RESUMEN
The potential of PACE4 as a pharmacological target in prostate cancer has been demonstrated as this proprotein convertase is strongly overexpressed in human prostate cancer tissues and its inhibition, using molecular or pharmacological approaches, results in reduced cell proliferation and tumor progression in mouse tumor xenograft models. We developed a PACE4 high-affinity peptide inhibitor, namely, the multi-leucine (ML), and sought to determine whether this peptide could be exploited for the targeting of prostate cancer for diagnostic or molecular imaging purposes. We conjugated a bifunctional chelator 1,4,7-triazacyclononane-1,4,7- triacetic acid (NOTA) to the ML peptide for copper-64 ((64)Cu) labeling and positron emission tomography (PET)- based prostate cancer detection. Enzyme kinetic assays against recombinant PACE4 showed that the NOTA-modified ML peptide displays identical inhibitory properties compared to the unmodified peptide. In vivo biodistribution of the (64)Cu/NOTA-ML peptide evaluated in athymic nude mice bearing xenografts of two human prostate carcinoma cell lines showed a rapid and high uptake in PACE4-expressing LNCaP tumor at an early time point and in PACE4-rich organs. Co-injection of unlabeled peptide confirmed that tumor uptake was target-specific. PACE4-negative tumors displayed no tracer uptake 15 minutes after injection, while the kidneys, demonstrated high uptake due to rapid renal clearance of the peptide. The present study supports the feasibility of using a (64)Cu/NOTA-ML peptide for PACE4-targeted prostate cancer detection and PACE4 status determination by PET imaging but also provides evidence that ML inhibitor-based drugs would readily reach tumor sites under in vivo conditions for pharmacological intervention or targeted radiation therapy.
Asunto(s)
Radioisótopos de Cobre , Péptidos/farmacología , Proproteína Convertasas/antagonistas & inhibidores , Neoplasias de la Próstata/diagnóstico por imagen , Neoplasias de la Próstata/patología , Inhibidores de Proteasas/farmacología , Animales , Línea Celular Tumoral , Radioisótopos de Cobre/química , Modelos Animales de Enfermedad , Estabilidad de Medicamentos , Humanos , Marcaje Isotópico , Masculino , Ratones , Terapia Molecular Dirigida , Péptidos/síntesis química , Tomografía de Emisión de Positrones , Proproteína Convertasas/metabolismo , Neoplasias de la Próstata/tratamiento farmacológico , Inhibidores de Proteasas/síntesis química , Serina Endopeptidasas/metabolismo , Distribución Tisular , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Proprotein convertases (PCs) are crucial in the processing and entry of viral or bacterial protein precursors and confer increased infectivity of pathogens bearing a PC activation site, which results in increased symptom severity and lethality. Previously, we developed a nanomolar peptide inhibitor of PCs to prevent PC activation of infectious agents. Herein, we describe a peptidomimetic approach that increases the stability of this inhibitor for use in vivo to prevent systemic infections and cellular damage, such as that caused by influenza H5N1 and Shiga toxin. The addition of azaß(3)-amino acids to both termini of the peptide successfully prevented influenza hemagglutinin 5 fusogenicity and Shiga toxin Vero toxicity in cell-based assays. The results from a cell-based model using stable shRNA-induced proprotein convertase knockdown indicate that only furin is the major proprotein convertase required for HA5 cleavage.
Asunto(s)
Furina/antagonistas & inhibidores , Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Peptidomiméticos , Toxina Shiga/metabolismo , Furina/fisiología , Relación Estructura-ActividadRESUMEN
Gene silencing strategies targeting mRNA are suitable methods to validate the functions of specific genes. In this chapter, we sought to compare two knockdown strategies for the study of proprotein convertases and proliferation in prostate cancer cells. We used both SOFA-HDV ribozyme and lentiviral-mediated shRNA delivery system to reduce PACE4 mRNA levels and validate its implication in the proliferation of DU145 prostate cancer cells. The cellular effects of PACE4 knockdown were assessed (1) in vitro using two tetrazolium salts (MTT and XTT assays) and (2) in vivo using a tumor xenograft approach in immunodeficient mice (Nu/Nu). Our results confirm the unique role of the proprotein convertase PACE4 in prostate cancer cell proliferation while demonstrating advantages and disadvantages of each approach. Achieving target validation in an effective manner is critical, as further development using a drug development approach is highly laborious and requires enormous resources.
