Lysosomal Proteolytic Activity Research Articles

Abstract 4454: Manipulation of the amino acid metabolism promotes therapeutic efficacy through targeting lysosome in glioblastoma

Abstract Nutrients are converted by the body to smaller molecules, which are utilized for both anabolic and catabolic reactions. Cooperative regulation of these metabolic processes is critical for maintaining tissue homeostasis. Based on these findings, our group has been focusing on how the regulation of nutrient-driven metabolism controls the malignancy of cancer cells. Metabolic regulation has been considered crucial for cellular adaptation to environmental changes and promoting survival and proliferation. Recent studies showed the successful applications of targeting specific nutrients for cancer therapy. In this study, we developed a new lysosome-targeting therapeutic strategy, centered around the manipulation of the amino acid metabolism for GBM. Using patient-derived GBM cells, we found that lysosomal proteolytic activity is a unique metabolic biomarker representing malignant phenotypes of GBM and that it is closely correlated with the efficacy of current conventional therapy for GBM patients. Furthermore, the MiT/TFE family members, TFEB and TFE3, which are the master regulators of lysosomal biogenesis, controlled malignant progression and therapeutic efficacy in vitro and vivo. Combinatory analyses with clinical patient samples and xenograft models demonstrated that the MiT/TFE family plays a crucial role as a regulator of malignant properties in GBM. Analysis of The Cancer Genome Atlas (TCGA) glioma dataset, which includes data from patients with Grade 2, 3, and 4 (GBM) gliomas, showed that higher expression of the MiT/TFE family was significantly associated with shorter survival. Interestingly, we discovered that an essential amino acid controls lysosomal biogenesis and membrane integrity by regulation of nitric oxide signaling pathway. Finally, we screened the potential anti-cancer drugs, including temozolomide, exhibiting the synergy effect in amino acid-restriction conditions by conducting a drug screening using a clinically available anti-cancer drug library. Our study demonstrated that lysosomal function, supported by amino acid metabolism, plays a critical role in the regulation of GBM malignant status and proposed precision nutrition as a promising therapeutic approach. Citation Format: Yongwei Jing, Masahiko Kobayashi, Atsushi Hirao. Manipulation of the amino acid metabolism promotes therapeutic efficacy through targeting lysosome in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4454.

Lysosomal BK channels facilitate silica-induced inflammation in macrophages

Background Lysosomal ion channels are proposed therapeutic targets for a number of diseases, including those driven by NLRP3 inflammasome-mediated inflammation. Here, the specific role of the lysosomal big conductance Ca2+-activated K+ (BK) channel was evaluated in a silica model of inflammation in murine macrophages. A specific-inhibitor of BK channel function, paxilline (PAX), and activators NS11021 and NS1619 were utilized to evaluate the role of lysosomal BK channel activity in silica-induced lysosomal membrane permeabilization (LMP) and NLRP3 inflammasome activation resulting in IL-1β release. Methods Murine macrophages were exposed in vitro to crystalline silica following pretreatment with BK channel inhibitors or activators and LMP, cell death, and IL-1β release were assessed. In addition, the effect of PAX treatment on silica-induced cytosolic K+ decrease was measured. Finally, the effects of BK channel modifiers on lysosomal pH, proteolytic activity, and cholesterol transport were also evaluated. Results PAX pretreatment significantly attenuated silica-induced cell death and IL-1β release. PAX caused an increase in lysosomal pH and decrease in lysosomal proteolytic activity. PAX also caused a significant accumulation of lysosomal cholesterol. BK channel activators NS11021 and NS1619 increased silica-induced cell death and IL-1β release. BK channel activation also caused a decrease in lysosomal pH and increase in lysosomal proteolytic function as well as a decrease in cholesterol accumulation. Conclusion Taken together, these results demonstrate that inhibiting lysosomal BK channel activity with PAX effectively reduced silica-induced cell death and IL-1β release. Blocking cytosolic K+ entry into the lysosome prevented LMP through the decrease of lysosomal acidification and proteolytic function and increase in lysosomal cholesterol.

