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1.
Int Immunopharmacol ; 136: 112284, 2024 Jul 30.
Article in English | MEDLINE | ID: mdl-38823179

ABSTRACT

Cathepsin B (CTSB) and inflammatory cytokines are critical in initiating and developing pancreatitis. Calcineurin, a central calcium (Ca2+)-responsive signaling molecule, mediates acinar cell death and inflammatory responses leading to pancreatitis. However, the detailed mechanisms for regulating CTSB activity and inflammatory cytokine production are unknown. Myricetin (MC) exhibits various biological activities, including anti-inflammatory effects. Here, we aimed to investigate MC effects on pancreatitis and the underlying mechanisms. Prophylactic and therapeutic MC treatment ameliorated the severity of cerulein-, L-arginine-, and PDL-induced acute pancreatitis (AP). The inhibition of CTSB activity by MC was mediated via decreased calcineurin activity and macrophage infiltration, not neutrophils infiltration, into the pancreas. Additionally, calcineurin activity inhibition by MC prevented the phosphorylation of Ca2+/CaM-dependent protein kinase kinase 2 (CaMKK2) during AP, resulting in the inhibition of CaMKIV phosphorylation and adenosine monophosphate-activated protein kinase (AMPK) dephosphorylation. Furthermore, MC reduced nuclear factor-κB activation by modulating the calcineurin-CaMKIV-IKKα/ß-Iκ-Bα and calcineurin-AMPK-sirtuin1 axes, resulting in reduced production of tumor necrosis factor-α, interleukin (IL)-1ß, and IL-6. Our results showed that MC alleviated AP severity by inhibiting acinar cell death and inflammatory responses, suggesting that MC as a calcineurin and CaMKK2 signaling modulator may be a potential treatment for AP.


Subject(s)
Calcineurin , Cathepsin B , Cytokines , Flavonoids , Mice, Inbred C57BL , Pancreatitis , Animals , Pancreatitis/drug therapy , Pancreatitis/immunology , Pancreatitis/pathology , Pancreatitis/chemically induced , Flavonoids/pharmacology , Flavonoids/therapeutic use , Cytokines/metabolism , Cathepsin B/metabolism , Mice , Male , Calcineurin/metabolism , Anti-Inflammatory Agents/therapeutic use , Anti-Inflammatory Agents/pharmacology , Calcium-Calmodulin-Dependent Protein Kinase Kinase/metabolism , Ceruletide , NF-kappa B/metabolism , Pancreas/pathology , Pancreas/drug effects , Pancreas/immunology , Signal Transduction/drug effects , Arginine/metabolism , Disease Models, Animal , AMP-Activated Protein Kinases/metabolism
2.
Int Immunopharmacol ; 134: 112190, 2024 Jun 15.
Article in English | MEDLINE | ID: mdl-38703569

ABSTRACT

Spinal cord injury (SCI) is a devastating condition for which effective clinical treatment is currently lacking. During the acute phase of SCI, myriad pathological changes give rise to subsequent secondary injury. The results of our previous studies indicated that treating rats post-SCI with nafamostat mesilate (NM) protected the blood-spinal cord barrier (BSCB) and exerted an antiapoptotic effect. However, the optimal dosage for mice with SCI and the underlying mechanisms potentially contributing to recovery, especially during the acute phase of SCI, have not been determined. In this study, we first determined the optimal dosage of NM for mice post-SCI (5 mg/kg/day). Subsequently, our RNA-seq findings revealed that NM has the potential to inhibit pyroptosis after SCI. These findings were further substantiated by subsequent Western blot (WB) and Immunofluorescence (IF) analyses in vivo. These results indicate that NM can alleviate NLRP3 (NOD-like receptor thermal protein domain associated protein 3)-mediated pyroptosis by modulating the NF-κB signaling pathway and reducing the protein expression levels of NIMA-related kinase 7 (NEK7) and cathepsin B (CTSB). In vitro experimental results supported our in vivo findings, revealing the effectiveness of NM in suppressing pyroptosis induced by adenosine triphosphate (ATP) and lipopolysaccharide (LPS) in BV2 cells. These results underscore the potential of NM to regulate NLRP3-mediated pyroptosis following SCI. Notably, compared with other synthetic compounds, NM exhibits greater versatility, suggesting that it is a promising clinical treatment option for SCI.


Subject(s)
Benzamidines , Guanidines , Mice, Inbred C57BL , NLR Family, Pyrin Domain-Containing 3 Protein , Pyroptosis , Spinal Cord Injuries , Animals , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Pyroptosis/drug effects , Spinal Cord Injuries/drug therapy , Spinal Cord Injuries/metabolism , Mice , Guanidines/pharmacology , Guanidines/therapeutic use , NF-kappa B/metabolism , Male , Signal Transduction/drug effects , Disease Models, Animal , Cathepsin B/metabolism
3.
Infect Immun ; 92(6): e0014124, 2024 Jun 11.
Article in English | MEDLINE | ID: mdl-38722166

