ABSTRACT
BAX is a pro-apoptotic protein that transforms from a cytosolic monomer into a toxic oligomer that permeabilizes the mitochondrial outer membrane. How BAX monomers assemble into a higher-order conformation, and the structural determinants essential to membrane permeabilization, remain a mechanistic mystery. A key hurdle has been the inability to generate a homogeneous BAX oligomer (BAXO) for analysis. Here, we report the production and characterization of a full-length BAXO that recapitulates physiologic BAX activation. Multidisciplinary studies revealed striking conformational consequences of oligomerization and insight into the macromolecular structure of oligomeric BAX. Importantly, BAXO enabled the assignment of specific roles to particular residues and α helices that mediate individual steps of the BAX activation pathway, including unexpected functionalities of BAX α6 and α9 in driving membrane disruption. Our results provide the first glimpse of a full-length and functional BAXO, revealing structural requirements for the elusive execution phase of mitochondrial apoptosis.
Subject(s)
Apoptosis , Mitochondria/pathology , Mitochondrial Membranes/metabolism , Protein Multimerization , bcl-2-Associated X Protein/chemistry , bcl-2-Associated X Protein/metabolism , Animals , Biological Transport , Cell Membrane Permeability , Cytosol/metabolism , Humans , Mice , Mitochondria/metabolism , Models, Molecular , Protein Conformation , Proto-Oncogene Proteins c-fosABSTRACT
Rapidly proliferating cells reshape their metabolism to satisfy their ever-lasting need for cellular building blocks. This phenomenon is exemplified in certain malignant conditions such as cancer but also during embryonic development when cells rely heavily on glycolytic metabolism to exploit its metabolic intermediates for biosynthetic processes. How cells reshape their metabolism is not fully understood. Here we report that loss of cathepsin L (Cts L) is associated with a fast proliferation rate and enhanced glycolytic metabolism that depend on lactate dehydrogenase A (LDHA) activity. Using mass spectrometry analysis of cells treated with a pan cathepsin inhibitor, we observed an increased abundance of proteins involved in central carbon metabolism. Further inspection of putative Cts L targets revealed an enrichment for glycolytic metabolism that was independently confirmed by metabolomic and biochemical analyses. Moreover, proteomic analysis of Cts L-knockout cells identified LDHA overexpression that was demonstrated to be a key metabolic junction in these cells. Lastly, we show that Cts L inhibition led to increased LDHA protein expression, suggesting a causal relationship between LDHA expression and function. In conclusion, we propose that Cts L regulates this metabolic circuit to keep cell division under control, suggesting the therapeutic potential of targeting this protein and its networks in cancer.
Subject(s)
Cathepsin L/metabolism , Metabolic Networks and Pathways , Animals , Cell Proliferation , Embryo, Mammalian/cytology , Fibroblasts/metabolism , Gene Deletion , Glycolysis , HeLa Cells , Humans , Lactate Dehydrogenase 5/genetics , Lactate Dehydrogenase 5/metabolism , Lipogenesis , Mass Spectrometry , Metabolomics , Mice , NIH 3T3 Cells , Phenotype , Proteomics , RNA, Messenger/genetics , RNA, Messenger/metabolismABSTRACT
BACKGROUND/AIMS: Atherosclerosis underlies the majority of cardiovascular events, consequent to non-resolving inflammation. Considerable evidence implicates autophagy dysfunction at the core of this inflammatory condition, but the basis of this dysfunction is not fully understood. METHODS: Using an in vitro model of lipid-laden macrophages, activity-based probes and high-throughput techniques, we studied the role of the cysteine proteases cathepsins in autophagy. RESULTS: We showed that cathepsin activity is suppressed by oxidized lipids and that cathepsin has an indispensable role in the autophagy-lysosomal degradation pathway. Accordingly, loss of cathepsin function resulted in autophagy derangement. Shotgun proteomics confirmed autophagy dysfunction and unveiled a pivotal role of cathepsin L in a putative cathepsin degradation network. At the physiological level, cathepsin inhibition resulted in mitochondrial stress, which translated into impaired oxidative metabolism, excessive production of reactive oxygen species and activation of the cellular stress response, driven by ATF4-CHOP transcription factors. In addition, transcriptomic analysis of these cells uncovered some genetic similarities with the inflammatory macrophage phenotype (a.k.a M1 macrophages) and increased expression of inflammatory cytokines. CONCLUSION: Our data highlight the importance of cathepsins for mitochondrial quality control mechanisms and amelioration of vascular inflammation.