Lysosomal membrane integrity in fibroblasts derived from patients with Gaucher disease.

Gaucher disease (GD) is a recessively inherited lysosomal storage disorder characterized by a deficiency of lysosomal glucocerebrosidase (GBA1). This deficiency results in the accumulation of its substrate, glucosylceramide (GlcCer), within lysosomes. Here, we investigated lysosomal abnormalities in fibroblasts derived from patients with GD. It is noteworthy that the cellular distribution of lysosomes and lysosomal proteolytic activity remained largely unaffected in GD fibroblasts. However, we found that lysosomal membranes of GD fibroblasts were susceptible to damage when exposed to a lysosomotropic agent. Moreover, the susceptibility of lysosomal membranes to a lysosomotropic agent could be partly restored by exogenous expression of wild-type GBA1. Here, we report that the lysosomal membrane integrity is altered in GD fibroblasts, but lysosomal distribution and proteolytic activity is not significantly altered.Key words: glucosylceramide, lysosome, Gaucher disease, lysosomotropic agent.

LRRK2 suppresses lysosome degradative activity in macrophages and microglia through MiT-TFE transcription factor inhibition

Cells maintain optimal levels of lysosome degradative activity to protect against pathogens, clear waste, and generate nutrients. Here, we show that LRRK2, a protein that is tightly linked to Parkinson's disease, negatively regulates lysosome degradative activity in macrophages and microglia via a transcriptional mechanism. Depletion of LRRK2 and inhibition of LRRK2 kinase activity enhanced lysosomal proteolytic activity and increased the expression of multiple lysosomal hydrolases. Conversely, the kinase hyperactive LRRK2 G2019S Parkinson's disease mutant suppressed lysosomal degradative activity and gene expression. We identified MiT-TFE transcription factors (TFE3, TFEB, and MITF) as mediators of LRRK2-dependent control of lysosomal gene expression. LRRK2 negatively regulated the abundance and nuclear localization of these transcription factors and their depletion prevented LRRK2-dependent changes in lysosome protein levels. These observations define a role for LRRK2 in controlling lysosome degradative activity and support a model wherein LRRK2 hyperactivity may increase Parkinson's disease risk by suppressing lysosome degradative activity.

Abstract LB258: Therapeutic advantage of targeting lysosome as signaling hub for metabolic conditions in malignant gliomas

Abstract Lysosome, as the digestive system of the cell, participated in numerous cell biological processes, such as macromolecular degradation, cell adhesion/migration, and apoptosis by regulating metabolic status and pro-growth signaling. Therefore, lysosome could be a promising therapeutic target for cancer therapy, but it remains unclear how lysosome is involved in cancer malignancy. In this study, to investigate the roles of function lysosomes in the regulation of malignant status, we evaluated the proteolytic activity of lysosomes using BODIPY-dye conjugated BSA (DQ-BSA) in malignant gliomas characterized by high aggressivity, destructivity, and invasiveness, glioblastoma multiforme (GBM). While most GBM patient-derived cells showed higher lysosomal proteolytic activity than an immortalized human neural progenitor cell line derived from the embryonic brain, levels of lysosome activity positively co-related with sphere formation. Multi-omics analysis demonstrated that the level of lysosomal proteolytic activity was co-related with metabolic status, including activated mitochondria biogenesis and amino acid transport. The orthotopic xenograft model revealed that lysosome activity in vitro is a critical biomarker representing malignant phenotypes of GBM. We found that the MiT/TFE family, a master regulator of lysosomal biogenesis, controlled the malignant progression of GBM, including therapy resistance, indicating that quality control of lysosomes is a critical determinant for the malignant properties of gliomas. To develop a novel therapeutic approach for targeting lysosomal function, we performed screening of FDA-approved compounds and found that lysosomotropic agents, which induced galectin-3 punctation as an indicator of lysosomal membrane damage, efficiently suppressed tumor growth. Ifenprodil, a clinically available drug that acts as a lysosomotropic agent, increased intracellular Ca2+ levels, resulting in mitochondrial reactive oxygen species-mediated cytotoxicity. Furthermore, we found that the efficacy of ifenprodil was remarkably enhanced by metabolic modifications, such as amino acid restriction, in vitro and in vivo. Collectively, these data indicate that lysosomes as signaling hubs for metabolic conditions play critical roles in the regulation of the malignant status of GBM patients. Our findings propose that lysosomal biogenesis is a promising target to develop a novel therapeutic approach for GBM. Citation Format: Yongwei Jing, Masahiko Kobayashi, Atsushi Hirao. Therapeutic advantage of targeting lysosome as signaling hub for metabolic conditions in malignant gliomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB258.

