RESUMEN
Hepatocellular carcinoma is the most frequent primary liver cancer. Macroautophagy/autophagy inhibitors have been extensively studied in cancer but, to date, none has reached efficacy in clinical trials. In this study, we demonstrated that GNS561, a new autophagy inhibitor, whose anticancer activity was previously linked to lysosomal cell death, displayed high liver tropism and potent antitumor activity against a panel of human cancer cell lines and in two hepatocellular carcinoma in vivo models. We showed that due to its lysosomotropic properties, GNS561 could reach and specifically inhibited its enzyme target, PPT1 (palmitoyl-protein thioesterase 1), resulting in lysosomal unbound Zn2+ accumulation, impairment of cathepsin activity, blockage of autophagic flux, altered location of MTOR (mechanistic target of rapamycin kinase), lysosomal membrane permeabilization, caspase activation and cell death. Accordingly, GNS561, for which a global phase 1b clinical trial in liver cancers was just successfully achieved, represents a promising new drug candidate and a hopeful therapeutic strategy in cancer treatment.Abbreviations: ANXA5:annexin A5; ATCC: American type culture collection; BafA1: bafilomycin A1; BSA: bovine serum albumin; CASP3: caspase 3; CASP7: caspase 7; CASP8: caspase 8; CCND1: cyclin D1; CTSB: cathepsin B; CTSD: cathepsin D; CTSL: cathepsin L; CQ: chloroquine; iCCA: intrahepatic cholangiocarcinoma; DEN: diethylnitrosamine; DMEM: Dulbelcco's modified Eagle medium; FBS: fetal bovine serum; FITC: fluorescein isothiocyanate; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; HCC: hepatocellular carcinoma; HCQ: hydroxychloroquine; HDSF: hexadecylsulfonylfluoride; IC50: mean half-maximal inhibitory concentration; LAMP: lysosomal associated membrane protein; LC3-II: phosphatidylethanolamine-conjugated form of MAP1LC3; LMP: lysosomal membrane permeabilization; MALDI: matrix assisted laser desorption ionization; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MKI67: marker of proliferation Ki-67; MTOR: mechanistic target of rapamycin kinase; MRI: magnetic resonance imaging; NH4Cl: ammonium chloride; NtBuHA: N-tert-butylhydroxylamine; PARP: poly(ADP-ribose) polymerase; PBS: phosphate-buffered saline; PPT1: palmitoyl-protein thioesterase 1; SD: standard deviation; SEM: standard error mean; vs, versus; Zn2+: zinc ion; Z-Phe: Z-Phe-Tyt(tBu)-diazomethylketone; Z-VAD-FMK: carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]- fluoromethylketone.
Asunto(s)
Antineoplásicos , Carcinoma Hepatocelular , Neoplasias Hepáticas , Antineoplásicos/farmacología , Autofagosomas/metabolismo , Autofagia/fisiología , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/metabolismo , Humanos , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/metabolismo , Lisosomas/metabolismo , Proteínas de la Membrana/metabolismo , Sirolimus/farmacología , Serina-Treonina Quinasas TOR/metabolismo , Tioléster Hidrolasas/metabolismo , Tioléster Hidrolasas/farmacologíaRESUMEN
Coccidiosis is a disease caused by Eimeria, which represents the first parasitic disease in poultry farming. Among them, E. tenella is a virulent species which specifically colonizes the caecum. The inflammatory response to infection is associated to numerous host proteases including cysteine cathepsins that can be deleterious for tissue and innate immunity integrity. Here, germ-free and conventional chickens were used as models to find out whether the microbiota could modify the intestinal expression of host cysteine cathepsins during coccidiosis. The basal caecal peptidase activity primarily relies on host proteases rather than proteases from the commensal flora. While mRNA levels of E. tenella cathepsins B and L remained unchanged in germ-free and conventional broilers, an overall increase in endopeptidase activity of cysteine cathepsins was found in E. tenella-infected caeca in both experimental models (P < 0.005). A significant decrease in avian cystatin C transcription was also observed in infected conventional, but not in infected germ-free broilers. Despite an unchanged mRNA level of avian cathepsin L (CatL), its protein expression raised following infection, in parallel with an increased transcription of antimicrobial ß-defensins (AvBD1, AvBD2, AvBD4, AvBD6, and AvBD7). Taken together, data support that host CatL is post-translationally upregulated during E. tenella infection, and thus may be involved in the alteration of the gut proteolytic balance. Furthermore, CatL may participate to inflammation occurring during coccidiosis through its known ability to proteolytically inactivates up-regulated avian ß-defensins that are key molecules of innate immunity.