Subject(s)
Anti-Inflammatory Agents/pharmacology , Cathepsin B/metabolism , Cathepsin L/metabolism , Cathepsins/metabolism , Macrophages/metabolism , Animals , Autophagy/drug effects , Bone Marrow Cells/cytology , Cathepsin B/antagonists & inhibitors , Cathepsin L/antagonists & inhibitors , Cells, Cultured , Cholesterol/metabolism , Humans , Macrophages/drug effects , Male , Mass Spectrometry , Mice , Mice, Knockout , Mitochondria/drug effects , Mitochondria/metabolism , Mitochondrial Dynamics , Oxidative Stress/drug effects , Proteomics/methods , RAW 264.7 Cells , Reactive Oxygen Species/metabolismABSTRACT
X-ray computed tomography (CT) is a robust, precise, fast, and reliable imaging method that enables excellent spatial resolution and quantification of contrast agents throughout the body. However, CT is largely inadequate for molecular imaging applications due mainly to its low contrast sensitivity that forces the use of large concentrations of contrast agents for detection. To overcome this limitation, we generated a new class of iodinated nanoscale activity-based probes (IN-ABPs) that sufficiently accumulates at the target site by covalently binding cysteine cathepsins that are exceptionally highly expressed in cancer. The IN-ABPs are comprised of a short targeting peptide selective to specific cathepsins, an electrophilic moiety that allows activity-dependent covalent binding, and tags containing dendrimers with up to 48 iodine atoms. IN-ABPs selectively bind and inhibit activity of recombinant and intracellular cathepsin B, L, and S. We compared the in vivo kinetics, biodistribution, and tumor accumulation of IN-ABPs bearing 18 and 48 iodine atoms each, and their control counterparts lacking the targeting moiety. Here we show that although both IN-ABPs bind specifically to cathepsins within the tumor and produce detectable CT contrast, the 48-iodine bearing IN-ABP was found to be optimal with signals over 2.1-fold higher than its nontargeted counterpart. In conclusion, this study shows the synthetic feasibility and potential utility of IN-ABPs as potent contrast agents that enable molecular imaging of tumors using CT.
ABSTRACT
Mammalian target of rapamycin complex 1 (mTORC1) is a key regulator of cell metabolism and lymphocyte proliferation. It is inhibited by the tuberous sclerosis complex (TSC), a heterodimer of TSC1 and TSC2. Deletion of either gene results in robust activation of mTORC1. Mature B cells reside in the spleen at two major anatomical locations, the marginal zone (MZ) and follicles. The MZ constitutes the first line of humoral response against blood-borne pathogens and undergoes atrophy in chronic inflammation. In previous work, we showed that mice deleted for TSC1 in their B cells (TSC1BKO ) have almost no MZ B cells, whereas follicular B cells are minimally affected. To explore potential underlying mechanisms for MZ B-cell loss, we have analysed the spleen MZ architecture of TSC1BKO mice and found it to be severely impaired. Examination of lymphotoxins (LTα and LTß) and lymphotoxin receptor (LTßR) expression indicated that LTßR levels in spleen stroma were reduced by TSC1 deletion in the B cells. Furthermore, LTα transcripts in B cells were reduced. Because LTßR is sensitive to proteolysis, we analysed cathepsin activity in TSC1BKO . A higher cathepsin activity, particularly of cathepsin B, was observed, which was reduced by mTORC1 inhibition with rapamycin in vivo. Remarkably, in vivo administration of a pan-cathepsin inhibitor restored LTßR expression, LTα mRNA levels and the MZ architecture. Our data identify a novel connection, although not elucidated at the molecular level, between mTORC1 and cathepsin activity in a manner relevant to MZ dynamics.
