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Background. Chronic obstructive pulmonary disease (COPD) is an irreversible disease mainly caused by smoking. COPD is characterized by emphysema and chronic bronchitis associated with enhanced epithelial permeability. Hypothesis. Lung biopsies from smokers revealed a decreased expression level of occludin, which is a protein involved in the cohesion of epithelial tight junctions. Moreover, the occludin level correlated negatively with smoking history (pack-years), COPD grades, and cathepsin S (CatS) activity. Thus, we examined whether CatS could participate in the modulation of the integrity of human lung epithelial barriers. Methods and results. Cigarette smoke extract (CSE) triggered the upregulation of CatS by THP-1 macrophages through the mTOR/TFEB signaling pathway. In a co-culture model, following the exposure of macrophages to CSE, an enhanced level of permeability of lung epithelial (16HBE and NHBE) cells towards FITC-Dextran was observed, which was associated with a decrease in occludin level. Similar results were obtained using 16HBE and NHBE cells cultured at the air–liquid interface. The treatment of THP-1 macrophages by CatS siRNAs or by a pharmacological inhibitor restored the barrier function of epithelial cells, suggesting that cigarette smoke-elicited CatS induced an alteration of epithelial integrity via the proteolytic injury of occludin. Conclusions. Alongside its noteworthy resistance to oxidative stress induced by cigarette smoke oxidants and its deleterious elastin-degrading potency, CatS may also have a detrimental effect on the barrier function of epithelial cells through the cleavage of occludin. The obtained data emphasize the emerging role of CatS in smoking-related lung diseases and strengthen the relevance of targeting CatS in the treatment of emphysema and COPD. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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-β1 (TGF-β1) 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-β1 maturation and autophagic processes. Moreover, GNS561 decreased HSC activation and extracellular matrix deposition by downregulating TGF-β1/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. [ABSTRACT FROM AUTHOR]