Preprint Highlight: LRRK2 Suppresses Lysosome Degradative Activity in Macrophages and Microglia via Transcription Factor E3 Inhibition

Mutations in the leucine-rich repeat kinase 2 (LRRK2) that increase kinase activity are associated with some forms of Parkinson's disease (PD). While increased LRRK2 activity is linked to impaired lysosomal function, the underlying molecular mechanisms are incompletely understood. Using mutant mouse models and human induced pluripotent stem cell–derived macrophages and microglia, the authors show an inverse correlation between LRRK2 activity and the mRNA levels of lysosomal hydrolases and cathepsins responsible for lysosomal proteolytic activity. Notably, the abundance and nuclear translocation of the transcription factor TFE3, one of the master lysosomal gene expression regulators, was found to be inhibited by LRRK2. This study reveals a new player that links LRRK2 to lysosomal function and positions the TFE family as a potentially promising target in the pathogenesis of PD. This preprint has been assigned the following badges: New Hypothesis, Cross-Species, New Materials, Cross-Validation. Read the preprint on bioRxiv ( Yadavalli and Ferguson, 2022 ): https://doi.org/10.1101/2022.12.17.520834 .

Exploring lysosomal defects and rescue strategies for Alzheimer’s disease using human transdifferentiated neurons

AbstractBackgroundAging is the critical factor of Alzheimer’s disease (AD), the most common neurodegenerative disease. Among the pathological changes in the brain associated with AD, protein aggregates are the characteristic pathological hallmark that is composed of misfolded and toxic proteins. A decline in lysosomal proteolytic activity has been linked to protein aggregation and neuronal death, whereas how the lysosomes become defective and exacerbate neurodegeneration in the brain of AD patients is not well understood.MethodOne of the hurdles is a lack of human neuron system to acquire deep knowledge of aging and AD. Using a powerful cell reprogramming approach, we developed a new cell model for aging and AD based on the transdifferentiation of human adult fibroblasts into cortical neurons (tNeurons) by bypassing stem‐cell stage. We leveraged cell reprogramming, quantitative proteomics, CRISPR genome editing and drug discovery techniques to achieve a better understanding of how aging and AD affect human neurons, with an emphasis on their impact on the lysosomal pathway. We further used tNeurons as a platform to identify pharmacological candidates that exert neuroprotective effect against lysosomal defects, protein aggregation and neurodegeneration in AD.ResultThe transcription factor‐mediated reprogramming approach generated >85% of tNeurons as cortical glutamatergic neurons after 35‐42 days in culture. The tNeurons retained age‐related changes in epigenetic state, DNA damage and protein homeostasis (proteostasis)(Figure 1). The AD tNeurons exhibited an increased levels of Aβ42 peptides and aberrant autophagosome‐lysosome formation in the soma and neurites. Quantitative proteomics further revealed differential expression of proteins in the endosome‐lysosome and autophagy pathway between aged and AD tNeurons. We performed phenotypic and CRISPR screens to identify pharmacological candidates that ameliorate lysosomal defects and protein aggregation in aged and AD tNeurons.ConclusionThis study is anticipated to highlight that the tNeurons provide a tractable system equipped with a capacity to capture signals of human neuronal aging and neurodegenerative diseases. The outcomes provide novel insights into the role of lysosomes in the aging and neurodegenerative process, and its therapeutic implications for AD.