Subject(s)
B-Lymphocytes/immunology , Cathepsins/metabolism , Mechanistic Target of Rapamycin Complex 1/metabolism , Spleen/immunology , Animals , CHO Cells , Cathepsins/antagonists & inhibitors , Cell Line , Cricetulus , Lymphotoxin beta Receptor/biosynthesis , Lymphotoxin-alpha/biosynthesis , Lymphotoxin-beta/biosynthesis , Mice , Mice, Transgenic , Sirolimus/pharmacology , Spleen/cytology , Tuberous Sclerosis Complex 1 Protein/genetics , Tuberous Sclerosis Complex 2 Protein/geneticsABSTRACT
X-ray CT instruments are among the most available, efficient, and cost-effective imaging modalities in hospitals. The field of CT molecular imaging is emerging which relies mainly on the detection of gold nanoparticles and iodine-containing compounds directed to tagging a variety of abundant biomolecules. Here for the first time we attempted to detect enzymatic activity, while the low sensitivity of CT scanners to contrast reagents made this a challenging task. Therefore, we developed a new class of nanosized cathepsin-targeted activity-based probes (ABPs) for functional CT imaging of cancer. ABPs are small molecules designed to covalently modify enzyme targets in an activity-dependent manner. Using a CT instrument, these novel probes enable detection of the elevated cathepsin activity within cancerous tissue, thus creating a direct link between biological processes and imaging signals. We present the generation and biochemical evaluation of a library of ABPs tagged with different sized gold nanoparticles (GNPs), with various ratios of cathepsin-targeting moiety and a combination of different polyethylene glycol (PEG) protective layers. The most potent and stable GNP-ABPs were applied for noninvasive cancer imaging in mice. Surprisingly, detection of CT contrast from the tumor had reverse correlation to GNP size and the amount of targeting moiety. Interestingly, TEM images of tumor sections show intercellular lysosomal subcellular localization of the GNP-ABPs. In conclusion, we demonstrate that the covalent linkage is key for detection using low sensitive imaging modalities and the utility of GNP-ABPs as a promising tool for enzymatic-based CT imaging.
Subject(s)
Cathepsin B/metabolism , Dipeptides/pharmacology , Enzyme Inhibitors/pharmacology , Metal Nanoparticles/chemistry , Neoplasms/metabolism , Animals , Cathepsin B/antagonists & inhibitors , Cell Line, Tumor , Dipeptides/chemical synthesis , Dipeptides/chemistry , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Gold/chemistry , Humans , Lysosomes/metabolism , Male , Mice , Mice, Inbred BALB C , NIH 3T3 Cells , Neoplasms/diagnostic imaging , Neoplasms/pathology , Polyethylene Glycols/chemistry , Tomography, X-Ray Computed/methodsABSTRACT
BACKGROUND AND PURPOSE: Atherosclerosis is a leading cause of mortality worldwide, contributing to both strokes and heart attacks. Macrophages are key players in atherogenesis, promoting vascular inflammation and arterial remodeling through cysteine cathepsin proteases. We used a cathepsin-targeted activity-based probe in human carotid plaque to assess its diagnostic potential and evaluate macrophage subtypes ex vivo. METHODS: Carotid plaque specimens surgically removed during endarterectomy from 62 patients (age range, 38% female, 28% symptomatic) were graded pathologically as either stable (Grade 1) or unstable (Grade 2 or 3). A cathepsin activity-based probe was used to quantify individual cathepsins in plaque tissue and macrophage subtypes. RESULTS: Cathepsin B and S activities were increased in unstable carotid plaques. They were quantified using the probe to biochemically investigate individual cathepsins (Cathepsin B and S: 0.97 and 0.90 for grade 3 versus 0.51 and 0.59 for grade 1; P=0.006 and P=0.03 arbitrary units (AU), respectively). Higher cathepsin activity was observed in carotid plaques from symptomatic patients (Cathepsin B and S: 0.65 and 0.77 for asymptomatic, 0.99 and 1.17 for symptomatic; P=0.008 and P=0.005 AU, respectively). Additionally, it was demonstrated that M2 macrophages from unstable plaques express cathepsin activity 5-fold higher than M2 macrophages from stable plaques (25.52 versus 5.22; P=0.008 AU). CONCLUSIONS: Targeting cathepsin activity in human carotid plaques may present a novel diagnostic tool for characterizing high-risk plaques. Novel cathepsin activity patterns within plaques and macrophage subpopulations suggest their involvement in the transition to active disease.
