Limits...
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.

Show MeSH

Related in: MedlinePlus

Inhibition of PC-3 colony formation by peptide P111-136 and association with the HARP receptors. (A), Western blot analysis of HARP in the conditioned media of PC-3 cells (PC-3), 20 ng of recombinant HARP was used as reference. (B), polyclonal anti-human HARP antibody or control IgG or (C), P111-136 peptide were added to the culture medium twice a week. After 12 days, colonies with diameters greater than 50 μm were identified using a phase-contrast microscope equipped with a reticule. Representative pictures of colonies are shown below each treatment. Scale bar, 100 μm. Asterisks denote statistically significant differences with the corresponding untreated cells. Data are the means +/- SD of two experiments, each carried out in triplicate. *0.01 <p < 0.1, **0.001 <p < 0.01, and *** p < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3118947&req=5

Figure 1: Inhibition of PC-3 colony formation by peptide P111-136 and association with the HARP receptors. (A), Western blot analysis of HARP in the conditioned media of PC-3 cells (PC-3), 20 ng of recombinant HARP was used as reference. (B), polyclonal anti-human HARP antibody or control IgG or (C), P111-136 peptide were added to the culture medium twice a week. After 12 days, colonies with diameters greater than 50 μm were identified using a phase-contrast microscope equipped with a reticule. Representative pictures of colonies are shown below each treatment. Scale bar, 100 μm. Asterisks denote statistically significant differences with the corresponding untreated cells. Data are the means +/- SD of two experiments, each carried out in triplicate. *0.01 <p < 0.1, **0.001 <p < 0.01, and *** p < 0.001.

Mentions: To confirm the HARP autocrine loop in supporting the growth of PC-3 cells, the presence of HARP in the conditioned media of PC-3 was firstly investigated by Western-blot. As shown in Figure 1A, a weak immunoreactive signal was observed in accord with previous observations [32]. Secondly, to prove that secreted HARP acted on PC-3 cells, the growth of these cells was studied in the soft agar colony-formation assay, a hallmark of malignant transformation, in absence or presence of a polyclonal anti-HARP antibody. As presented in Figure 1B, the polyclonal anti-HARP antibody induced a dose-dependent decrease in colony formation, whereas no effect has been observed using the idiotypic immunoglobulins as the control. These data confirmed the existence of an autocrine HARP-signalling loop for PC-3 cells and prompted us to investigate the effect of P111-136 on the proliferation of this cell line. P111-136 dose-dependently decreased colony formation (Figure 1C). In the presence of 1 μM P111-136, the colonies were smaller and 47% less numerous, compared to the control performed without peptide. In this respect, it is noteworthy that when tested in the same conditions, P122-131, which is another peptide derived from the C-terminal part of HARP inhibiting the growth of DU145 and LNCap prostate cancer cells [33], acted also on PC-3 cells but in a lesser extent with only 22% inhibition for 1 μM (data not shown).


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 PC-3 colony formation by peptide P111-136 and association with the HARP receptors. (A), Western blot analysis of HARP in the conditioned media of PC-3 cells (PC-3), 20 ng of recombinant HARP was used as reference. (B), polyclonal anti-human HARP antibody or control IgG or (C), P111-136 peptide were added to the culture medium twice a week. After 12 days, colonies with diameters greater than 50 μm were identified using a phase-contrast microscope equipped with a reticule. Representative pictures of colonies are shown below each treatment. Scale bar, 100 μm. Asterisks denote statistically significant differences with the corresponding untreated cells. Data are the means +/- SD of two experiments, each carried out in triplicate. *0.01 <p < 0.1, **0.001 <p < 0.01, and *** p < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Inhibition of PC-3 colony formation by peptide P111-136 and association with the HARP receptors. (A), Western blot analysis of HARP in the conditioned media of PC-3 cells (PC-3), 20 ng of recombinant HARP was used as reference. (B), polyclonal anti-human HARP antibody or control IgG or (C), P111-136 peptide were added to the culture medium twice a week. After 12 days, colonies with diameters greater than 50 μm were identified using a phase-contrast microscope equipped with a reticule. Representative pictures of colonies are shown below each treatment. Scale bar, 100 μm. Asterisks denote statistically significant differences with the corresponding untreated cells. Data are the means +/- SD of two experiments, each carried out in triplicate. *0.01 <p < 0.1, **0.001 <p < 0.01, and *** p < 0.001.
Mentions: To confirm the HARP autocrine loop in supporting the growth of PC-3 cells, the presence of HARP in the conditioned media of PC-3 was firstly investigated by Western-blot. As shown in Figure 1A, a weak immunoreactive signal was observed in accord with previous observations [32]. Secondly, to prove that secreted HARP acted on PC-3 cells, the growth of these cells was studied in the soft agar colony-formation assay, a hallmark of malignant transformation, in absence or presence of a polyclonal anti-HARP antibody. As presented in Figure 1B, the polyclonal anti-HARP antibody induced a dose-dependent decrease in colony formation, whereas no effect has been observed using the idiotypic immunoglobulins as the control. These data confirmed the existence of an autocrine HARP-signalling loop for PC-3 cells and prompted us to investigate the effect of P111-136 on the proliferation of this cell line. P111-136 dose-dependently decreased colony formation (Figure 1C). In the presence of 1 μM P111-136, the colonies were smaller and 47% less numerous, compared to the control performed without peptide. In this respect, it is noteworthy that when tested in the same conditions, P122-131, which is another peptide derived from the C-terminal part of HARP inhibiting the growth of DU145 and LNCap prostate cancer cells [33], acted also on PC-3 cells but in a lesser extent with only 22% inhibition for 1 μM (data not shown).

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