ABSTRACT

The human-specific bacterial pathogen group A Streptococcus (GAS) is a significant cause of morbidity and mortality. Macrophages are important to control GAS infection, but previous data indicate that GAS can persist in macrophages. In this study, we detail the molecular mechanisms by which GAS survives in THP-1 macrophages. Our fluorescence microscopy studies demonstrate that GAS is readily phagocytosed by macrophages, but persists within phagolysosomes. These phagolysosomes are not acidified, which is in agreement with our findings that GAS cannot survive in low pH environments. We find that the secreted pore-forming toxin Streptolysin O (SLO) perforates the phagolysosomal membrane, allowing leakage of not only protons but also large proteins including the lysosomal protease cathepsin B. Additionally, GAS recruits CD63/LAMP-3, which may contribute to lysosomal permeabilization, especially in the absence of SLO. Thus, although GAS does not inhibit fusion of the lysosome with the phagosome, it has multiple mechanisms to prevent proper phagolysosome function, allowing for persistence of the bacteria within the macrophage. This has important implications for not only the initial response but also the overall functionality of the macrophages, which may lead to the resulting pathologies in GAS infection. Our data suggest that therapies aimed at improving macrophage function may positively impact patient outcomes in GAS infection.


Subject(s)
Bacterial Proteins , Lysosomes , Macrophages , Streptococcus pyogenes , Streptolysins , Streptococcus pyogenes/immunology , Humans , Macrophages/microbiology , Macrophages/immunology , Macrophages/metabolism , Lysosomes/metabolism , Lysosomes/microbiology , Streptolysins/metabolism , Bacterial Proteins/metabolism , Bacterial Proteins/genetics , Phagosomes/microbiology , Phagosomes/metabolism , THP-1 Cells , Phagocytosis , Streptococcal Infections/immunology , Streptococcal Infections/microbiology , Streptococcal Infections/metabolism , Cathepsin B/metabolism , Hydrogen-Ion Concentration
4.
Int J Biol Macromol ; 272(Pt 1): 132684, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38810845

ABSTRACT

The emergence of cathepsins as a potential target for anticancer drugs has led to extensive research in the development of their inhibitors. In the present study, we designed, synthesized, and characterized several cinnamaldehyde schiff bases employing diverse hydrazines, as potential cathepsin B inhibitors. The parallel studies on cathepsin B isolated from liver and cerebrospinal fluid unveiled the significance of the synthesized compounds as cathepsin B inhibitors at nanomolar concentrations. The compound, 7 exhibited the highest inhibition of 83.48 % and 82.96 % with an IC50 value of 0.06 nM and 0.09 nM for liver and cerebrospinal fluid respectively. The inhibitory potential of synthesized compounds has been extremely effective in comparison to previous reports. With the help of molecular docking studies using iGEMDOCK software, we found that the active site -CH2SH group is involved in the case of α-N-benzoyl-D, l-arginine-b-naphthylamide (BANA), curcumin 2, 3, 6, and 7. For toxicity prediction, ADMET studies were conducted and the synthesized compounds emerged to be non-toxic. The results obtained from the in vitro studies were supported with in silico studies. The synthesized cinnamaldehyde schiff bases can be considered promising drug candidates in conditions with elevated cathepsin B levels.


Subject(s)
Acrolein , Cathepsin B , Hydrazones , Liver , Molecular Docking Simulation , Cathepsin B/antagonists & inhibitors , Cathepsin B/metabolism , Acrolein/analogs & derivatives , Acrolein/chemistry , Acrolein/pharmacology , Liver/drug effects , Liver/metabolism , Humans , Hydrazones/pharmacology , Hydrazones/chemistry , Hydrazones/chemical synthesis , Catalytic Domain , Animals
5.
ACS Infect Dis ; 10(6): 1935-1948, 2024 Jun 14.
Article in English | MEDLINE | ID: mdl-38757505

ABSTRACT

Schistosomiasis, caused by a parasitic blood fluke of the genus Schistosoma, is a global health problem for which new chemotherapeutic options are needed. We explored the scaffold of gallinamide A, a natural peptidic metabolite of marine cyanobacteria that has previously been shown to inhibit cathepsin L-type proteases. We screened a library of 19 synthetic gallinamide A analogs and identified nanomolar inhibitors of the cathepsin B-type protease SmCB1, which is a drug target for the treatment of schistosomiasis mansoni. Against cultured S. mansoni schistosomula and adult worms, many of the gallinamides generated a range of deleterious phenotypic responses. Imaging with a fluorescent-activity-based probe derived from gallinamide A demonstrated that SmCB1 is the primary target for gallinamides in the parasite. Furthermore, we solved the high-resolution crystal structures of SmCB1 in complex with gallinamide A and its two analogs and describe the acrylamide covalent warhead and binding mode in the active site. Quantum chemical calculations evaluated the contribution of individual positions in the peptidomimetic scaffold to the inhibition of the target and demonstrated the importance of the P1' and P2 positions. Our study introduces gallinamides as a powerful chemotype that can be exploited for the development of novel antischistosomal chemotherapeutics.