Subject(s)
Carotid Arteries/metabolism , Carotid Artery Diseases/metabolism , Cathepsins/metabolism , Macrophages/metabolism , Plaque, Atherosclerotic/metabolism , Aged , Aged, 80 and over , Carotid Arteries/pathology , Carotid Arteries/surgery , Carotid Artery Diseases/pathology , Carotid Artery Diseases/surgery , Endarterectomy, Carotid , Female , Humans , Male , Middle Aged , Plaque, Atherosclerotic/pathology , Plaque, Atherosclerotic/surgeryABSTRACT
MCL-1 is a high-priority target due to its dominant role in the pathogenesis and chemoresistance of cancer, yet clinical trials of MCL-1 inhibitors are revealing toxic side effects. MCL-1 biology is complex, extending beyond apoptotic regulation and confounded by its multiple isoforms, its domains of unresolved structure and function, and challenges in distinguishing noncanonical activities from the apoptotic response. We find that, in the presence or absence of an intact mitochondrial apoptotic pathway, genetic deletion or pharmacologic targeting of MCL-1 induces DNA damage and retards cell proliferation. Indeed, the cancer cell susceptibility profile of MCL-1 inhibitors better matches that of anti-proliferative than pro-apoptotic drugs, expanding their potential therapeutic applications, including synergistic combinations, but heightening therapeutic window concerns. Proteomic profiling provides a resource for mechanistic dissection and reveals the minichromosome maintenance DNA helicase as an interacting nuclear protein complex that links MCL-1 to the regulation of DNA integrity and cell-cycle progression.
Subject(s)
Antineoplastic Agents , Proto-Oncogene Proteins c-bcl-2 , Proto-Oncogene Proteins c-bcl-2/metabolism , Myeloid Cell Leukemia Sequence 1 Protein/metabolism , Apoptosis , Proteomics , Antineoplastic Agents/pharmacology , DNA Damage , Cell Line, TumorABSTRACT
p53 is a critical tumor-suppressor protein that guards the human genome against mutations by inducing cell-cycle arrest or apoptosis. Cancer cells subvert p53 by deletion, mutation, or overexpression of the negative regulators HDM2 and HDMX. For tumors that retain wild-type p53, its reactivation by pharmacologic targeting of HDM2 and/or HDMX represents a promising strategy, with a series of selective small-molecule HDM2 inhibitors and a dual HDM2/HDMX stapled-peptide inhibitor being evaluated in clinical trials. Because selective HDM2 targeting can cause hematologic toxicity, selective HDMX inhibitors could provide an alternative p53-reactivation strategy, but clinical candidates remain elusive. Here, we applied a mutation-scanning approach to uncover p53-based stapled peptides that are selective for HDMX. Crystal structures of stapled-peptide/HDMX complexes revealed a molecular mechanism for the observed specificity, which was validated by HDMX mutagenesis. Thus, we provide a blueprint for the development of HDMX-selective inhibitors to dissect and target the p53/HDMX interaction.
