Prokineticin 1 stimulates prostate epithelial cell migration and proliferation in vitro
Daniela Visonti1, Giuseppe Bellastella1, Valentina Rossi1, Paolo Chieffi2, Luigi Maione1, Paola Punzo1, Antonio Bellastella1 & Antonio Agostino Sinisi1
Prokineticin 1 (Prok1), the product of EGVEGF/PROK 1 gene, acts through two G-coupled receptors (PKR1 and PKR2) and is involved in a wide spectrum of actions, including tumorigenesis. Increased Prok1 expression has been found in prostate hyperplasia and cancer, suggesting a role in prostate cancer and BPH. Aim of this study was to elucidate the role of Prok1 on prostate cell function and growth. We evaluated the effects of Prok1 on epithelial prostate cell (PC) migration and proliferation, using two in vitro models: the androgen-dependent epithelial PC line EPN, and a stabilized PC line derived from prostate cancer (CPEC).
Methods: Semiconfluent starved cultures were treated with recombinant Prok1 (5 nM) alone or associated with antiProk1 MoAb or solvent. Cells were harvested 48 h after the treatment and stained with propidium iodide for flow cytometry of cell cycle by FACS caliber or recovered for protein extraction for western-blot analysis, or for mRNA extraction for semiquantitative RT-PCR. Cells grown on slides were also treated ad harvested after 46 h for TUNEL assay. A wound assay was performed for the evaluation of cell motility after overnight incubation. For ERK phosphorylation assay cell cultures were recovered after 5, 10, 20 and 60 min following treatment.
Results: An increase of the cell number in S phase, with a decrease of cell counts in pre-G1 and G0/G1, and a significant reduction of percent of apoptotic nuclei was found after Prok1 treatment (P<0.05 versus control). Treatment induced an increase of migration in CEPC only. All these effects were abolished when antiProk1 MoAb was added. Prok1 induced a rapid and transient phosphorylation of ERK in EPN and more sustained effects on CEPC; these effects were abolished by pretreatment with PD98059 (50 nM). Semiquantitative PCR showed an increase of Prok-R2 transcript in treated cells.
Conclusions: Our study demonstrates that Prok1 has stimulating effects on prostate epithelial tumor cells growth and migration in vitro, suggesting a role in the neoplastic progression. These effects are specifically mediated by receptor activation and induction. These data suggest that Prok1-ProkR signaling pathway may be target of new therapeutic approach for the control of prostate tumor development and/or progression.