Subject(s)
Cathepsin B , Schistosoma mansoni , Cathepsin B/antagonists & inhibitors , Cathepsin B/metabolism , Animals , Schistosoma mansoni/enzymology , Schistosoma mansoni/drug effects , Crystallography, X-Ray , Schistosomicides/pharmacology , Schistosomicides/chemistry , Protein Binding , Models, Molecular
6.
Cell Mol Life Sci ; 81(1): 207, 2024 May 06.
Article in English | MEDLINE | ID: mdl-38709385

ABSTRACT

The co-localization of the lysosomal protease cathepsin B (CTSB) and the digestive zymogen trypsinogen is a prerequisite for the initiation of acute pancreatitis. However, the exact molecular mechanisms of co-localization are not fully understood. In this study, we investigated the role of lysosomes in the onset of acute pancreatitis by using two different experimental approaches. Using an acinar cell-specific genetic deletion of the ras-related protein Rab7, important for intracellular vesicle trafficking and fusion, we analyzed the subcellular distribution of lysosomal enzymes and the severity of pancreatitis in vivo and ex vivo. Lysosomal permeabilization was performed by the lysosomotropic agent Glycyl-L-phenylalanine 2-naphthylamide (GPN). Acinar cell-specific deletion of Rab7 increased endogenous CTSB activity and despite the lack of re-distribution of CTSB from lysosomes to the secretory vesicles, the activation of CTSB localized in the zymogen compartment still took place leading to trypsinogen activation and pancreatic injury. Disease severity was comparable to controls during the early phase but more severe at later time points. Similarly, GPN did not prevent CTSB activation inside the secretory compartment upon caerulein stimulation, while lysosomal CTSB shifted to the cytosol. Intracellular trypsinogen activation was maintained leading to acute pancreatitis similar to controls. Our results indicate that initiation of acute pancreatitis seems to be independent of the presence of lysosomes and that fusion of lysosomes and zymogen granules is dispensable for the disease onset. Intact lysosomes rather appear to have protective effects at later disease stages.


Subject(s)
Cathepsin B , Lysosomes , Pancreatitis , Secretory Vesicles , rab GTP-Binding Proteins , rab7 GTP-Binding Proteins , Animals , Lysosomes/metabolism , Pancreatitis/metabolism , Pancreatitis/pathology , Pancreatitis/genetics , Cathepsin B/metabolism , Cathepsin B/genetics , Mice , Secretory Vesicles/metabolism , rab GTP-Binding Proteins/metabolism , rab GTP-Binding Proteins/genetics , rab7 GTP-Binding Proteins/metabolism , Acute Disease , Acinar Cells/metabolism , Acinar Cells/pathology , Trypsinogen/metabolism , Trypsinogen/genetics , Ceruletide , Enzyme Precursors/metabolism , Enzyme Precursors/genetics , Mice, Inbred C57BL , Mice, Knockout
7.
Bioorg Chem ; 147: 107370, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38621338

ABSTRACT

Here, we introduce a novel and effective approach utilizing a cathepsin B cleavage albumin-binding SN38 prodrug specifically designed for the treatment of metastatic breast cancer. Termed Mal-va-mac-SN38, our prodrug exhibits a unique ability to rapidly and covalently bind with endogenous albumin, resulting in the formation of HSA-va-mac-SN38. This prodrug demonstrates exceptional stability in human plasma. Importantly, HSA-va-mac-SN38 showcases an impressive enhancement in cellular uptake by 4T1 breast cancer cells, primarily facilitated through caveolin-mediated endocytosis. Intriguingly, the release of the active SN38, is triggered by the enzymatic activity of cathepsin B within the lysosomal environment. In vivo studies employing a lung metastasis 4T1 breast cancer model underscore the potency of HSA-va-mac-SN38. Histological immunohistochemical analyses further illuminate the multifaceted impact of our prodrug, showcasing elevated levels of apoptosis, downregulated expression of matrix metalloproteinases, and inhibition of angiogenesis, all critical factors contributing to the anti-metastatic effect observed. Biodistribution studies elucidate the capacity of Mal-va-mac-SN38 to augment tumor accumulation through covalent binding to serum albumin, presenting a potential avenue for targeted therapeutic interventions. Collectively, our findings propose a promising therapeutic avenue for metastatic breast cancer, through the utilization of a cathepsin B-cleavable albumin-binding prodrug.


Subject(s)
Antineoplastic Agents , Breast Neoplasms , Cathepsin B , Drug Design , Prodrugs , Prodrugs/chemical synthesis , Prodrugs/chemistry , Prodrugs/pharmacology , Cathepsin B/metabolism , Humans , Female , Breast Neoplasms/drug therapy , Breast Neoplasms/pathology , Breast Neoplasms/metabolism , Antineoplastic Agents/pharmacology , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Animals , Mice , Molecular Structure , Structure-Activity Relationship , Drug Screening Assays, Antitumor , Cell Proliferation/drug effects , Mice, Inbred BALB C , Dose-Response Relationship, Drug , Apoptosis/drug effects
8.
J Photochem Photobiol B ; 255: 112919, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38677261

ABSTRACT

Endolysosomes perform a wide range of cellular functions, including nutrient sensing, macromolecule digestion and recycling, as well as plasma membrane repair. Because of their high activity in cancerous cells, endolysosomes are attractive targets for the development of novel cancer treatments. Light-activated compounds termed photosensitizers (PS) can catalyze the oxidation of specific biomolecules and intracellular organelles. To selectively damage endosomes and lysosomes, HT-29 colorectal cancer cells were incubated with nanomolar concentrations of meso-tetraphenylporphine disulfonate (TPPS2a), an amphiphilic PS taken up via endocytosis and activated by green light (522 nm, 2.1 J.cm-1). Several cellular responses were characterized by a combination of immunofluorescence and immunoblotting assays. We showed that TPPS2a photosensitization blocked autophagic flux without extensive endolysosomal membrane rupture. Nevertheless, there was a severe functional failure of endolysosomes due to a decrease in CTSD (cathepsin D, 55%) and CTSB (cathepsin B, 52%) maturation. PSAP (prosaposin) processing (into saposins) was also considerably impaired, a fact that could be detrimental to glycosphingolipid homeostasis. Therefore, photosensitization of HT-29 cells previously incubated with a low concentration of TPPS2a promotes endolysosomal dysfunction, an effect that can be used to improve cancer therapies.