Asunto(s)
Coccidiosis , Eimeria tenella , Enfermedades de las Aves de Corral , Animales , Catepsina L/genética , Pollos , Coccidiosis/veterinaria , Regulación hacia ArribaRESUMEN
BACKGROUND: Hepatic fibrosis is the result of chronic liver injury that can progress to cirrhosis and lead to liver failure. Nevertheless, there are no anti-fibrotic drugs licensed for human use. Here, we investigated the anti-fibrotic activity of GNS561, a new lysosomotropic molecule with high liver tropism. METHODS: The anti-fibrotic effect of GNS561 was determined in vitro using LX-2 hepatic stellate cells (HSCs) and primary human HSCs by studying cell viability, activity of caspases 3/7, autophagic flux, cathepsin maturation and activity, HSC activation and transforming growth factor-ß1 (TGF-ß1) maturation and signaling. The contribution of GNS561 lysosomotropism to its anti-fibrotic activity was assessed by increasing lysosomal pH. The potency of GNS561 on fibrosis was evaluated in vivo in a rat model of diethylnitrosamine-induced liver fibrosis. RESULTS: GNS561 significantly decreased cell viability and promoted apoptosis. Disrupting the lysosomal pH gradient impaired its pharmacological effects, suggesting that GNS561 lysosomotropism mediated cell death. GNS561 impaired cathepsin activity, leading to defective TGF-ß1 maturation and autophagic processes. Moreover, GNS561 decreased HSC activation and extracellular matrix deposition by downregulating TGF-ß1/Smad and mitogen-activated proteine kinase signaling and inducing fibrolysis. Finally, oral administration of GNS561 (15 mg/kg per day) was well tolerated and attenuated diethylnitrosamine-induced liver fibrosis in this rat model (decrease of collagen deposition and of pro-fibrotic markers and increase of fibrolysis). CONCLUSION: GNS561 is a new potent lysosomotropic compound that could represent a valid medicinal option for hepatic fibrosis treatment through both its anti-fibrotic and its pro-fibrolytic effects. In addition, this study provides a rationale for targeting lysosomes as a promising therapeutic strategy in liver fibrosis.
RESUMEN
Pulmonary fibrosis is a progressive disease characterized by a widespread accumulation of myofibroblasts and extracellular matrix components. Growing evidences support that cysteine cathepsins, embracing cathepsin B (CatB) that affects TGF-ß1-driven Smad pathway, along with their extracellular inhibitor cystatin C, participate in myofibrogenesis. Here we established that curcumin, a potent antifibrotic drug used in traditional Asian medicine, impaired the expression of both α-smooth muscle actin and mature TGF-ß1 and inhibited the differentiation of human lung fibroblasts (CCD-19Lu cells). Curcumin induced a compelling upregulation of CatB and CatL. Conversely cystatin C was downregulated, which allowed the recovery of the peptidase activity of secreted cathepsins and the restoration of the proteolytic balance. Consistently, the amount of both insoluble and soluble type I collagen decreased, reaching levels similar to those observed for undifferentiated fibroblasts. The signaling pathways activated by curcumin were further examined. Curcumin triggered the expression of nuclear peroxisome proliferator-activated receptor γ (PPARγ). Contrariwise PPARγ inhibition, either by an antagonist (2-chloro-5-nitro-N-4-pyridinyl-benzamide) or by RNA silencing, restored TGF-ß1-driven differentiation of curcumin-treated CCD-19Lu cells. PPARγ response element (PPRE)-like sequences were identified in the promoter regions of both CatB and CatL. Finally, we established that the transcriptional induction of CatB and CatL depends on the binding of PPARγ to PPRE sequences as a PPARγ/Retinoid X Receptor-α heterodimer.