Abstract 6227: An optimized preclinical antibody-drug conjugate against cancers with cKIT overexpression or activating mutations

Abstract cKIT overexpression and activating mutations have been observed in many types of cancer, including small cell lung cancer (SCLC), gastrointestinal stromal tumor (GIST), and acute myeloid leukemia (AML), and are known to be predictive of poor prognosis. In previously reported work, NN2101-DM1, a fully human, anti-cKIT antibody-drug conjugate (ADC), was developed as a proof-of-concept (PoC) entity by conjugating the site-nonspecific and uncleavable linker SMCC with the microtubule inhibitory payload DM1. This linker-payload system is the same used in ado-trastuzumab emtansine, an approved ADC for HER2-positive breast cancer. NN2101-DM1 showed therapeutic efficacy against SCLC and GIST both in vitro and in xenograft models in vivo, demonstrating the feasibility of ADC development using NN2101. Despite the potent efficacy of NN2101-DM1, there are inherent limitations to SMCC linker and DM1 payload systems. The nonspecific attachment of the linker to the antibody produces highly heterogeneous drug-antibody ratios (DARs) with unpredictable attachment. Additionally, impaired lysosomal proteolytic activity has been shown to contribute to resistance to SMCC-DM1 cleavage. To optimize the ADC configuration for advancement to a clinical candidate, four types of ADC with different linker-payload combinations and DARs were designed, and their efficacy and safety were evaluated in vitro and in vivo. Among them, NN3201, a conjugation of the antimitotic payload MMAE to the cKIT-targeting antibody NN2101 via a site-specific, cleavable peptide linker, with a homogenous (>95%) DAR of 4 was selected as a lead candidate. This linker system utilizes a cysteine re-bridging technology to produce site-specific conjugation with high DAR homogeneity. NN3201 showed consistent sub-nanomolar IC50s in several in vitro cell lines and demonstrated complete remission as monotherapy in SCLC (H526), GIST (GIST-T1), AML (Kasumi-1), and mastocytoma (HMC 1.2) cell-derived xenograft models in doses ranging from 1.0-3.0 mg/kg. There were no significant toxicities detected at doses up to 60 mg/kg in normal mice, suggesting a therapeutic index greater than 20 times. In conclusion, NN3201 showed potent anti-tumor activity and was selected as preclinical candidate. Citation Format: Jin-Ock Kim, Han-Jik Ko, Jae-Won Kim, Sang Gyu Park. An optimized preclinical antibody-drug conjugate against cancers with cKIT overexpression or activating mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6227.

Urocortin 2 promotes hypertrophy and enhances skeletal muscle function through cAMP and insulin/IGF-1 signaling pathways

ObjectiveAlthough it is well established that urocortin 2 (Ucn2), a peptide member of the corticotrophin releasing factor (CRF) family, and its specific corticotrophin-releasing factor 2 receptor (CRF2R) are highly expressed in skeletal muscle, the role of this peptide in the regulation of skeletal muscle mass and protein metabolism remains elusive. MethodsTo elucidate the mechanisms how Ucn2 directly controls protein metabolism in skeletal muscles of normal mice, we carried out genetic tools, physiological and molecular analyses of muscles in vivo and in vitro. ResultsHere, we demonstrated that Ucn2 overexpression activated cAMP signaling and promoted an expressive muscle hypertrophy associated with higher rates of protein synthesis and activation of Akt/mTOR and ERK1/2 signaling pathways. Furthermore, Ucn2 induced a decrease in mRNA levels of atrogin-1 and in autophagic flux inferred by an increase in the protein content of LC3-I, LC3-II and p62. Accordingly, Ucn2 reduced both the transcriptional activity of FoxO in vivo and the overall protein degradation in vitro through an inhibition of lysosomal proteolytic activity. In addition, we demonstrated that Ucn2 induced a fast-to-slow fiber type shift and improved fatigue muscle resistance, an effect that was completely blocked in muscles co-transfected with mitogen-activated protein kinase phosphatase 1 (MKP-1), but not with dominant-negative Akt mutant (Aktmt). ConclusionsThese data suggest that Ucn2 triggers an anabolic and anti-catabolic response in skeletal muscle of normal mice probably through the activation of cAMP cascade and participation of Akt and ERK1/2 signaling. These findings open new perspectives in the development of therapeutic strategies to cope with the loss of muscle mass.