Asunto(s)
Proproteína Convertasas/genética , Neoplasias de la Próstata/genética , ARN Catalítico/genética , ARN Mensajero/genética , Serina Endopeptidasas/genética , Animales , Línea Celular Tumoral , Proliferación Celular , Técnicas de Silenciamiento del Gen , Silenciador del Gen , Humanos , Masculino , Ratones , Biología Molecular/métodos , Proproteína Convertasas/metabolismo , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , ARN Interferente Pequeño/genética , Serina Endopeptidasas/metabolismoRESUMEN
Better understanding of the distinct and redundant functions of the proprotein convertase (PC) enzyme family within pathophysiological states has a great importance for potential therapeutic strategies. In this study, we investigated the functional redundancy of PCs in prostate cancer in the commonly used androgen-sensitive LNCaP and the androgen-independent DU145 human cell lines. Using a lentiviral-based shRNA delivery system, we examined in vitro and in vivo cell proliferation characteristics of knockdown cell lines for the endogenous PCs furin, PACE4, and PC7 in both cell lines. Of the three PCs, only PACE4 was essential to maintain a high-proliferative status, as determined in vitro using XTT proliferation assays and in vivo using tumor xenografts in nude mice. Furin knockdowns in both cell lines had no effects on cell proliferation or tumor xenograft growth. Paradoxically, PC7 knockdowns reduced in vitro cellular proliferation but had no effect in vivo. Because PCs act within secretion pathways, we showed that conditioned media derived from PACE4 knockdown cells had very poor cell growth-stimulating effects in vitro. Immunohistochemistry of PACE4 knockdown tumors revealed reduced Ki67 and higher p27(KIP) levels (proliferation and cell cycle arrest markers, respectively). Interestingly, we determined that the epidermal growth factor receptor signaling pathway was activated in PC7 knockdown tumors only, providing some explanations of the paradoxical effects of PC7 silencing in prostate cancer cell lines. We conclude that PACE4 has a distinct role in maintaining proliferation and tumor progression in prostate cancer and this positions PACE4 as a relevant therapeutic target for this disease.
Asunto(s)
Proproteína Convertasas/metabolismo , Neoplasias de la Próstata/enzimología , Serina Endopeptidasas/metabolismo , Animales , Ciclo Celular/genética , Línea Celular Tumoral , Proliferación Celular , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Técnicas de Silenciamiento del Gen , Silenciador del Gen , Humanos , Masculino , Ratones , Neovascularización Patológica/genética , Neovascularización Patológica/metabolismo , Proproteína Convertasas/genética , Neoplasias de la Próstata/genética , Serina Endopeptidasas/genética , Trasplante HeterólogoRESUMEN
The proprotein convertases (PCs) play an important role in protein precursor activation through processing at paired basic residues. However, significant substrate cleavage redundancy has been reported between PCs. The question remains whether specific PC inhibitors can be designed. This study describes the identification of the sequence LLLLRVKR, named Multi-Leu (ML)-peptide, that displayed a 20-fold selectivity on PACE4 over furin, two enzymes with similar structural characteristics. We have previously demonstrated that PACE4 plays an important role in prostate cancer and could be a druggable target. The present study demonstrates that the ML-peptide significantly reduced the proliferation of DU145 and LNCaP prostate cancer-derived cell lines and induced G0/G1 cell cycle arrest. However, the ML-peptide must enter the cell to inhibit proliferation. It is concluded that peptide-based inhibitors can yield specific PC inhibitors and that the ML-peptide is an important lead compound that could potentially have applications in prostate cancer.