Subject(s)
Autophagy , Lysosomes , Photosensitizing Agents , Humans , HT29 Cells , Lysosomes/metabolism , Lysosomes/drug effects , Autophagy/drug effects , Autophagy/radiation effects , Photosensitizing Agents/pharmacology , Photosensitizing Agents/chemistry , Endosomes/metabolism , Endosomes/drug effects , Cathepsins/metabolism , Cathepsins/antagonists & inhibitors , Light , Porphyrins/pharmacology , Porphyrins/chemistry , Cathepsin D/metabolism , Cathepsin B/metabolism
9.
ACS Infect Dis ; 10(5): 1664-1678, 2024 05 10.
Article in English | MEDLINE | ID: mdl-38686397

ABSTRACT

In this study, we have identified and optimized two lead structures from an in-house screening, with promising results against the parasitic flatworm Schistosoma mansoni and its target protease S. mansoni cathepsin B1 (SmCB1). Our correlation analysis highlighted the significance of physicochemical properties for the compounds' in vitro activities, resulting in a dual approach to optimize the lead structures, regarding both phenotypic effects in S. mansoni newly transformed schistosomula (NTS), adult worms, and SmCB1 inhibition. The optimized compounds from both approaches ("phenotypic" vs "SmCB1" approach) demonstrated improved efficacy against S. mansoni NTS and adult worms, with 2h from the "SmCB1" approach emerging as the most potent compound. 2h displayed nanomolar inhibition of SmCB1 (Ki = 0.050 µM) while maintaining selectivity toward human off-target cathepsins. Additionally, the greatly improved efficacy of compound 2h toward S. mansoni adults (86% dead worms at 10 µM, 68% at 1 µM, 35% at 0.1 µM) demonstrates its potential as a new therapeutic agent for schistosomiasis, underlined by its improved permeability.


Subject(s)
Cathepsin B , Schistosoma mansoni , Schistosoma mansoni/drug effects , Schistosoma mansoni/enzymology , Schistosoma mansoni/genetics , Animals , Cathepsin B/antagonists & inhibitors , Cathepsin B/metabolism , Schistosomiasis mansoni/drug therapy , Drug Design , Humans , Phenotype , Structure-Activity Relationship , Anthelmintics/pharmacology , Anthelmintics/chemistry , Helminth Proteins/antagonists & inhibitors
10.
J Microbiol ; 62(4): 315-325, 2024 Apr.
Article in English | MEDLINE | ID: mdl-38451450

ABSTRACT

Acinetobacter baumannii (A. baumannii) causes autophagy flux disorder by degrading STX17, resulting in a serious inflammatory response. It remains unclear whether STX17 can alter the inflammatory response process by controlling autolysosome function. This study aimed to explore the role of STX17 in the regulation of pyroptosis induced by A. baumannii. Our findings indicate that overexpression of STX17 enhances autophagosome degradation, increases LAMP1 expression, reduces Cathepsin B release, and improves lysosomal function. Conversely, knockdown of STX17 suppresses autophagosome degradation, reduces LAMP1 expression, augments Cathepsin B release, and accelerates lysosomal dysfunction. In instances of A. baumannii infection, overexpression of STX17 was found to improve lysosomal function and reduce the expression of mature of GSDMD and IL-1ß, along with the release of LDH, thus inhibiting pyroptosis caused by A. baumannii. Conversely, knockdown of STX17 led to increased lysosomal dysfunction and further enhanced the expression of mature of GSDMD and IL-1ß, and increased the release of LDH, exacerbating pyroptosis induced by A. baumannii. These findings suggest that STX17 regulates pyroptosis induced by A. baumannii by modulating lysosomal function.


Subject(s)
Acinetobacter baumannii , Interleukin-1beta , Lysosomes , Pyroptosis , Qa-SNARE Proteins , Lysosomes/metabolism , Acinetobacter baumannii/metabolism , Acinetobacter baumannii/genetics , Interleukin-1beta/metabolism , Interleukin-1beta/genetics , Humans , Qa-SNARE Proteins/metabolism , Qa-SNARE Proteins/genetics , Phosphate-Binding Proteins/metabolism , Phosphate-Binding Proteins/genetics , Autophagy , Animals , Cathepsin B/metabolism , Cathepsin B/genetics , Acinetobacter Infections/microbiology , Mice , Intracellular Signaling Peptides and Proteins/metabolism , Intracellular Signaling Peptides and Proteins/genetics , Autophagosomes/metabolism , Lysosomal-Associated Membrane Protein 1/metabolism , Gasdermins
11.
FEBS J ; 291(11): 2372-2387, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38431778

