly inhibits both the PC-3 tumour growth and the associated angiogenesis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratoire de Recherche sur la Croissance Cellulaire, la Réparation et la Régénération Tissulaires, Université Paris Est Créteil, CNRS, avenue du Général de Gaulle, 94010 Créteil Cedex, France.

ABSTRACT

Background: Heparin affin regulatory peptide (HARP), also called pleiotrophin, is a heparin-binding, secreted factor that is overexpressed in several tumours and associated to tumour growth, angiogenesis and metastasis. The C-terminus part of HARP composed of amino acids 111 to 136 is particularly involved in its biological activities and we previously established that a synthetic peptide composed of the same amino acids (P111-136) was capable of inhibiting the biological activities of HARP. Here we evaluate the ability of P111-136 to inhibit in vitro and in vivo the growth of a human tumour cell line PC-3 which possess an HARP autocrine loop.

Methods: A total lysate of PC-3 cells was incubated with biotinylated P111-136 and pulled down for the presence of the HARP receptors in Western blot. In vitro, the P111-136 effect on HARP autocrine loop in PC-3 cells was determined by colony formation in soft agar. In vivo, PC-3 cells were inoculated in the flank of athymic nude mice. Animals were treated with P111-136 (5 mg/kg/day) for 25 days. Tumour volume was evaluated during the treatment. After the animal sacrifice, the tumour apoptosis and associated angiogenesis were evaluated by immunohistochemistry. In vivo anti-angiogenic effect was confirmed using a mouse Matrigel™ plug assay.

Results: Using pull down experiments, we identified the HARP receptors RPTPβ/ζ, ALK and nucleolin as P111-136 binding proteins. In vitro, P111-136 inhibits dose-dependently PC-3 cell colony formation. Treatment with P111-136 inhibits significantly the PC-3 tumour growth in the xenograft model as well as tumour angiogenesis. The angiostatic effect of P111-136 on HARP was also confirmed using an in vivo Matrigel™ plug assay in mice

Conclusions: Our results demonstrate that P111-136 strongly inhibits the mitogenic effect of HARP on in vitro and in vivo growth of PC-3 cells. This inhibition could be linked to a direct or indirect binding of this peptide to the HARP receptors (ALK, RPTPβ/ζ, nucleolin). In vivo, the P111-136 treatment significantly inhibits both the PC-3 tumour growth and the associated angiogenesis. Thus, P111-136 may be considered as an interesting pharmacological tool to interfere with tumour growth that has now to be evaluated in other cancer types.

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Inhibition of HARP-induced in vivo angiogenesis by peptide P111-136. Liquid Matrigel™ at 4°C was injected subcutaneously into Swiss mice. The Matrigel™ was used alone or after incorporation of HARP, P111-136, or both. The animals were sacrificed 8 days later; the Matrigel™ plugs were removed, sectioned, stained using the Gomori-Trichrome method, and examined under the microscope. Scale bar, 100 μm. Micrographs of Gomori-Trichrome stained Matrigel™ plug (A) alone, (B) containing 1 μM of P111-136, (C), containing 5 nM of HARP, or (D) containing 5 nM of HARP and 1 μM of P111-136. (E) endothelial-cell migration into the Matrigel™ was quantified as the mean cell count in six fields in each of three Matrigel™ plug sections per mouse, data are means +/- SD of values in four mice per group. **p < 0.01.
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Figure 5: Inhibition of HARP-induced in vivo angiogenesis by peptide P111-136. Liquid Matrigel™ at 4°C was injected subcutaneously into Swiss mice. The Matrigel™ was used alone or after incorporation of HARP, P111-136, or both. The animals were sacrificed 8 days later; the Matrigel™ plugs were removed, sectioned, stained using the Gomori-Trichrome method, and examined under the microscope. Scale bar, 100 μm. Micrographs of Gomori-Trichrome stained Matrigel™ plug (A) alone, (B) containing 1 μM of P111-136, (C), containing 5 nM of HARP, or (D) containing 5 nM of HARP and 1 μM of P111-136. (E) endothelial-cell migration into the Matrigel™ was quantified as the mean cell count in six fields in each of three Matrigel™ plug sections per mouse, data are means +/- SD of values in four mice per group. **p < 0.01.

Mentions: To investigate a direct effect of P111-136 on angiogenesis processes, we questioned whether the peptide inhibits the normal angiogenesis induced by HARP. This study was performed using in vivo mouse Matrigel™ plug assay. Incorporating 5 nM of HARP into the Matrigel™ resulted in a 3.7-fold increase in endothelial-cell infiltration (Figure 5C and 5E) compared to the untreated control plug, which contained only a few endothelial cells (Figure 5A and 5E). Addition of 1 μM of P111-136 to the HARP-containing Matrigel™ inhibited the effect of HARP on endothelial cell infiltration by 72% (Figure 5C, D and 5E). P111-136 had no effect on endothelial-cell infiltration using Matrigel™ alone (Figure 5A, B and 5E). These data indicate that P111-136 inhibited the angiogenesis induced by HARP.


The synthetic peptide P111-136 derived from the C-terminal domain of heparin affin regulatory peptide inhibits tumour growth of prostate cancer PC-3 cells.

