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PACE4 inhibitors and their peptidomimetic analogs block prostate cancer tumor progression through quiescence induction, increased apoptosis and impaired neovascularisation.

Levesque C, Couture F, Kwiatkowska A, Desjardins R, Guérin B, Neugebauer WA, Day R - Oncotarget (2015)

Bottom Line: We developed an inhibitor named the Multi-Leu peptide, with potent in vitro anti-proliferative effects.Using a peptidomimetic approach, we modified the initial scaffold, generating the analog Ac-[DLeu]LLLRVK-Amba, which demonstrates increased inhibitory potency and stability.Pharmacokinetic and biodistribution profiles of this inhibitor confirm adequate tumor delivery properties of the compound.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery/Urology Division and Faculté de Médecine et Sciences de la Santé, Université de Sherbrooke, Sherbrooke, Québec, J1H 5N4, Canada.

ABSTRACT
Prostate cancer is the leading cancer in North American men. Current pharmacological treatments are limited to anti-androgen strategies and the development of new therapeutic approaches remains a challenge. As a fundamentally new approach, we propose the inhibition of PACE4, a member of the proprotein convertases family of enzymes, as a therapeutic target in prostate cancer. We developed an inhibitor named the Multi-Leu peptide, with potent in vitro anti-proliferative effects. However, the Multi-Leu peptide has not been tested under in vivo conditions and its potency under such conditions is most likely limited, due to the labile characteristics of peptides in general. Using a peptidomimetic approach, we modified the initial scaffold, generating the analog Ac-[DLeu]LLLRVK-Amba, which demonstrates increased inhibitory potency and stability. The systemic administration of this peptidomimetic significantly inhibits tumor progression in the LNCaP xenograft model of prostate cancer by inducing tumor cell quiescence, increased apoptosis and neovascularization impairment. Pharmacokinetic and biodistribution profiles of this inhibitor confirm adequate tumor delivery properties of the compound. We conclude that PACE4 peptidomimetic inhibitors could result in stable and potent drugs for a novel therapeutic strategy for prostate cancer.

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Immunohistochemistry analyses on harvested tumorsImmunohistochemistry analyses for (A) proliferation marker Ki-67 and (B) quiescence marker p27KIP reveals a cell cycle arrest in the Ac-[DLeu]LLLRVK-Amba treated tumors. (C) Apoptosis index was evaluated from cleaved PARP at Asp214 marker and (D) micro vascularization was assessed from CD34 immunostaining. Statistical significance was established from unpaired two-tailed student T tests. *p < 0.05; **p < 0.01; ***p < 0.001; n = 12–15 tumor per group, and 3 to 5 area were evaluated for each tumor. Data in histograms are mean ± SEM and pictures are representative area. Scale bar represent 25 μm (Ki-67; PARP Asp214) and 100 μm (p27KIP; CD34).
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Figure 6: Immunohistochemistry analyses on harvested tumorsImmunohistochemistry analyses for (A) proliferation marker Ki-67 and (B) quiescence marker p27KIP reveals a cell cycle arrest in the Ac-[DLeu]LLLRVK-Amba treated tumors. (C) Apoptosis index was evaluated from cleaved PARP at Asp214 marker and (D) micro vascularization was assessed from CD34 immunostaining. Statistical significance was established from unpaired two-tailed student T tests. *p < 0.05; **p < 0.01; ***p < 0.001; n = 12–15 tumor per group, and 3 to 5 area were evaluated for each tumor. Data in histograms are mean ± SEM and pictures are representative area. Scale bar represent 25 μm (Ki-67; PARP Asp214) and 100 μm (p27KIP; CD34).

Mentions: At the completion of the experiment, tumors were harvested and histological analyses were performed using well-established biomarkers to determine the effects of the peptide on cell proliferation and progression through cell cycle within tumors. Immunostaining for Ki-67, a proliferation marker, revealed a significant 20% reduction of cells progressing through cell cycle, with a relative count of 81 ± 3% of Ki-67+ cells from treated animals when compared to control tumors (100 ± 2%) (Figure 6A). The p27KIP immunostaining in tumors, which is indicative of quiescent cells, demonstrated a significant increase among treated tumors with a relative staining of 130 ± 10% versus 100 ± 8% for control tumors (Figure 6B). From the cleaved PARP (Asp214) apoptosis marker immunostaining, a significant increase in the rate of cells under apoptosis was observed (Figure 6C). From the baseline 2.4 ± 0.2% of apoptotic cells within control tumor, this rate increased to 6 ± 1% in treated tumors, which represent a 250% increase. The decreased cell proliferation rate and increased in cell quiescence along with the induction of apoptosis in tumors of treated animals is consistent with data obtained from in vitro cell cycle assay of LNCaP cells treated with peptide Ac-[DLeu]LLLRVK-Amba (Figure 1B), indicating that the molecular effects of this peptide on LNCaP cells in vitro are preserved in vivo. The far-reaching effects that result from PACE4 inhibition are consistent with the role of PACE4 as a hub protein for the activation of cancer promoting factors in prostate cancer. Since LNCaP xenografts are well known for their high rate of angiogenesis, histological analysis was performed with the endothelial cell marker CD34 (Figure 6D). Tumor micro-vascularization was significantly decreased in Ac-[DLeu]LLLRVK-Amba treated animals when compared to controls with a relative microvessel count of 60 ± 10% compared to 100 ± 20% in control animals. Various pro-angiogenesis factors require processing by the PCs (e.g.: vascular endothelial growth factor, basic fibroblast growth factor, transforming growth factor-β, platelet-derived endothelial growth factor).