ABSTRACT

Vitamin B6 is a critical molecule for metabolism, development, and stress sensitivity in plants. It is a cofactor for numerous biochemical reactions, can serve as an antioxidant, and has the potential to increase tolerance against both biotic and abiotic stressors. Due to the importance of vitamin B6, its biosynthesis is likely tightly regulated. Plants can synthesize vitamin B6 de novo via the concerted activity of Pyridoxine Biosynthesis Protein 1 (PDX1) and PDX2. Previously, PDX proteins have been identified as targets for ubiquitination, indicating they could be marked for degradation by two highly conserved pathways: the Ubiquitin Proteasome Pathway (UPP) and the autophagy pathway. Initial experiments show that PDXs are in fact degraded, but surprisingly, in a ubiquitin-independent manner. Inhibitor studies pointed toward cathepsin B, a conserved lysosomal cysteine protease, which is implicated in both programed cell death and autophagy in humans and plants. In plants, cathepsin Bs are poorly described, and no confirmed substrates have been identified. Here, we present PDX proteins from Arabidopsis thaliana as interactors and substrates of a plant Cathepsin B. These findings not only describe a novel cathepsin B substrate in plants, but also provide new insights into how plants regulate de novo biosynthesis of vitamin B6.


Subject(s)
Arabidopsis Proteins , Arabidopsis , Cathepsin B , Vitamin B 6 , Cathepsin B/metabolism , Cathepsin B/genetics , Arabidopsis/metabolism , Arabidopsis/genetics , Vitamin B 6/metabolism , Vitamin B 6/biosynthesis , Arabidopsis Proteins/metabolism , Arabidopsis Proteins/genetics , Substrate Specificity , Ubiquitination , Gene Expression Regulation, Plant , Carbon-Nitrogen Lyases
12.
J Pharm Sci ; 113(7): 1927-1933, 2024 Jul.
Article in English | MEDLINE | ID: mdl-38555998

ABSTRACT

Although gemcitabine (GEM) is a first-line chemotherapeutic drug in treating pancreatic cancer, the therapeutic efficacy of GEM is relatively poor. One main reason is that GEM can be easily deaminated to inactive 2',2'-difluorodeoxyuridine (dFdU) by cytidine deaminase (CDA). In order to improve the antitumor activity of GEM, a polypeptide modified GEM prodrug RGDGFLG-GEM (GEM-RGD) is designed. Because the amino group of GEM is protected by RGDGFLG peptide sequence, the in vivo stability of GEM-RGD can be significantly improved since the deamination of GEM can be avoided. GEM-RGD shows enhanced uptake by pancreatic cancer cells due to the active targeting RGD group. The cathepsin B-sensitive GFLG sequence endows GEM-RGD with specific release of GEM in pancreatic cancer cells. Compared to free GEM and non-targeted GEM prodrug RDGGFLG-GEM (GEM-RDG), GEM-RGD exhibits enhanced antitumor activity and reduced systemic toxicity. These results implies that GEM-RGD is a promising candidate in treating pancreatic cancer.


Subject(s)
Cathepsin B , Deoxycytidine , Gemcitabine , Pancreatic Neoplasms , Prodrugs , Prodrugs/pharmacology , Prodrugs/therapeutic use , Cathepsin B/metabolism , Cathepsin B/antagonists & inhibitors , Deoxycytidine/analogs & derivatives , Deoxycytidine/pharmacology , Pancreatic Neoplasms/drug therapy , Pancreatic Neoplasms/metabolism , Pancreatic Neoplasms/pathology , Humans , Animals , Cell Line, Tumor , Mice , Oligopeptides/pharmacology , Antimetabolites, Antineoplastic/pharmacology , Antimetabolites, Antineoplastic/therapeutic use , Mice, Nude
13.
Part Fibre Toxicol ; 21(1): 16, 2024 Mar 21.
Article in English | MEDLINE | ID: mdl-38509617