Asunto(s)
Furina/antagonistas & inhibidores , Oligopéptidos/farmacología , Proproteína Convertasas/antagonistas & inhibidores , Neoplasias de la Próstata/patología , Secuencia de Aminoácidos , Línea Celular Tumoral , Humanos , Masculino , Modelos Moleculares , Datos de Secuencia Molecular , Oligopéptidos/química , Neoplasias de la Próstata/enzimología , Proteínas Recombinantes/farmacología , Homología de Secuencia de Aminoácido , Serina EndopeptidasasRESUMEN
Ovarian cancer (OVC) is the fourth leading cause of cancer mortality among women in Europe and the United States. Its early detection is difficult due to the lack of specificity of clinical symptoms. Unfortunately, late diagnosis is a major contributor to the poor survival rates for OVC, which can be attributed to the lack of specific sets of markers. Aside from patients sharing a strong family history of ovarian and breast cancer, including the BRCA1 and BRCA2 tumor suppressor genes mutations, the most used biomarker is the Cancer-antigen 125 (CA-125). CA-125 has a sensitivity of 80 % and a specificity of 97 % in epithelial cancer (stage III or IV). However, its sensitivity is 30 % in stage I cancer, as its increase is linked to several physiological phenomena and benign situations. CA-125 is particularly useful for at-risk population diagnosis and to assess response to treatment. It is clear that alone, CA-125 is inadequate as a biomarker for OVC diagnosis. There is an unmet need to identify additional biomarkers. Novel and more sensitive proteomic strategies such as MALDI mass spectrometry imaging studies are well suited to identify better markers for both diagnosis and prognosis. In the present review, we will focus on such proteomic strategies in regards to OVC signaling pathways, OVC development and escape from the immune response.
Asunto(s)
Neoplasias Ováricas/patología , Animales , Antígeno Ca-125/sangre , Proliferación Celular , Femenino , Humanos , Quinasa I-kappa B/fisiología , Lisofosfolípidos/fisiología , Sistema de Señalización de MAP Quinasas , FN-kappa B/fisiología , Metástasis de la Neoplasia , Neoplasias Ováricas/diagnóstico , Fosfatidilinositol 3-Quinasas/fisiología , Proteómica , Receptores de Estrógenos/fisiología , Transducción de Señal/fisiologíaRESUMEN
The proprotein convertase 1/3 is expressed in the regulated secretory pathway of neural and endocrine cells. Its major function is in the post-translational processing and activation of precursor proteins. The PC1/3 knock-out (KO) mouse model has allowed us to elucidate its physiological functions in studies focused primarily on neuroendocrine tissues. However, PC1/3 is also expressed in cells of the immune system, mainly in macrophages. The present study explores the effects of innate immune challenge in the PC1/3 KO mouse. PC1/3 KO mice have an enlarged spleen with marked disorganization of the marginal zone and red pulp. Immunohistochemical studies using various markers demonstrate a depletion of dendritic cells in PC1/3 KO spleens. When challenged with lipopolysaccharide, PC1/3 KO mice are more susceptible to septic shock than wild-type controls or other PC KO mice, such as PC2 and PC7 null mice. Plasma levels of proinflammatory cytokines (IL-6, IL-1ß, and TNF-α) were very significantly elevated in PC1/3 KO mice, consistent with a hypercytokinemia, i.e. indicative of a major systemic uncontrolled inflammatory response or cytokine storm. Peritoneal macrophages isolated from PC1/3 KO mice also demonstrate elevated cytokine secretion when treated with LPS. Electron micrographs show morphological features indicating a prolonged activation of these cells following LPS stimulation. We also present evidence that the proinflammatory T(h)1 pathway is dominant in the PC1/3 KO mouse model. We conclude that aside from its important role in neuroendocrine functions PC1/3 also has an important role in the regulation of the innate immune system, most likely through the regulation of cytokine secretion in macrophages.