Imatinib disturbs lysosomal function and morphology and impairs the activity of mTORC1 in human hepatocyte cell lines

The tyrosine kinase inhibitors (TKIs) imatinib and lapatinib are associated with severe hepatotoxicity, whose mechanisms are currently under investigation. As amphiphilic drugs, imatinib and lapatinib enrich in lysosomes. In the present study, we investigated their effects on lysosomal morphology and function in HepG2 and HuH-7 cells and explored possible links between lysosomal dysfunction and hepatotoxicity. Both TKIs increased the lysosomal volume time and concentration-dependently in HepG2 and HuH-7 cells. In HepG2 cells, lapatinib and imatinib raised the lysosomal pH and destabilized the lysosomal membrane, thereby impairing lysosomal proteolytic activity such as cathepsin B processing. Imatinib activated the transcription factor EB (TFEB), a regulator of lysosomal biogenesis and function, as demonstrated by nuclear TFEB accumulation and increased expression of TFEB-target genes. Because of lysosomal dysfunction, imatinib impaired mTORC1 activation, a protein complex activated on the lysosomal surface, which explained TFEB activation. HepG2 cells treated with imatinib showed increased levels of MAP1LC3A/B-II and of ATG13 (S318) phosphorylation, indicating induction of autophagy due to TFEB activation. Finally, imatinib induced apoptosis in HepG2 cells in a time and concentration-dependent manner, explained by lysosomal and mitochondrial toxicity. Our findings provide a new lysosome-centered mechanism for imatinib-induced hepatotoxicity that could be extended to other lysosomotropic drugs.

Autophagy of mucin granules contributes to resolution of airway mucous metaplasia

Exacerbations of muco-obstructive airway diseases such as COPD and asthma are associated with epithelial changes termed mucous metaplasia (MM). Many molecular pathways triggering MM have been identified; however, the factors that regulate resolution are less well understood. We hypothesized that the autophagy pathway is required for resolution of MM by eliminating excess non-secreted intracellular mucin granules. We found increased intracellular levels of mucins Muc5ac and Muc5b in mice deficient in autophagy regulatory protein, Atg16L1, and that this difference was not due to defects in the known baseline or stimulated mucin secretion pathways. Instead, we found that, in mucous secretory cells, Lc3/Lamp1 vesicles colocalized with mucin granules particularly adjacent to the nucleus, suggesting that some granules were being eliminated in the autophagy pathway rather than secreted. Using a mouse model of MM resolution, we found increased lysosomal proteolytic activity that peaked in the days after mucin production began to decline. In purified lysosomal fractions, Atg16L1-deficient mice had reduced proteolytic degradation of Lc3 and Sqstm1 and persistent accumulation of mucin granules associated with impaired resolution of mucous metaplasia. In normal and COPD derived human airway epithelial cells (AECs), activation of autophagy by mTOR inhibition led to a reduction of intracellular mucin granules in AECs. Our findings indicate that during peak and resolution phases of MM, autophagy activity rather than secretion is required for elimination of some remaining mucin granules. Manipulation of autophagy activation offers a therapeutic target to speed resolution of MM in airway disease exacerbations.

Polyethylenimine-Modified Mesoporous Silica Nanoparticles Induce a Survival Mechanism in Vascular Endothelial Cells via Microvesicle-Mediated Autophagosome Release.