ABSTRACT

BACKGROUND: Organomodified nanoclays (ONC), two-dimensional montmorillonite with organic coatings, are increasingly used to improve nanocomposite properties. However, little is known about pulmonary health risks along the nanoclay life cycle even with increased evidence of airborne particulate exposures in occupational environments. Recently, oropharyngeal aspiration exposure to pre- and post-incinerated ONC in mice caused low grade, persistent lung inflammation with a pro-fibrotic signaling response with unknown mode(s) of action. We hypothesized that the organic coating presence and incineration status of nanoclays determine the inflammatory cytokine secretary profile and cytotoxic response of macrophages. To test this hypothesis differentiated human macrophages (THP-1) were acutely exposed (0-20 µg/cm2) to pristine, uncoated nanoclay (CloisNa), an ONC (Clois30B), their incinerated byproducts (I-CloisNa and I-Clois30B), and crystalline silica (CS) followed by cytotoxicity and inflammatory endpoints. Macrophages were co-exposed to lipopolysaccharide (LPS) or LPS-free medium to assess the role of priming the NF-κB pathway in macrophage response to nanoclay treatment. Data were compared to inflammatory responses in male C57Bl/6J mice following 30 and 300 µg/mouse aspiration exposure to the same particles. RESULTS: In LPS-free media, CloisNa exposure caused mitochondrial depolarization while Clois30B exposure caused reduced macrophage viability, greater cytotoxicity, and significant damage-associated molecular patterns (IL-1α and ATP) release compared to CloisNa and unexposed controls. LPS priming with low CloisNa doses caused elevated cathepsin B/Caspage-1/IL-1ß release while higher doses resulted in apoptosis. Clois30B exposure caused dose-dependent THP-1 cell pyroptosis evidenced by Cathepsin B and IL-1ß release and Gasdermin D cleavage. Incineration ablated the cytotoxic and inflammatory effects of Clois30B while I-CloisNa still retained some mild inflammatory potential. Comparative analyses suggested that in vitro macrophage cell viability, inflammasome endpoints, and pro-inflammatory cytokine profiles significantly correlated to mouse bronchioalveolar lavage inflammation metrics including inflammatory cell recruitment. CONCLUSIONS: Presence of organic coating and incineration status influenced inflammatory and cytotoxic responses following exposure to human macrophages. Clois30B, with a quaternary ammonium tallow coating, induced a robust cell membrane damage and pyroptosis effect which was eliminated after incineration. Conversely, incinerated nanoclay exposure primarily caused elevated inflammatory cytokine release from THP-1 cells. Collectively, pre-incinerated nanoclay displayed interaction with macrophage membrane components (molecular initiating event), increased pro-inflammatory mediators, and increased inflammatory cell recruitment (two key events) in the lung fibrosis adverse outcome pathway.


Subject(s)
Cathepsin B , Lipopolysaccharides , Male , Humans , Mice , Animals , Cathepsin B/metabolism , Cathepsin B/pharmacology , Lipopolysaccharides/pharmacology , High-Throughput Screening Assays , Inflammation/chemically induced , Inflammation/metabolism , Macrophages , Cytokines/metabolism , Interleukin-1beta/metabolism
14.
Eur J Med Chem ; 269: 116329, 2024 Apr 05.
Article in English | MEDLINE | ID: mdl-38508117

ABSTRACT

Cathepsin B (CTSB) is a key lysosomal protease that plays a crucial role in the development of cancer. This article elucidates the relationship between CTSB and cancer from the perspectives of its structure, function, and role in tumor growth, migration, invasion, metastasis, angiogenesis and autophagy. Further, we summarized the research progress of cancer treatment related drugs targeting CTSB, as well as the potential and advantages of Traditional Chinese medicine in treating tumors by regulating the expression of CTSB.


Subject(s)
Cathepsin B , Cathepsin B/metabolism , Endopeptidases/chemistry , Endopeptidases/metabolism , Lysosomes/chemistry , Lysosomes/metabolism
15.
Neurobiol Dis ; 194: 106468, 2024 May.
Article in English | MEDLINE | ID: mdl-38460801

ABSTRACT

Intracerebral hemorrhage (ICH) is a subtype of stroke marked by elevated mortality and disability rates. Recently, mounting evidence suggests a significant role of ferroptosis in the pathogenesis of ICH. Through a combination of bioinformatics analysis and basic experiments, our goal is to identify the primary cell types and key molecules implicated in ferroptosis post-ICH. This aims to propel the advancement of ferroptosis research, offering potential therapeutic targets for ICH treatment. Our study reveals pronounced ferroptosis in microglia and identifies the target gene, cathepsin B (Ctsb), by analyzing differentially expressed genes following ICH. Ctsb, a cysteine protease primarily located in lysosomes, becomes a focal point in our investigation. Utilizing in vitro and in vivo models, we explore the correlation between Ctsb and ferroptosis in microglia post-ICH. Results demonstrate that ICH and hemin-induced ferroptosis in microglia coincide with elevated levels and activity of Ctsb protein. Effective alleviation of ferroptosis in microglia after ICH is achieved through the inhibition of Ctsb protease activity and protein levels using inhibitors and shRNA. Additionally, a notable increase in m6A methylation levels of Ctsb mRNA post-ICH is observed, suggesting a pivotal role of m6A methylation in regulating Ctsb translation. These research insights deepen our comprehension of the molecular pathways involved in ferroptosis after ICH, underscoring the potential of Ctsb as a promising target for mitigating brain damage resulting from ICH.


Subject(s)
Brain Injuries , Cathepsin B , Ferroptosis , Microglia , Humans , Brain Injuries/metabolism , Cathepsin B/genetics , Cathepsin B/metabolism , Cerebral Hemorrhage/pathology , Microglia/metabolism , Animals , Mice
16.
Bioconjug Chem ; 35(2): 132-139, 2024 02 21.
Article in English | MEDLINE | ID: mdl-38345213

ABSTRACT

Targeted drug delivery approaches that selectively and preferentially deliver therapeutic agents to specific tissues are of great interest for safer and more effective pharmaceutical treatments. We investigated whether cathepsin B cleavage of a valine-citrulline [VC(S)]-containing linker is required for the release of monomethyl auristatin E (MMAE) from albumin-drug conjugates. In this study, we used an engineered version of human serum albumin, Veltis High Binder II (HBII), which has enhanced binding to the neonatal Fc (fragment crystallizable) receptor (FcRn) to improve drug release upon binding and FcRn-mediated recycling. The linker-payload was conjugated to cysteine 34 of albumin using a carbonylacrylic (caa) reagent which produced homogeneous and plasma stable conjugates that retained FcRn binding. Two caa-linker-MMAE reagents were synthesized─one with a cleavable [VC(S)] linker and one with a noncleavable [VC(R)] linker─to question whether protease-mediated cleavage is needed for MMAE release. Our findings demonstrate that cathepsin B is required to achieve efficient and selective antitumor activity. The conjugates equipped with the cleavable [VC(S)] linker had potent antitumor activity in vivo facilitated by the release of free MMAE upon FcRn binding and internalization. In addition to the pronounced antitumor activity of the albumin conjugates in vivo, we also demonstrated their preferable tumor biodistribution and biocompatibility with no associated toxicity or side effects. These results suggest that the use of engineered albumins with high FcRn binding combined with protease cleavable linkers is an efficient strategy to target delivery of drugs to solid tumors.


