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

Induction of apoptosis in xenograft tumour by P111-136. PC-3 tumour apoptosis was evaluated on tumour sections using immunohistochemistry with antibody directed against cleaved caspase 3. (A), untreated tumours and (B), tumours treated with P111-136. Scale bar, 100 μm. (C), apoptosis quantified by image analysis of caspase 3-labelled cells on the whole tumour sections. The data are mean areas +/- SD obtained from 5 control mice and 5 mice treated with P111-136. **p < 0.01 versus control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Induction of apoptosis in xenograft tumour by P111-136. PC-3 tumour apoptosis was evaluated on tumour sections using immunohistochemistry with antibody directed against cleaved caspase 3. (A), untreated tumours and (B), tumours treated with P111-136. Scale bar, 100 μm. (C), apoptosis quantified by image analysis of caspase 3-labelled cells on the whole tumour sections. The data are mean areas +/- SD obtained from 5 control mice and 5 mice treated with P111-136. **p < 0.01 versus control.

Mentions: To further investigate the effect of P111-136 on HARP-induced PC-3 proliferation in vivo, athymic mice were subcutaneously injected with PC-3 cells, which consistently led to tumour development within 2 weeks. P111-136 treatment was initiated at the end of the second week, when the tumours were well established, in order to simulate a curative treatment. Peritumoral injection of P111-136 (5 mg/kg/day) significantly reduced tumour growth as soon as the first week of treatment, compared to PBS used as the control (Figure 2A). After 25 days, tumour size was reduced by 61% in the P111-136 group. P111-136 treatment had no effect on body weight (data not shown) and induced no evidence of toxicity such as diarrhoea, infection, weakness, or lethargy. As expected, control treatment with Taxol® (10 mg/kg twice a week) strongly inhibited tumour growth, by 71% compared to PBS, after 25 days of treatment (Figure 2A). At the end of the study, the animals were sacrificed and tumour weight was determined. Both P111-136 and Taxol® significantly decreased tumour weight, by more than 65%, compared with PBS, supporting the tumour-size data (Figure 2B). In order to investigate whether P111-136 treatment was associated with apoptosis of PC-3 cells, cleaved caspase 3 immunostaining was performed on tumour sections and quantified by software analysis. As shown in Figure 3, a two-fold increase in cleaved caspase 3 labelling was observed in tumour treated with P111-136 compare to the untreated tumours. In third investigation, to determine whether angiogenesis associated with tumour growth was also affected by P111-136 treatment, we used CD31 immunostaining to quantify blood vessels. Compared to the untreated tumours (Figure 4A and 4C), peritumoral injections of P111-136 significantly decreased endothelial-cell density (Figure 4B and 4C). The mean percentage of endothelial cells in viable fields of tumours treated with 5 mg/kg/day of P111-136 was inhibited by 64% compared to the control tumour value (1.7 ± 0.58 vs. 4.8 ± 2, based on 25 fields in each of four tumours). These results suggested that the antitumoral effect of P111-136 could also act through direct inhibition of angiogenesis.


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)

Induction of apoptosis in xenograft tumour by P111-136. PC-3 tumour apoptosis was evaluated on tumour sections using immunohistochemistry with antibody directed against cleaved caspase 3. (A), untreated tumours and (B), tumours treated with P111-136. Scale bar, 100 μm. (C), apoptosis quantified by image analysis of caspase 3-labelled cells on the whole tumour sections. The data are mean areas +/- SD obtained from 5 control mice and 5 mice treated with P111-136. **p < 0.01 versus control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Induction of apoptosis in xenograft tumour by P111-136. PC-3 tumour apoptosis was evaluated on tumour sections using immunohistochemistry with antibody directed against cleaved caspase 3. (A), untreated tumours and (B), tumours treated with P111-136. Scale bar, 100 μm. (C), apoptosis quantified by image analysis of caspase 3-labelled cells on the whole tumour sections. The data are mean areas +/- SD obtained from 5 control mice and 5 mice treated with P111-136. **p < 0.01 versus control.
Mentions: To further investigate the effect of P111-136 on HARP-induced PC-3 proliferation in vivo, athymic mice were subcutaneously injected with PC-3 cells, which consistently led to tumour development within 2 weeks. P111-136 treatment was initiated at the end of the second week, when the tumours were well established, in order to simulate a curative treatment. Peritumoral injection of P111-136 (5 mg/kg/day) significantly reduced tumour growth as soon as the first week of treatment, compared to PBS used as the control (Figure 2A). After 25 days, tumour size was reduced by 61% in the P111-136 group. P111-136 treatment had no effect on body weight (data not shown) and induced no evidence of toxicity such as diarrhoea, infection, weakness, or lethargy. As expected, control treatment with Taxol® (10 mg/kg twice a week) strongly inhibited tumour growth, by 71% compared to PBS, after 25 days of treatment (Figure 2A). At the end of the study, the animals were sacrificed and tumour weight was determined. Both P111-136 and Taxol® significantly decreased tumour weight, by more than 65%, compared with PBS, supporting the tumour-size data (Figure 2B). In order to investigate whether P111-136 treatment was associated with apoptosis of PC-3 cells, cleaved caspase 3 immunostaining was performed on tumour sections and quantified by software analysis. As shown in Figure 3, a two-fold increase in cleaved caspase 3 labelling was observed in tumour treated with P111-136 compare to the untreated tumours. In third investigation, to determine whether angiogenesis associated with tumour growth was also affected by P111-136 treatment, we used CD31 immunostaining to quantify blood vessels. Compared to the untreated tumours (Figure 4A and 4C), peritumoral injections of P111-136 significantly decreased endothelial-cell density (Figure 4B and 4C). The mean percentage of endothelial cells in viable fields of tumours treated with 5 mg/kg/day of P111-136 was inhibited by 64% compared to the control tumour value (1.7 ± 0.58 vs. 4.8 ± 2, based on 25 fields in each of four tumours). These results suggested that the antitumoral effect of P111-136 could also act through direct inhibition of angiogenesis.

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