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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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Interaction of P111-136 with HARP receptors. Whole PC-3 cell lysates were incubated or not with biot-P111-136 immobilized on streptavidin-Sepharose beads, or with streptavidin-Sepharose beads alone. The lysates or the precipitates were analyzed by SDS-PAGE and electroblotted and the membrane was probed against ALK (A), RPTPβ/ζ (B) and nucleolin (C) using specific antibodies. U87 MG and DU145 cell lysates were respectively used as positive controls for the presence of ALK and RPTPβ/ζ receptors.
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Figure 6: Interaction of P111-136 with HARP receptors. Whole PC-3 cell lysates were incubated or not with biot-P111-136 immobilized on streptavidin-Sepharose beads, or with streptavidin-Sepharose beads alone. The lysates or the precipitates were analyzed by SDS-PAGE and electroblotted and the membrane was probed against ALK (A), RPTPβ/ζ (B) and nucleolin (C) using specific antibodies. U87 MG and DU145 cell lysates were respectively used as positive controls for the presence of ALK and RPTPβ/ζ receptors.

Mentions: Since P111-136 was previously described to compete with HARP for the binding of the ALK receptor [26] in a cell free assay, we then questioned whether P111-136 could bind in situ, using PC-3 cells, ALK and also the other molecular targets involved in the biological activity of HARP including RPTPβ/ζ and nucleolin [34]. Pull-down experiments using biotinylated P111-136 (biot-P111-136) and Western blot analysis were performed to answer this question. Firstly, Western blot analysis performed from whole-cell extracts of PC-3 indicated that this cell line expressed the 140 and 220 kDa forms of ALK (Figure 6A), only the 240 kDa form of RPTPβ/ζ (Figure 6B) and the 100 kDa nucleolin and its degradation products (Figure 6C), as previously described [35]. For expression of ALK and RPTPβ/ζ, U87 MG and DU145 cell lysates were used respectively as control (Figure 6A and 6B). These different HARP interacting proteins were also detected in biot-P111-136 pull down experiments (Figure 6A, B and 6C) in which these proteins were identified by Western blot analysis while no band was detected when bio-P111-136 was omitted from the assay. It is noteworthy that only the 140 kDa isoform of ALK was detected in the biot-P111-136 pull down (Figure 6A). All together these results demonstrate that the inhibition of PC-3 proliferation observed with P111-136 could be link to a direct or indirect binding of this peptide to the different HARP interacting proteins.


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)

Interaction of P111-136 with HARP receptors. Whole PC-3 cell lysates were incubated or not with biot-P111-136 immobilized on streptavidin-Sepharose beads, or with streptavidin-Sepharose beads alone. The lysates or the precipitates were analyzed by SDS-PAGE and electroblotted and the membrane was probed against ALK (A), RPTPβ/ζ (B) and nucleolin (C) using specific antibodies. U87 MG and DU145 cell lysates were respectively used as positive controls for the presence of ALK and RPTPβ/ζ receptors.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Interaction of P111-136 with HARP receptors. Whole PC-3 cell lysates were incubated or not with biot-P111-136 immobilized on streptavidin-Sepharose beads, or with streptavidin-Sepharose beads alone. The lysates or the precipitates were analyzed by SDS-PAGE and electroblotted and the membrane was probed against ALK (A), RPTPβ/ζ (B) and nucleolin (C) using specific antibodies. U87 MG and DU145 cell lysates were respectively used as positive controls for the presence of ALK and RPTPβ/ζ receptors.
Mentions: Since P111-136 was previously described to compete with HARP for the binding of the ALK receptor [26] in a cell free assay, we then questioned whether P111-136 could bind in situ, using PC-3 cells, ALK and also the other molecular targets involved in the biological activity of HARP including RPTPβ/ζ and nucleolin [34]. Pull-down experiments using biotinylated P111-136 (biot-P111-136) and Western blot analysis were performed to answer this question. Firstly, Western blot analysis performed from whole-cell extracts of PC-3 indicated that this cell line expressed the 140 and 220 kDa forms of ALK (Figure 6A), only the 240 kDa form of RPTPβ/ζ (Figure 6B) and the 100 kDa nucleolin and its degradation products (Figure 6C), as previously described [35]. For expression of ALK and RPTPβ/ζ, U87 MG and DU145 cell lysates were used respectively as control (Figure 6A and 6B). These different HARP interacting proteins were also detected in biot-P111-136 pull down experiments (Figure 6A, B and 6C) in which these proteins were identified by Western blot analysis while no band was detected when bio-P111-136 was omitted from the assay. It is noteworthy that only the 140 kDa isoform of ALK was detected in the biot-P111-136 pull down (Figure 6A). All together these results demonstrate that the inhibition of PC-3 proliferation observed with P111-136 could be link to a direct or indirect binding of this peptide to the different HARP interacting proteins.

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