Hamma-Kourbali Y, Bermek O, Bernard-Pierrot I, Karaky R, Martel-Renoir D, Frechault S, Courty J, Delbé J - BMC Cancer (2011)

Inhibition of HARP-induced in vivo angiogenesis by peptide P111-136. Liquid Matrigel™ at 4°C was injected subcutaneously into Swiss mice. The Matrigel™ was used alone or after incorporation of HARP, P111-136, or both. The animals were sacrificed 8 days later; the Matrigel™ plugs were removed, sectioned, stained using the Gomori-Trichrome method, and examined under the microscope. Scale bar, 100 μm. Micrographs of Gomori-Trichrome stained Matrigel™ plug (A) alone, (B) containing 1 μM of P111-136, (C), containing 5 nM of HARP, or (D) containing 5 nM of HARP and 1 μM of P111-136. (E) endothelial-cell migration into the Matrigel™ was quantified as the mean cell count in six fields in each of three Matrigel™ plug sections per mouse, data are means +/- SD of values in four mice per group. **p < 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3118947&req=5

Figure 5: Inhibition of HARP-induced in vivo angiogenesis by peptide P111-136. Liquid Matrigel™ at 4°C was injected subcutaneously into Swiss mice. The Matrigel™ was used alone or after incorporation of HARP, P111-136, or both. The animals were sacrificed 8 days later; the Matrigel™ plugs were removed, sectioned, stained using the Gomori-Trichrome method, and examined under the microscope. Scale bar, 100 μm. Micrographs of Gomori-Trichrome stained Matrigel™ plug (A) alone, (B) containing 1 μM of P111-136, (C), containing 5 nM of HARP, or (D) containing 5 nM of HARP and 1 μM of P111-136. (E) endothelial-cell migration into the Matrigel™ was quantified as the mean cell count in six fields in each of three Matrigel™ plug sections per mouse, data are means +/- SD of values in four mice per group. **p < 0.01.
Mentions: To investigate a direct effect of P111-136 on angiogenesis processes, we questioned whether the peptide inhibits the normal angiogenesis induced by HARP. This study was performed using in vivo mouse Matrigel™ plug assay. Incorporating 5 nM of HARP into the Matrigel™ resulted in a 3.7-fold increase in endothelial-cell infiltration (Figure 5C and 5E) compared to the untreated control plug, which contained only a few endothelial cells (Figure 5A and 5E). Addition of 1 μM of P111-136 to the HARP-containing Matrigel™ inhibited the effect of HARP on endothelial cell infiltration by 72% (Figure 5C, D and 5E). P111-136 had no effect on endothelial-cell infiltration using Matrigel™ alone (Figure 5A, B and 5E). These data indicate that P111-136 inhibited the angiogenesis induced by HARP.

Bottom Line: In vivo anti-angiogenic effect was confirmed using a mouse Matrigel™ plug assay.The angiostatic effect of P111-136 on HARP was also confirmed using an in vivo Matrigel™ plug assay in mice Our results demonstrate that P111-136 strongly inhibits the mitogenic effect of HARP on in vitro and in vivo growth of PC-3 cells.In vivo, the P111-136 treatment significantly inhibits both the PC-3 tumour growth and the associated angiogenesis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratoire de Recherche sur la Croissance Cellulaire, la Réparation et la Régénération Tissulaires, Université Paris Est Créteil, CNRS, avenue du Général de Gaulle, 94010 Créteil Cedex, France.

ABSTRACT

Background: Heparin affin regulatory peptide (HARP), also called pleiotrophin, is a heparin-binding, secreted factor that is overexpressed in several tumours and associated to tumour growth, angiogenesis and metastasis. The C-terminus part of HARP composed of amino acids 111 to 136 is particularly involved in its biological activities and we previously established that a synthetic peptide composed of the same amino acids (P111-136) was capable of inhibiting the biological activities of HARP. Here we evaluate the ability of P111-136 to inhibit in vitro and in vivo the growth of a human tumour cell line PC-3 which possess an HARP autocrine loop.

Methods: A total lysate of PC-3 cells was incubated with biotinylated P111-136 and pulled down for the presence of the HARP receptors in Western blot. In vitro, the P111-136 effect on HARP autocrine loop in PC-3 cells was determined by colony formation in soft agar. In vivo, PC-3 cells were inoculated in the flank of athymic nude mice. Animals were treated with P111-136 (5 mg/kg/day) for 25 days. Tumour volume was evaluated during the treatment. After the animal sacrifice, the tumour apoptosis and associated angiogenesis were evaluated by immunohistochemistry. In vivo anti-angiogenic effect was confirmed using a mouse Matrigel™ plug assay.

Results: Using pull down experiments, we identified the HARP receptors RPTPβ/ζ, ALK and nucleolin as P111-136 binding proteins. In vitro, P111-136 inhibits dose-dependently PC-3 cell colony formation. Treatment with P111-136 inhibits significantly the PC-3 tumour growth in the xenograft model as well as tumour angiogenesis. The angiostatic effect of P111-136 on HARP was also confirmed using an in vivo Matrigel™ plug assay in mice

Conclusions: Our results demonstrate that P111-136 strongly inhibits the mitogenic effect of HARP on in vitro and in vivo growth of PC-3 cells. This inhibition could be linked to a direct or indirect binding of this peptide to the HARP receptors (ALK, RPTPβ/ζ, nucleolin). In vivo, the P111-136 treatment significantly inhibits both the PC-3 tumour growth and the associated angiogenesis. Thus, P111-136 may be considered as an interesting pharmacological tool to interfere with tumour growth that has now to be evaluated in other cancer types.

Show MeSH
Related in: MedlinePlus