Subject(s)
Antineoplastic Agents , Immunoconjugates , Neoplasms , Humans , Infant, Newborn , Albumins/metabolism , Cathepsin B/metabolism , Cell Line, Tumor , Immunoconjugates/pharmacology , Immunoconjugates/therapeutic use , Immunoconjugates/metabolism , Neoplasms/drug therapy , Peptide Hydrolases , Tissue Distribution
17.
ACS Nano ; 18(8): 6445-6462, 2024 Feb 27.
Article in English | MEDLINE | ID: mdl-38358804

ABSTRACT

Tumor-associated macrophages (TAMs) are closely related to the progression of glioblastoma multiform (GBM) and its development of therapeutic resistance to conventional chemotherapy. TAM-targeted therapy combined with conventional chemotherapy has emerged as a promising strategy to combat GBM. However, the presence of the blood-brain barrier (BBB) severely limits the therapeutic efficacy. Meanwhile, the lack of ability to distinguish different targeted cells also poses a challenge for precise therapy. Herein, we propose a cathepsin B (CTSB)-responsive programmed brain-targeted delivery system (D&R-HM-MCA) for simultaneous TAM-targeted and GBM-targeted delivery. D&R-HM-MCA could cross the BBB via low density lipoprotein receptor-associated protein 1 (LRP1)-mediated transcytosis. Upon reaching the GBM site, the outer angiopep-2 modification could be detached from D&R-HM-MCA via cleavage of the CTSB-responsive peptide, which could circumvent abluminal LRP1-mediated efflux. The exposed p-aminophenyl-α-d-mannopyranoside (MAN) modification could further recognize glucose transporter-1 (GLUT1) on GBM and macrophage mannose receptor (MMR) on TAMs. D&R-HM-MCA could achieve chemotherapeutic killing of GBM and simultaneously induce TAM polarization from anti-inflammatory M2 phenotype to pro-inflammatory M1 phenotype, thus resensitizing the chemotherapeutic response and improving anti-GBM immune response. This CTSB-responsive brain-targeted delivery system not only can improve brain delivery efficiency, but also can enable the combination of chemo-immunotherapy against GBM. The effectiveness of this strategy may provide thinking for designing more functional brain-targeted delivery systems and more effective therapeutic regimens.


Subject(s)
Brain Neoplasms , Glioblastoma , Humans , Glioblastoma/drug therapy , Glioblastoma/metabolism , Cathepsin B/metabolism , Brain Neoplasms/drug therapy , Brain/metabolism , Immunotherapy , Cell Line, Tumor , Tumor Microenvironment
18.
Sci Rep ; 14(1): 4517, 2024 02 24.
Article in English | MEDLINE | ID: mdl-38402255

ABSTRACT

PURPOSE Cathepsin B (Cat B) is a cysteine lysosomal protease that is upregulated in many inflammatory diseases and widely expressed in the brain. Here, we used a Cat B activatable near-infrared (NIR) imaging probe to measure glial activation in vivo in the formalin test, a standard orofacial inflammatory pain model. The probe's efficacy was quantified with immunohistochemical analysis of the somatosensory cortex. PROCEDURES Three different concentrations of Cat B imaging probe (30, 50, 100 pmol/200 g bodyweight) were injected intracisternally into the foramen magnum of rats under anesthesia. Four hours later formalin (1.5%, 50 µl) was injected into the upper lip and the animal's behaviors recorded for 45 min. Subsequently, animals were repeatedly scanned using the IVIS Spectrum (8, 10, and 28 h post imaging probe injection) to measure extracellular Cat B activity. Aldehyde fixed brain sections were immunostained with antibodies against microglial marker Iba1 or astrocytic GFAP and detected with fluorescently labeled secondary antibodies to quantify co-localization with the fluorescent probe. RESULTS The Cat B imaging probe only slightly altered the formalin test results. Nocifensive behavior was only reduced in phase 1 in the 100 pmol group. In vivo measured fluorescence efficiency was highest in the 100 pmol group 28 h post imaging probe injection. Post-mortem immunohistochemical analysis of the somatosensory cortex detected the greatest amount of NIR fluorescence localized on microglia and astrocytes in the 100 pmol imaging probe group. Sensory neuron neuropeptide and cell injury marker expression in ipsilateral trigeminal ganglia was not altered by the presence of fluorescent probe. CONCLUSIONS These data demonstrate a concentration- and time-dependent visualization of extracellular Cat B in activated glia in the formalin test using a NIR imaging probe. Intracisternal injections are well suited for extracellular CNS proteinase detection in conditions when the blood-brain barrier is intact.