Subject(s)
Antineoplastic Agents/chemistry , Cell Cycle Proteins/chemistry , Oligopeptides/chemistry , Proto-Oncogene Proteins/chemistry , Tumor Suppressor Protein p53/chemistry , Amino Acid Substitution , Antineoplastic Agents/pharmacology , Binding Sites , Cell Cycle Proteins/antagonists & inhibitors , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Line, Tumor , Humans , Molecular Docking Simulation , Oligopeptides/pharmacology , Protein Binding , Proto-Oncogene Proteins/antagonists & inhibitors , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-mdm2/antagonists & inhibitors , Proto-Oncogene Proteins c-mdm2/chemistry , Proto-Oncogene Proteins c-mdm2/metabolism , Tumor Suppressor Protein p53/metabolismABSTRACT
Despite the common use of lipid-lowering medications, cardiovascular diseases continue to be a significant health concern. Atherosclerosis, one of the most frequent causes of cardiovascular morbidity, involves extensive inflammatory activity and remodeling of the vascular endothelium. This relentless inflammatory condition can ultimately give rise to clinical manifestations, such as ischemic heart disease or stroke. Accumulating evidence over the past decades implicates cysteine protease cathepsins in cardiovascular disorders. In particular, Cathepsins B, L, and S are over-expressed during vascular inflammation, and their activity is associated with impaired clinical outcomes. Here we took advantage of these molecular events to introduce a non-invasive detection and treatment approach to modulate vascular inflammation using a Photosensitizing quenched Activity-Based Probed (PS-qABP) that targets these proteases. Methods: We tested the application of this approach in LDL receptor-deficient mice and used non-invasive imaging and heart cross-section staining to assess the theranostic efficacy of this probe. Moreover, we used fresh human endarterectomy tissues to analyze cathepsin signals on gel, and verified cathepsin identity by mass spectrometry. Results: We showed that our PS-qABP can rapidly accumulate in areas of inflammatory atheromas in vivo, and application of light therapy profoundly reduced lesional immune cell content without affecting smooth muscle cell and collagen contents. Lastly, using human tissue samples we provided proof-of-concept for future clinical applications of this technology. Conclusions: Photodynamic therapy guided by cysteine cathepsin activity is an effective approach to reduce vascular inflammation and attenuate atherosclerosis progression. This approach could potentially be applied in clinical settings.
Subject(s)
Cardiovascular Diseases/metabolism , Cardiovascular Diseases/therapy , Cathepsins/metabolism , Animals , Atherosclerosis/metabolism , Atherosclerosis/therapy , Collagen/metabolism , Female , Fluorescent Antibody Technique , Macrophages/metabolism , Mass Spectrometry , Mice , Mice, Mutant Strains , Photochemotherapy , Receptors, LDL/deficiency , Receptors, LDL/genetics , Receptors, LDL/metabolismABSTRACT
Complete tumor removal during surgery has a great impact on patient survival. To that end, the surgeon should detect the tumor, remove it and validate that there are no residual cancer cells left behind. Residual cells at the incision margin of the tissue removed during surgery are associated with tumor recurrence and poor prognosis for the patient. In order to remove the tumor tissue completely with minimal collateral damage to healthy tissue, there is a need for diagnostic tools that will differentiate between the tumor and its normal surroundings. Methods: We designed, synthesized and characterized three novel polymeric Turn-ON probes that will be activated at the tumor site by cysteine cathepsins that are highly expressed in multiple tumor types. Utilizing orthotopic breast cancer and melanoma models, which spontaneously metastasize to the brain, we studied the kinetics of our polymeric Turn-ON nano-probes. Results: To date, numerous low molecular weight cathepsin-sensitive substrates have been reported, however, most of them suffer from rapid clearance and reduced signal shortly after administration. Here, we show an improved tumor-to-background ratio upon activation of our Turn-ON probes by cathepsins. The signal obtained from the tumor was stable and delineated the tumor boundaries during the whole surgical procedure, enabling accurate resection. Conclusions: Our findings show that the control groups of tumor-bearing mice, which underwent either standard surgery under white light only or under the fluorescence guidance of the commercially-available imaging agents ProSense® 680 or 5-aminolevulinic acid (5-ALA), survived for less time and suffered from tumor recurrence earlier than the group that underwent image-guided surgery (IGS) using our Turn-ON probes. Our "smart" polymeric probes can potentially assist surgeons' decision in real-time during surgery regarding the tumor margins needed to be removed, leading to improved patient outcome.