PACE4 inhibitors and their peptidomimetic analogs block prostate cancer tumor progression through quiescence induction, increased apoptosis and impaired neovascularisation.

Levesque C, Couture F, Kwiatkowska A, Desjardins R, Guérin B, Neugebauer WA, Day R - Oncotarget (2015)

Immunohistochemistry analyses on harvested tumorsImmunohistochemistry analyses for (A) proliferation marker Ki-67 and (B) quiescence marker p27KIP reveals a cell cycle arrest in the Ac-[DLeu]LLLRVK-Amba treated tumors. (C) Apoptosis index was evaluated from cleaved PARP at Asp214 marker and (D) micro vascularization was assessed from CD34 immunostaining. Statistical significance was established from unpaired two-tailed student T tests. *p < 0.05; **p < 0.01; ***p < 0.001; n = 12–15 tumor per group, and 3 to 5 area were evaluated for each tumor. Data in histograms are mean ± SEM and pictures are representative area. Scale bar represent 25 μm (Ki-67; PARP Asp214) and 100 μm (p27KIP; CD34).
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Related In: Results  -  Collection

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Figure 6: Immunohistochemistry analyses on harvested tumorsImmunohistochemistry analyses for (A) proliferation marker Ki-67 and (B) quiescence marker p27KIP reveals a cell cycle arrest in the Ac-[DLeu]LLLRVK-Amba treated tumors. (C) Apoptosis index was evaluated from cleaved PARP at Asp214 marker and (D) micro vascularization was assessed from CD34 immunostaining. Statistical significance was established from unpaired two-tailed student T tests. *p < 0.05; **p < 0.01; ***p < 0.001; n = 12–15 tumor per group, and 3 to 5 area were evaluated for each tumor. Data in histograms are mean ± SEM and pictures are representative area. Scale bar represent 25 μm (Ki-67; PARP Asp214) and 100 μm (p27KIP; CD34).
Mentions: At the completion of the experiment, tumors were harvested and histological analyses were performed using well-established biomarkers to determine the effects of the peptide on cell proliferation and progression through cell cycle within tumors. Immunostaining for Ki-67, a proliferation marker, revealed a significant 20% reduction of cells progressing through cell cycle, with a relative count of 81 ± 3% of Ki-67+ cells from treated animals when compared to control tumors (100 ± 2%) (Figure 6A). The p27KIP immunostaining in tumors, which is indicative of quiescent cells, demonstrated a significant increase among treated tumors with a relative staining of 130 ± 10% versus 100 ± 8% for control tumors (Figure 6B). From the cleaved PARP (Asp214) apoptosis marker immunostaining, a significant increase in the rate of cells under apoptosis was observed (Figure 6C). From the baseline 2.4 ± 0.2% of apoptotic cells within control tumor, this rate increased to 6 ± 1% in treated tumors, which represent a 250% increase. The decreased cell proliferation rate and increased in cell quiescence along with the induction of apoptosis in tumors of treated animals is consistent with data obtained from in vitro cell cycle assay of LNCaP cells treated with peptide Ac-[DLeu]LLLRVK-Amba (Figure 1B), indicating that the molecular effects of this peptide on LNCaP cells in vitro are preserved in vivo. The far-reaching effects that result from PACE4 inhibition are consistent with the role of PACE4 as a hub protein for the activation of cancer promoting factors in prostate cancer. Since LNCaP xenografts are well known for their high rate of angiogenesis, histological analysis was performed with the endothelial cell marker CD34 (Figure 6D). Tumor micro-vascularization was significantly decreased in Ac-[DLeu]LLLRVK-Amba treated animals when compared to controls with a relative microvessel count of 60 ± 10% compared to 100 ± 20% in control animals. Various pro-angiogenesis factors require processing by the PCs (e.g.: vascular endothelial growth factor, basic fibroblast growth factor, transforming growth factor-β, platelet-derived endothelial growth factor).

Bottom Line: We developed an inhibitor named the Multi-Leu peptide, with potent in vitro anti-proliferative effects.Using a peptidomimetic approach, we modified the initial scaffold, generating the analog Ac-[DLeu]LLLRVK-Amba, which demonstrates increased inhibitory potency and stability.Pharmacokinetic and biodistribution profiles of this inhibitor confirm adequate tumor delivery properties of the compound.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery/Urology Division and Faculté de Médecine et Sciences de la Santé, Université de Sherbrooke, Sherbrooke, Québec, J1H 5N4, Canada.

ABSTRACT
Prostate cancer is the leading cancer in North American men. Current pharmacological treatments are limited to anti-androgen strategies and the development of new therapeutic approaches remains a challenge. As a fundamentally new approach, we propose the inhibition of PACE4, a member of the proprotein convertases family of enzymes, as a therapeutic target in prostate cancer. We developed an inhibitor named the Multi-Leu peptide, with potent in vitro anti-proliferative effects. However, the Multi-Leu peptide has not been tested under in vivo conditions and its potency under such conditions is most likely limited, due to the labile characteristics of peptides in general. Using a peptidomimetic approach, we modified the initial scaffold, generating the analog Ac-[DLeu]LLLRVK-Amba, which demonstrates increased inhibitory potency and stability. The systemic administration of this peptidomimetic significantly inhibits tumor progression in the LNCaP xenograft model of prostate cancer by inducing tumor cell quiescence, increased apoptosis and neovascularization impairment. Pharmacokinetic and biodistribution profiles of this inhibitor confirm adequate tumor delivery properties of the compound. We conclude that PACE4 peptidomimetic inhibitors could result in stable and potent drugs for a novel therapeutic strategy for prostate cancer.

Show MeSH
Related in: MedlinePlus