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Prostate cancer is the most common cancer in men. For patients with advanced or metastatic prostate cancer, available treatments can slow down its progression but cannot cure it. The development of innovative drugs resulting from the exploration of biodiversity could open new therapeutic alternatives. Dermaseptin-B2, a natural multifunctional antimicrobial peptide isolated from Amazonian frog skin, has been reported to possess antitumor activity. To improve its pharmacological properties and to decrease its peripheral toxicity and lethality we developed a hormonotoxin molecule composed of dermaseptin-B2 combined with d-Lys6-LHRH to target the LHRH receptor. This hormonotoxin has a significant antiproliferative effect on the PC3 tumor cell line, with an IC50 value close to that of dermaseptin-B2. Its antitumor activity has been confirmed in vivo in a xenograft mouse model with PC3 tumors and appears to be better tolerated than dermaseptin-B2. Biophysical experiments showed that the addition of LHRH to dermaseptin-B2 did not alter its secondary structure or biological activity. The combination of different experimental approaches indicated that this hormonotoxin induces cell death by an apoptotic mechanism instead of necrosis, as observed for dermaseptin-B2. These results could explain the lower toxicity observed for this hormonotoxin compared to dermaseptin-B2 and may represent a promising targeting approach for cancer therapy.

Pleiotrophin (PTN) is a pleiotropic growth factor that exhibits angiogenic properties and is involved in tumor growth and metastasis. Although it has been shown that PTN is expressed in tumor cells, few studies have investigated its receptors and their involvement in cell migration and invasion. Neuropilin-1 (NRP-1) is a receptor for multiple growth factors that mediates cell motility and plays an important role in angiogenesis and tumor progression. Here we provide evidence for the first time that NRP-1 is crucial for biological activities of PTN. We found that PTN interacted directly with NRP-1 through its thrombospondin type-I repeat domains. Importantly, binding of PTN to NRP-1 stimulated the internalization and recycling of NRP-1 at the cell surface. Invalidation of NRP-1 by RNA interference in human carcinoma cells inhibited PTN-induced intracellular signaling of the serine-threonine kinase, mitogen-activated protein MAP kinase, and focal adhesion kinase pathways. Accordingly, NRP-1 silencing or blocking by antibody inhibited PTN-induced human umbilical vein endothelial cell migration and tumor cell invasion. These results suggest that NRP-1/PTN interaction provides a novel mechanism for controlling the response of endothelial and tumoral cells to PTN and may explain, at least in part, how PTN contributes to tumor angiogenesis and cancer progression.

Dermaseptin-B2 (DRS-B2) is a multifunctional cationic antimicrobial peptide (CAP) isolated from frog skin secretion. We previously reported that DRS-B2 possesses anticancer and antiangiogenic activities in vitro and in vivo. In the present study, we evaluated the antiproliferative activity of DRS-B2 on numerous tumor cell lines, its cell internalization and studies of its molecular partners as well as their influences on its structure. Confocal microscopy using ([Alexa594]-(Cys0)-DRS-B2) shows that in sensitive human tumor cells (PC3), DRS-B2 seems to accumulate rapidly at the cytoplasmic membranes and enters the cytoplasm and the nucleus, while in less sensitive tumor cells (U87MG), DRS-B2 is found packed in vesicles at the cell membrane. Furthermore FACS analysis shows that PC3 cells viability decreases after DRS-B2 treatment while U87 MG seems to be unaffected. However, "pull down" experiments performed with total protein pools from PC3 or U87MG cells and the comparison between the antiproliferative effect of DRS-B2 and its synthetic analog containing all D-amino acids suggest the absence of a stereo-selective protein receptor. Pretreatment of PC3 cells with sodium chlorate, decreases the antiproliferative activity of DRS-B2. This activity is partially restored after addition of exogenous chondroitin sulfate C (CS-C). Moreover, we demonstrate that at nanomolar concentrations CS-C potentiates the antiproliferative effect of DRS-B2. These results highlight the partial implication of glycosaminoglycans in the mechanism of antiproliferative action of DRS-B2. Structural analysis of DRS-B2 by circular dichroism in the presence of increasing concentration of CS-C shows that DRS-B2 adopts an α-helical structure. Finally, structure-activity-relationship studies suggest a key role of the W residue in position 3 of the DRS-B2 sequence for its antiproliferative activity.

Recently, we have found that the skin secretions of the Amazonian tree frog Phyllomedusa bicolor contains molecules with antitumor and angiostatic activities and identified one of them as the antimicrobial peptide dermaseptin (Drs) B2. In the present study we further explored the in vitro and in vivo antitumor activity of this molecule and investigated its mechanism of action. We showed that Drs B2 inhibits the proliferation and colony formation of various human tumor cell types, and the proliferation and capillary formation of endothelial cells in vitro. Furthermore, Drs B2 inhibited tumor growth of the human prostate adenocarcinoma cell line PC3 in a xenograft model in vivo. Research on the mechanism of action of Drs B2 on tumor PC3 cells demonstrated a rapid increasing amount of cytosolic lactate dehydrogenase, no activation of caspase-3, and no changes in mitochondrial membrane potential. Confocal microscopy analysis revealed that Drs B2 can interact with the tumor cell surface, aggregate and penetrate the cells. These data together indicate that Drs B2 does not act by apoptosis but possibly by necrosis. In conclusion, Drs B2 could be considered as an interesting and promising pharmacological and therapeutic leader molecule for the treatment of cancer.

BACKGROUND: Emerging evidences suggest that nucleolin expressed on the cell surface is implicated in growth of tumor cells and angiogenesis. Nucleolin is one of the major proteins of the nucleolus, but it is also expressed on the cell surface where is serves as a binding protein for variety of ligands implicated in cell proliferation, differentiation, adhesion, mitogenesis and angiogenesis. METHODOLOGY/PRINCIPAL FINDINGS: By using a specific antagonist that binds the C-terminal tail of nucleolin, the HB-19 pseudopeptide, here we show that the growth of tumor cells and angiogenesis are suppressed in various in vitro and in vivo experimental models. HB-19 inhibited colony formation in soft agar of tumor cell lines, impaired migration of endothelial cells and formation of capillary-like structures in collagen gel, and reduced blood vessel branching in the chick embryo chorioallantoic membrane. In athymic nude mice, HB-19 treatment markedly suppressed the progression of established human breast tumor cell xenografts in nude mice, and in some cases eliminated measurable tumors while displaying no toxicity to normal tissue. This potent antitumoral effect is attributed to the direct inhibitory action of HB-19 on both tumor and endothelial cells by blocking and down regulating surface nucleolin, but without any apparent effect on nucleolar nucleolin. CONCLUSION/SIGNIFICANCE: Our results illustrate the dual inhibitory action of HB-19 on the tumor development and the neovascularization process, thus validating the cell-surface expressed nucleolin as a strategic target for an effective cancer drug. Consequently, the HB-19 pseudopeptide provides a unique candidate to consider for innovative cancer therapy.