Subject(s)
Breast Neoplasms/surgery , Melanoma/surgery , Nanoparticles/administration & dosage , Optical Imaging/methods , Staining and Labeling/methods , Surgery, Computer-Assisted/methods , Animals , Cathepsins/metabolism , Disease Models, Animal , Fluorescent Dyes/administration & dosage , Fluorescent Dyes/metabolism , Humans , Mice , Nanoparticles/metabolism , Treatment OutcomeABSTRACT
The development of reactive drug carriers that could actively respond to biological signals is a challenging task. Different peptides can self-assemble into biocompatible nanostructures of various functionalities, including drugs carriers. Minimal building blocks, such as diphenylalanine, readily form ordered nanostructures. Here we present the development of self-assembled tetra-peptides that include the diphenylalanine motif, serving as substrates of the cathepsin proteases. This is of great clinical importance as cathepsins, whose activity and expression are highly elevated in cancer and other pathologies, have been shown to serve as efficient enzymes for therapeutic release. Based on the cathepsins affinity around the active site, we generated a library of Phe-Phe-Lys-Phe (FFKF) tetra-peptide substrates (TPSs). We inserted various N-termini capping groups with different chemical properties to investigate the effect on protease affinity and self-assembly. All nine TPSs were cleaved by their targets, cathepsins B and L. However, solvent switching led to nanofibers self-assembly of only seven of them. Due to its rapid self-assembly and complete degradation by cathepsin B, we focused on TPS4, Cbz-FFKF-OH. Degradation of TPS4 nanofibers by cathepsin B led to the release of 91.8±0.3% of the incorporated anti-cancerous drug Doxorubicin from the nanofibers within 8h while only 55±0.2% was released without enzyme treatment. Finally, we demonstrated that tumor lysates fully degraded TPS4 nanofibers. Collectively, these results suggest that tetra-peptide substrates that form nanostructures could serve as a promising platform for targeted drug delivery to pathologies in which protease activity is highly elevated.
Subject(s)
Cathepsins/metabolism , Drug Carriers/metabolism , Drug Delivery Systems , Oligopeptides/metabolism , Antibiotics, Antineoplastic/administration & dosage , Cell Line, Tumor , Doxorubicin/administration & dosage , Drug Carriers/chemistry , Humans , Nanofibers/chemistry , Nanofibers/ultrastructure , Neoplasms/drug therapy , Neoplasms/metabolism , Oligopeptides/chemistryABSTRACT
BACKGROUND AND PURPOSE: Cardiovascular disease is the leading cause of death worldwide, mainly due to an increasing prevalence of atherosclerosis characterized by inflammatory plaques. Plaques with high levels of macrophage infiltration are considered "vulnerable" while those that do not have significant inflammation are considered stable; cathepsin protease activity is highly elevated in macrophages of vulnerable plaques and contributes to plaque instability. Establishing novel tools for non-invasive molecular imaging of macrophages in plaques could aid in preclinical studies and evaluation of therapeutics. Furthermore, compounds that reduce the macrophage content within plaques should ultimately impact care for this disease. METHODS: We have applied quenched fluorescent cathepsin activity-based probes (ABPs) to a murine atherosclerosis model and evaluated their use for in vivo imaging using fluorescent molecular tomography (FMT), as well as ex vivo fluorescence imaging and fluorescent microscopy. Additionally, freshly dissected human carotid plaques were treated with our potent cathepsin inhibitor and macrophage apoptosis was evaluated by fluorescent microscopy. RESULTS: We demonstrate that our ABPs accurately detect murine atherosclerotic plaques non-invasively, identifying cathepsin activity within plaque macrophages. In addition, our cathepsin inhibitor selectively induced cell apoptosis of 55%±10% of the macrophage within excised human atherosclerotic plaques. CONCLUSIONS: Cathepsin ABPs present a rapid diagnostic tool for macrophage detection in atherosclerotic plaque. Our inhibitor confirms cathepsin-targeting as a promising approach to treat atherosclerotic plaque inflammation.