Asunto(s)
Catepsina B/biosíntesis , Catepsina L/biosíntesis , Diferenciación Celular/efectos de los fármacos , Curcumina/farmacología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Pulmón/metabolismo , PPAR gamma/metabolismo , Factor de Crecimiento Transformador beta1/farmacología , Regulación hacia Arriba/efectos de los fármacos , Línea Celular , HumanosRESUMEN
The cysteine protease cathepsin C (CatC) activates granule-associated proinflammatory serine proteases in hematopoietic precursor cells. Its early inhibition in the bone marrow is regarded as a new therapeutic strategy for treating proteolysis-driven chronic inflammatory diseases, but its complete inhibition is elusive in vivo Controlling the activity of CatC may be achieved by directly inhibiting its activity with a specific inhibitor or/and by preventing its maturation. We have investigated immunochemically and kinetically the occurrence of CatC and its proform in human hematopoietic precursor cells and in differentiated mature immune cells in lung secretions. The maturation of proCatC obeys a multistep mechanism that can be entirely managed by CatS in neutrophilic precursor cells. CatS inhibition by a cell-permeable inhibitor abrogated the release of the heavy and light chains from proCatC and blocked â¼80% of CatC activity. Under these conditions the activity of neutrophil serine proteases, however, was not abolished in precursor cell cultures. In patients with neutrophilic lung inflammation, mature CatC is found in large amounts in sputa. It is secreted by activated neutrophils as confirmed through lipopolysaccharide administration in a nonhuman primate model. CatS inhibitors currently in clinical trials are expected to decrease the activity of neutrophilic CatC without affecting those of elastase-like serine proteases.
Asunto(s)
Catepsina C/metabolismo , Pulmón/enzimología , Neutrófilos/enzimología , Neumonía/enzimología , Animales , Catepsina C/genética , Modelos Animales de Enfermedad , Células HL-60 , Humanos , Pulmón/patología , Macaca fascicularis , Ratones , Ratones Endogámicos BALB C , Neutrófilos/patología , Neumonía/inducido químicamente , Neumonía/patología , Ratas Sprague-Dawley , Esputo/metabolismoRESUMEN
Papillon-Lefèvre syndrome (PLS) (OMIM: 245000) is a rare disease characterized by severe periodontitis and palmoplantar keratoderma. It is caused by mutations in both alleles of the cathepsin C (CatC) gene CTSC that completely abrogate the proteolytic activity of this cysteine proteinase. Most often, a genetic analysis to enable early and rapid diagnosis of PLS is unaffordable or unavailable. In this study, we tested the hypothesis that active CatC is constitutively excreted and can be easily traced in the urine of normal subjects. If this is true, determining its absence in the urine of patients would be an early, simple, reliable, low-cost and easy diagnostic technique. All 75 urine samples from healthy control subjects (aged 3 months to 80 years) contained proteolytically active CatC and its proform, as revealed by kinetic analysis and immunochemical detection. Of the urine samples of 31 patients with a PLS phenotype, 29 contained neither proteolytically active CatC nor the CatC antigen, so that the PLS diagnosis was confirmed. CatC was detected in the urine of the other two patients, and genetic analysis revealed no loss-of-function mutation in CTSC, indicating that they suffer from a PLS-like condition but not from PLS. Screening for the absence of urinary CatC activity soon after birth and early treatment before the onset of PLS manifestations will help to prevent aggressive periodontitis and loss of many teeth, and should considerably improve the quality of life of PLS patients.