A Pleiotrophin C-terminus peptide induces anti-cancer effects through RPTPβ/ζ

Abstract

Background: Pleiotrophin, also known as HARP (Heparin Affin Regulatory Peptide) is a growth factor expressed in various tissues and cell lines. Pleiotrophin participates in multiple biological actions including the induction of cellular proliferation, migration and angiogenesis, and is involved in carcinogenesis. Recently, we identified and characterized several pleiotrophin proteolytic fragments with biological activities similar or opposite to that of pleiotrophin. Here, we investigated the biological actions of P(122-131), a synthetic peptide corresponding to the carboxy terminal region of this growth factor.

Results: Our results show that P(122-131) inhibits in vitro adhesion, anchorage-independent proliferation, and migration of DU145 and LNCaP cells, which express pleiotrophin and its receptor RPTPβ/ζ. In addition, P(122-131) inhibits angiogenesis in vivo, as determined by the chicken embryo CAM assay. Investigation of the transduction mechanisms revealed that P(122-131) reduces the phosphorylation levels of Src, Pten, Fak, and Erk1/2. Finally, P(122-131) not only interacts with RPTPβ/ζ, but also interferes with other pleiotrophin receptors, as demonstrated by selective knockdown of pleiotrophin or RPTPβ/ζ expression with the RNAi technology.

Conclusions: In conclusion, our results demonstrate that P(122-131) inhibits biological activities that are related to the induction of a transformed phenotype in PCa cells, by interacing with RPTPβ/ζ and interfering with other pleiotrophin receptors. Cumulatively, these results indicate that P(122-131) may be a potential anticancer agent, and they warrant further study of this peptide.

Figure 1

P(122-131) promotes anti-tumor phenotypes in DU145 and LNCaP cells and inhibits angiogenesis in vivo. (A) Number of adherent DU145 and LNCaP cells in the presence of increasing concentrations of P(122-131). An equal number of DU145 or LNCaP cells was incubated with increasing concentrations of P(122-131) for 30 min before seeding. After a 10-min incubation period, adherent cells were measured by the crystal violet assay. (B) Soft agar growth assays showing anchorage-independent proliferation. An equal number of DU145 or LNCaP cells was resuspended in growth medium containing 10% FBS, 0.3% agar, and increasing concentrations of P(122-131), and seeded onto the bottom agar, which consisted of growth medium containing 10% FBS and 0.8% agar. The top agar was allowed to solidify, and standard growth media supplemented with peptide was added to each well. The cells were incubated 12 days, after which cell colonies larger than 50 μm were quantified by counting the entire area of each well. (C) Migration of cells through Transwell filters. The lower compartment of Transwell filters (8 μm pores) was filled with growth media containing 2.5% FBS, 0.5% BSA, and increasing concentrations of P(122-131). An equal number of DU145 or LNCaP cells was resuspended in growth medium containing 2.5% FBS and 0.5% BSA, and transferred into Transwell inserts. Cells that successfully migrated through the filter pores, were fixed, stained and quantified by counting the entire area of each filter. (D) Effect of P(122-131) on angiogenesis, as measured by the chicken embryo CAM assay. An 1 cm² area of chicken embryo CAM, restricted by a silicon ring, was incubated with increasing concentrations of P(122-131). 48 h later total vessels length was quantified as described in Materials and Methods. Results are mean values ± SE from at least 3 independent experiments.

Figure 2

P(122-131) inhibitory effect is attributed to its specific amino acid sequence and charge. (A) Number of adherent DU145 and LNCaP cells in the presence of 20 μM P(122-131), AAD, or 5K, as determined by the crystal violet assay. (B) Soft agar growth assays showing anchorage-independent proliferation. (C) Migration of cells through Transwell filters. (D) Effect of 2 nmol P(122-131), AAD, or 5K on angiogenesis as measured by the chicken embryo CAM assay. Results are mean values ± SE from at least 3 independent experiments.

Figure 3

P(122-131) binds to RPTPβ/ζ. Merged images of overlapping co-labelling for biotinylated P(122-131), [B(122-131)] (green) and RPTPβ/ζ (red). Incubation of DU145 cells with B(122-131) for 30 sec (A) or for 20 min (B). Incubation of DU145 cells with B(122-131) and pleiotrophin for 20 min (C). Overlapping labelling appears yellow. B(122-131) was visualized using streptavidin-FITC, while RPTPβ/ζ using specific monoclonal antibody and rhodamine conjugated secondary antibody.

Figure 4

Elucidation of the mechanism through which P(122-131) exerts its biological actions. Effect of P(122-131) on adhesion (A), anchorage-independent proliferation (B), and migration (C) of DU145, DU145-HM2, and DU145-RM6 cells. DU145-HM2 cells transfected with pleiotrophin antisense RNA, DU145-RM6 cells transfected with shRNA targeting RPTPβ/ζ. The last two bars of each diagram indicate DU145-HM2 cells that were transiently transfected with siRNA targeting RPTPβ/ζ. Results are mean values ± SE from at least 3 independent experiments.

Figure 5

Src, Fak, and Erk¹/₂ are inactivated by P(122-131), while PTEN is activated. Western blot analysis of phosphorylated Src(A), Fak (B) Pten (C), and Erk¹/₂ (D), in cells stimulated with increasing concentrations of P(122-131) for 3, 15, and 45 minutes. The blots were stripped and reprobed for HSC70. Results are expressed as % change relative to control and are mean values ± SE from at least 3 independent experiments.

Figure 6

Effect of pleiotrophin or RPTPβ/ζ knockdown on P(122-131)-induced kinase signaling. Western blot analysis of phosphorylated Src, Fak, Pten, and Erk¹/₂ in DU145-HM2 (A) and DU145-RM6 cells (B) stimulated with 20 μM P(122-131) for 3 minutes (Src) or 15 minutes (Fak, Pten, and Erk¹/₂). The blots were stripped and reprobed for HSC70. Results are expressed as% change relative to control and are mean values ± SE from at least 3 independent experiments.

Figure 7

Suggested mechanism of P(122-131) action on DU145 cells that express only RPTPβ/ζ and N-Syndecan.