Subject(s)
Atherosclerosis/diagnostic imaging , Cathepsins/metabolism , Fluorescent Dyes , Macrophages/enzymology , Animals , Apoptosis/drug effects , Carotid Stenosis/diagnostic imaging , Cathepsins/antagonists & inhibitors , Humans , Macrophages/drug effects , Macrophages/pathology , Male , Mice , Microscopy, Fluorescence , Molecular Imaging/methods , Plaque, Atherosclerotic/diagnostic imaging , Protease Inhibitors/pharmacologyABSTRACT
While chemotherapy strongly restricts or reverses tumor growth, the response of host tissue to therapy can counteract its anti-tumor activity by promoting tumor re-growth and/or metastases, thus limiting therapeutic efficacy. Here, we show that vascular endothelial growth factor receptor 3 (VEGFR3)-expressing macrophages infiltrating chemotherapy-treated tumors play a significant role in metastasis. They do so in part by inducing lymphangiogenesis as a result of cathepsin release, leading to VEGF-C upregulation by heparanase. We found that macrophages from chemotherapy-treated mice are sufficient to trigger lymphatic vessel activity and structure in naive tumors in a VEGFR3-dependent manner. Blocking VEGF-C/VEGFR3 axis inhibits the activity of chemotherapy-educated macrophages, leading to reduced lymphangiogenesis in treated tumors. Overall, our results suggest that disrupting the VEGF-C/VEGFR3 axis not only directly inhibits lymphangiogenesis but also blocks the pro-metastatic activity of macrophages in chemotherapy-treated mice.
Subject(s)
Lymphangiogenesis , Macrophages/pathology , Paclitaxel/pharmacology , Vascular Endothelial Growth Factor Receptor-3/metabolism , Animals , Cathepsins/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Endothelial Cells/drug effects , Endothelial Cells/metabolism , Female , Glucuronidase/metabolism , Humans , Lymphangiogenesis/drug effects , Lymphatic Vessels/metabolism , Macrophages/drug effects , Macrophages/metabolism , Mice, Inbred BALB C , Neoplasm Metastasis , Phenotype , Up-Regulation/drug effects , Vascular Endothelial Growth Factor C/blood , Vascular Endothelial Growth Factor C/metabolismABSTRACT
Elevated cathepsins levels and activities are found in several types of human cancer, making them valuable biomarkers for detection and targeting therapeutics. We designed small molecule quenched activity-based probes (qABPs) that fluoresce upon activity-dependent covalent modification, yielding cell killing by Photodynamic Therapy (PDT). These novel molecules are highly selective theranostic probes that enable both detection and treatment of cancer with minimal side effects. Our qABPs carry a photosensitizer (PS), which is activated by light, resulting in oxidative stress and subsequent cell ablation, and a quencher that when removed by active cathepsins allow the PS to fluoresce and demonstrate PD properties. Our most powerful and stable PS-qABP, YBN14, consists of a selective cathepsin recognition sequence, a QC-1 quencher and a new bacteriochlorin derivative as a PS. YBN14 allowed rapid and selective non-invasive in vivo imaging of subcutaneous tumors and induced specific tumor macrophage apoptosis by light treatment, resulting in a substantial tumor shrinkage in an aggressive breast cancer mouse model. These results demonstrate for the first time that the PS-qABPs technology offers a functional theranostic tool, which can be applied to numerous tumor types and other inflammation-associated diseases.