Surface-modified mesoporous silica nanoparticles (MSNs) have attracted more and more attention as promising materials for biomolecule delivery. However, the lack of detailed evaluation relevant to the potential cytotoxicity of these MSNs is still a major obstacle for their applications. Unlike the bare MSNs and amino- or liposome-modified MSNs, we found that polyethylenimine-modified MSNs (MSNs-PEI) had no obvious toxicity to human umbilical vein endothelial cells (HUVECs) at the concentrations up to 100 μg/mL. However, MSNs-PEI induced autophagosomes accumulation by blocking their fusion with lysosomes, an essential mechanism for the cytotoxicity of many nanoparticles (NPs). Thus, we predicted that an alternative pathway for autophagosome clearance exists in HUVECs to relieve autophagic stress induced by MSNs-PEI. We found that MSNs-PEI prevented STX17 loading onto autophagosomes instead of influencing lysosomal pH or proteolytic activity. MSNs-PEI induced the structural alternation of the cytoskeleton but did not cause endoplasmic reticulum stress. The accumulated autophagosomes were released to the extracellular space via microvesicles (MVs) when the autophagic degradation was blocked by MSNs-PEI. More importantly, blockade of either autophagosome formation or release caused the accumulation of damaged mitochondria and excessive ROS production in the MSNs-PEI-treated HUVECs, which in turn led to cell death. Thus, we propose here that the MV-mediated autophagosome release, a compensation mechanism, allows the vascular endothelial cell survival when the degradation of autophagosomes is blocked by MSNs-PEI. Accordingly, promoting the release of accumulated autophagosomes may be a protective strategy against the endothelial toxicity of NPs.

The role of mitophagy in the regulation of mitochondrial energetic status in neurons

ABSTRACT Mitochondria are the main cellular energy powerhouses and supply most of the energy in the form of ATP to fuel essential neuronal functions through oxidative phosphorylation (OXPHOS). In Alzheimer disease (AD), metabolic and mitochondrial disruptions are an early feature preceding any histopathological and clinical manifestations. Mitochondrial malfunction is also linked to synaptic defects in early AD. Mitophagy serves as a key cellular quality control mechanism involving sequestration of damaged mitochondria within autophagosomes and their subsequent degradation in lysosomes. However, it remains largely unknown whether mitophagy is involved in the regulation of energy metabolism in neurons, and if so, whether metabolic deficiency in AD is attributed to mitophagy dysfunction. Here we reveal that mitophagy is broadly activated in metabolically enhanced neurons upon OXPHOS stimulation, which sustains high energetic activity by increasing mitochondrial turnover and hence facilitating mitochondrial maintenance. Unexpectedly, in AD-related mutant HsAPP Tg mouse brains, early stimulation of OXPHOS activity fails to correct energy deficits but exacerbates synapse loss as a consequence of mitophagy failure. Excitingly, lysosomal enhancement in AD neurons restores impaired metabolic function by promoting elimination of damaged mitochondria, protecting against synaptic damage in AD mouse brains. Taken together, we propose a new mechanism by which mitophagy controls bioenergetic status in neurons, furthering our understanding of the direct impact of mitophagy defects on AD-linked metabolic deficits and shedding light on the development of novel therapeutic strategies to treat AD by the early stimulation of mitochondrial metabolism combined with elevation of lysosomal proteolytic activity. Abbreviations: AD: Alzheimer disease; Aβ: amyloid-β; APP: amyloid beta precursor protein; AV: autophagic vacuole; CHX: cycloheximide; CYCS: cytochrome c, somatic; DIV: days in vitro; FRET: Förster resonance energy transfer; Gln, glutamine; LAMP1: lysosomal associated membrane protein 1; LE: late endosome; Mito: mitochondria; Δψm: mitochondrial membrane potential; OCR: oxygen consumption rate; OXPHOS: oxidative phosphorylation; SQSTM1/p62: sequestosome 1; RHEB: Ras homolog, mTORC1 binding; ROS: reactive oxygen species; STX1: syntaxin 1; SYP: synaptophysin; Tg: transgenic; TMRE: tetramethylrhodamine ethyl ester; TEM: transmission electron microscopy; WT: wild type

Cobalt oxide nanoparticle-synergized protein degradation and phototherapy for enhanced anticancer therapeutics