Subject(s)
Cathepsin B , Fluorescent Dyes , Rats , Animals , Cathepsin B/metabolism , Pain Measurement , Fluorescent Dyes/metabolism , Brain/diagnostic imaging , Brain/metabolism , Microglia/metabolism , Facial Pain/metabolism , Formaldehyde/metabolism
19.
Int Immunopharmacol ; 129: 111645, 2024 Mar 10.
Article in English | MEDLINE | ID: mdl-38354512

ABSTRACT

Metabolic changes have been linked to the development of inflammatory bowel disease (IBD), which includes colitis. Allulose, an endogenous bioactive monosaccharide, is vital to the synthesis of numerous compounds and metabolic processes within living organisms. Nevertheless, the precise biochemical mechanism by which allulose inhibits colitis remains unknown. Allulose is an essential and intrinsic protector of the intestinal mucosal barrier, as it maintains the integrity of tight junctions in the intestines, according to the current research. It is also important to know that there is a link between the severity of inflammatory bowel disease (IBD) and colorectal cancer (CRC), chemically-induced colitis in rodents, and lower levels of allulose in the blood. Mice with colitis, either caused by dextran sodium sulphate (DSS) or naturally occurring colitis in IL-10-/- mice, had less damage to their intestinal mucosa after being given allulose. Giving allulose to a colitis model starts a chain of reactions because it stops cathepsin B from ejecting and helps lysosomes stick together. This system effectively stops the activity of myosin light chain kinase (MLCK) when intestinal epithelial damage happens. This stops the breakdown of tight junction integrity and the start of mitochondrial dysfunction. To summarise, the study's findings have presented data that supports the advantageous impact of allulose in reducing the advancement of colitis. Its ability to stop the disruption of the intestinal barrier enables this. Therefore, allulose has potential as a medicinal supplement for treating colitis.


Subject(s)
Colitis , Enteritis , Fructose , Inflammatory Bowel Diseases , Mitochondrial Diseases , Humans , Mice , Animals , Cathepsin B/metabolism , Caco-2 Cells , Inflammatory Bowel Diseases/metabolism , Colitis/chemically induced , Colitis/drug therapy , Colitis/metabolism , Intestinal Mucosa , Tight Junctions , Mitochondrial Diseases/metabolism , Dextran Sulfate/pharmacology , Mice, Inbred C57BL , Disease Models, Animal
20.
Cells ; 13(3)2024 Feb 04.
Article in English | MEDLINE | ID: mdl-38334675

ABSTRACT

Cathepsin B (CatB) is thought to be essential for the induction of Porphyromonas gingivalis lipopolysaccharide (Pg LPS)-induced Alzheimer's disease-like pathologies in mice, including interleukin-1ß (IL-1ß) production and cognitive decline. However, little is known about the role of CatB in Pg virulence factor-induced IL-1ß production by microglia. We first subjected IL-1ß-luciferase reporter BV-2 microglia to inhibitors of Toll-like receptors (TLRs), IκB kinase, and the NLRP3 inflammasome following stimulation with Pg LPS and outer membrane vesicles (OMVs). To clarify the involvement of CatB, we used several known CatB inhibitors, including CA-074Me, ZRLR, and human ß-defensin 3 (hBD3). IL-1ß production in BV-2 microglia induced by Pg LPS and OMVs was significantly inhibited by the TLR2 inhibitor C29 and the IκB kinase inhibitor wedelolactonne, but not by the NLRPs inhibitor MCC950. Both hBD3 and CA-074Me significantly inhibited Pg LPS-induced IL-1ß production in BV-2 microglia. Although CA-074Me also suppressed OMV-induced IL-1ß production, hBD3 did not inhibit it. Furthermore, both hBD3 and CA-074Me significantly blocked Pg LPS-induced nuclear NF-κB p65 translocation and IκBα degradation. In contrast, hBD3 and CA-074Me did not block OMV-induced nuclear NF-κB p65 translocation or IκBα degradation. Furthermore, neither ZRLR, a specific CatB inhibitor, nor shRNA-mediated knockdown of CatB expression had any effect on Pg virulence factor-induced IL-1ß production. Interestingly, phagocytosis of OMVs by BV-2 microglia induced IL-1ß production. Finally, the structural models generated by AlphaFold indicated that hBD3 can bind to the substrate-binding pocket of CatB, and possibly CatL as well. These results suggest that Pg LPS induces CatB/CatL-dependent synthesis and processing of pro-IL-1ß without activation of the NLRP3 inflammasome. In contrast, OMVs promote the synthesis and processing of pro-IL-1ß through CatB/CatL-independent phagocytic mechanisms. Thus, hBD3 can improve the IL-1ß-associated vicious inflammatory cycle induced by microglia through inhibition of CatB/CatL.


Subject(s)
Microglia , beta-Defensins , Humans , beta-Defensins/metabolism , Cathepsin B/metabolism , I-kappa B Kinase/metabolism , Inflammasomes/metabolism , Interleukin-1beta/metabolism , Lipopolysaccharides , Microglia/metabolism , NF-kappa B/metabolism , NF-KappaB Inhibitor alpha/metabolism , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Virulence Factors/metabolism
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