Asunto(s)
Citocinas/inmunología , Regulación Enzimológica de la Expresión Génica/inmunología , Inmunidad Innata , Macrófagos Peritoneales/inmunología , Proproteína Convertasa 1/inmunología , Animales , Citocinas/metabolismo , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/genética , Enfermedades del Sistema Inmune/genética , Enfermedades del Sistema Inmune/inmunología , Enfermedades del Sistema Inmune/metabolismo , Enfermedades del Sistema Inmune/patología , Inflamación/genética , Inflamación/inmunología , Inflamación/patología , Lipopolisacáridos/farmacología , Macrófagos Peritoneales/metabolismo , Macrófagos Peritoneales/ultraestructura , Ratones , Ratones Noqueados , Proproteína Convertasa 1/biosíntesis , Proproteína Convertasa 1/genética , Células TH1/enzimología , Células TH1/metabolismoRESUMEN
Prostate cancer remains the single most prevalent cancer in men. Standard therapies are still limited and include androgen ablation that initially causes tumor regression. However, tumor cells eventually relapse and develop into a hormone-refractory prostate cancer. One of the current challenges in this disease is to define new therapeutic targets, which have been virtually unchanged in the past 30 years. Recent studies have suggested that the family of enzymes known as the proprotein convertases (PCs) is involved in various types of cancers and their progression. The present study examined PC expression in prostate cancer and validates one PC, namely PACE4, as a target. The evidence includes the observed high expression of PACE4 in all different clinical stages of human prostate tumor tissues. Gene silencing studies targeting PACE4 in the DU145 prostate cancer cell line produced cells (cell line 4-2) with slower proliferation rates, reduced clonogenic activity, and inability to grow as xenografts in nude mice. Gene expression and proteomic profiling of the 4-2 cell line reveals an increased expression of known cancer-related genes (e.g., GJA1, CD44, IGFBP6) that are downregulated in prostate cancer. Similarly, cancer genes whose expression is decreased in the 4-2 cell line were upregulated in prostate cancer (e.g., MUC1, IL6). The direct role of PACE4 in prostate cancer is most likely through the upregulated processing of growth factors or through the aberrant processing of growth factors leading to sustained cancer progression, suggesting that PACE4 holds a central role in prostate cancer.
RESUMEN
There is increasing interest in the therapeutic targeting of proteases for the treatment of important diseases. Additionally new protein-based therapeutic strategies have the potential to widen the available treatments against these pathologies. In the last decade, accumulated evidence has confirmed that the family of proteases known as proprotein convertases (PCs) are potential targets for viral infections, osteoarthritis, cancer and cardiovascular disease, among others. Nevertheless, there are still many unanswered questions about the relevance of targeting PCs in a therapeutic context, especially regarding the anticipated secondary effects of treatment, considering the observed embryonic lethality of some PC knockout mice. In this review, the benefits of PCs as pharmacological targets will be discussed, with focus on concepts and strategies, as well as on the state of advancement of actual and future inhibitors.
RESUMEN
Endoproteolytic processing is carried out by subtilase-like pro-protein convertases in mammalian cells. In order to understand the distinct roles of a member of this family (SPC2), gene silencing in cultured cells is an ideal approach. Previous studies showed limited success in either the degree of inhibition obtained or the stability of the cell lines. Here we demonstrate the high potential of delta ribozyme as a post-transcriptional gene silencing tool in cultured cells. We used an expression vector based on the RNA polymerase III promoter to establish betaTC-3 stable cell lines expressing the chimeric tRNA(Val)-delta ribozyme transcript targeting SPC2 mRNA. Northern and Western blot hybridizations showed a specific reduction of SPC2 mRNA and protein. Validation of processing effects was tested by measuring the levels of dynorphin A-(1-8), which are present in betaTC-3 cells as a result of the unique cleavage of dynorphin A-(1-17) by SPC2. Moreover, a differential proteomic analysis confirmed these results and allowed identification of secretogranin II as a potential substrate of SPC2. The development of efficient, specific, and durable silencing tools, such as described in the present work, will be of great importance in elucidating the functions of the subtilase-like pro-protein convertases in regard to peptide processing and derived cellular events.