Subject(s)
Cathepsins/metabolism , Macrophages/immunology , Photosensitizing Agents/therapeutic use , Phototherapy/methods , Skin Neoplasms/drug therapy , Soft Tissue Neoplasms/drug therapy , Theranostic Nanomedicine , Animals , Light , Male , Mice, Inbred BALB C , Neoplasms, Experimental/diagnosis , Neoplasms, Experimental/drug therapy , Porphyrins/therapeutic use , Skin Neoplasms/diagnosis , Soft Tissue Neoplasms/diagnosisABSTRACT
INTRODUCTION: Lysosomal cathepsins have been reported to contribute to Osteoarthritis (OA) pathophysiology due to their increase in pro-inflammatory conditions. Given the causal role of cathepsins in OA, monitoring their specific activity could provide means for assessing OA severity. To this end, we herein sought to assess a cathepsin activity-based probe (ABP), GB123, in vitro and in vivo. METHODS: Protein levels and activity of cathepsins B and S were monitored by immunoblot analysis and GB123 labeling in cultured primary chondrocytes and conditioned media, following stimuli with tumor necrosis factor alpha (TNFα) and/or Interleukin 1 beta (IL-1ß). Similarly, cathepsin activity was examined in sections of intact cartilage (IC) and degraded cartilage (DC) regions of OA. Finally, synovial fluid (SF) and serum from donors with no signs of diseases, early OA, late OA and rheumatoid arthritis (RA) patients were analyzed with GB123 to detect distinct activity levels of cathepsin B and S. RESULTS: Cathepsin activity in cell lysates, conditioned media explants and DC sections showed enhanced enzymatic activity of cathepsins B and S. Further histological analysis revealed that cathepsin activity was found higher in superficial zones of DC than in IC. Examining serum and SF revealed that cathepsin B is significantly elevated with OA severity in serum and SF, yet levels of cathepsin S are more correlated with synovitis and RA. CONCLUSIONS: Based on our data, cathepsin activity monitored by ABPs correlated well with OA severity and joint inflammation, directing towards a novel etiological target for OA, which possesses significant translational potential in developing means for non-invasive detection of early signs of OA.
Subject(s)
Cathepsin B/metabolism , Osteoarthritis/enzymology , Osteoarthritis/pathology , Severity of Illness Index , Staining and Labeling/methods , Aged , Cathepsin B/analysis , Cells, Cultured , Chondrocytes/chemistry , Chondrocytes/metabolism , Enzyme Activation/physiology , Female , Humans , Male , Young AdultABSTRACT
One of the noncellular microenvironmental factors that contribute to malignancy of solid tumors is acidic peritumoral pH. We have previously demonstrated that extracellular acidosis leads to localization of the cysteine pro-tease cathepsin B on the tumor cell membrane and its secretion. The objective of the present study was to determine if an acidic extracellular pH such as that observed in vivo (i.e., pHe 6.8) affects the activity of proteases, e.g., cathepsin B, that contribute to degradation of collagen IV by tumor cells when grown in biologically relevant three-dimensional (3D) cultures. For these studies, we used 1) 3D reconstituted basement membrane overlay cultures of human carcinomas, 2) live cell imaging assays to assess proteolysis, and 3) in vivo imaging of active tumor proteases. At pHe 6.8, there were increases in pericellular active cysteine cathepsins and in degradation of dye-quenched collagen IV, which was partially blocked by a cathepsin B inhibitor. Imaging probes for active cysteine cathepsins localized to tumors in vivo. The amount of bound probe decreased in tumors in bicarbonate-treated mice, a treatment previously shown to increase peritumoral pHe and reduce local invasion of the tumors. Our results are consistent with the acid-mediated invasion hypothesis and with a role for cathepsin B in promoting degradation of a basement membrane protein substrate, i.e., type IV collagen, in an acidic peritumoral environment.