How to enable protein degradation pathways including the autophagy-lysosome pathway (ALP) and the ubiquitin-proteasome system (UPS) to enhance the efficacy of anticancer treatments remains a substantial challenge. Cobalt oxide nanoparticles (Co3O4 NPs) have attracted interest in recent years for their potential use as a synergistic anticancer treatment, although their therapeutic mechanisms of action are still poorly understood. Here, we describe the synergistic use of Co3O4 NPs as an autophagy inhibitor, chemosensitizer and photosensitizer, which manipulate protein degradation pathways (ALP and UPS) and photothermal therapy for enhanced anticancer treatments both in vitro and in vivo. We show that Co3O4 NPs can induce autolysosome accumulation and lysosomal functions damage by inhibiting lysosomal proteolytic activity and reducing intracellular ATP levels. Notably, Co3O4 NPs can be combined with the proteasome inhibitor, Carfilzomib (Cfz), to promote the accumulation of autophagic substrates, protein ubiquitination, and endoplasmic reticulum stress, and in doing so, inhibit cancer progression. By taking advantage of their photothermal conversion efficiency, Co3O4 NPs can also serve as photothermal sensitizer, which synergistically enhances the anticancer efficacy of Cfz both in vitro and in vivo. In summary, we provide evidence of a nanomaterial-synergized, photothermal anticancer strategy that synergistically targets cancer cell survival pathways and may eventually serve to enhance the anticancer efficacy of established cancer therapeutics.

Cobalt Oxide Nanoparticle-Synergized Strategy Manipulating Autophagy, Ubiquitin-Proteasome and Photothermal Therapy for Enhanced Anticancer Therapeutics

How to effectively enable both protein degradation pathways, which include autophagy-lysosome pathway (ALP) and the ubiquitin-proteasome system (UPS), with photothermal therapy synergistically for enhanced anticancer treatment in vivo remains a big challenge. Cobalt oxide nanoparticles (Co3O4 NPs) have attracted great interests for their biological application, including potential use for anticancer treatment although their exact mechanisms are still poorly understood. Here, we firstly report on the synergistic use of Co3O4 NPs as autophagy inhibitor, chemosensitizer and photosensitizer to manipulate protein degradation pathways (ALP and UPS) and photothermal therapy for enhanced anticancer treatments both in vitro and in vivo . Co3O4 NPs are found to block autophagic flux to induce autophagosome accumulation and lysosomal functions damage by inhibiting the lysosomal proteolytic activity and reducing the intracellular ATP level. Notably, Co3O4 NPs can further sensitize proteasome inhibitor Carfilzomib (Cfz) to promote autophagic substrates and ubiquitinated protein accumulation with increased endoplasmic reticulum stress for enhanced cancer inhibition. More importantly, taking advantages of the excellent photothermal conversion efficiency, Co3O4 NPs can further serve as photothermal sensitizer for photothermal therapy, which synergistically enhanced the anticancer efficacy of Cfz both in vitro and in vivo . This novel nanomaterial-synergized anticancer strategy synergistically manipulating autophagy, ubiquitin-proteasome system and photothermal therapy may potentially serve in more effective therapeutics against cancer.

The Impact of Modern Therapeutic Methods on the Oxidant-Antioxidant Equilibrium and Activities of Selected Lysosomal Enzymes and Serine Protease Inhibitor in Amateur Athletes.

Deep electromagnetic stimulation (DEMS) and low-frequency ultrasound (US) are new physical therapy methods used in the rehabilitation of the musculoskeletal system and wound healing. They are applied locally to treat the injured tissues. The beneficial effects of these methods in supportive care have been documented, but accurate biochemical effects are not known. The goal was to assess the effect of single DEMS and US sessions on the oxidant-antioxidant equilibrium, as well as the activities of lysosomal hydrolases and α1-antitrypsin (AAT) in peripheral blood of juvenile injured amateur athletes. In the athletes with low back pain (DEMS treated, N = 16) and pain in the shoulder or ankle joint (US treated, N = 14), as well as in healthy control amateur athletes (DEMS treated, N = 14; US treated, N = 17), before the sessions and 30 minutes and 24 hours after them, the levels of the following parameters were determined: thiobarbituric acid reactive substances (TBARS) in erythrocytes and plasma, superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) in erythrocytes, as well as acid phosphatase (AcP), arylsulfatase (ASA), cathepsin D (CTS D), and α1-antitrypsin (AAT) in serum. After both procedures, the levels of parameters changed in a negligible manner, excluding the cathepsin D activity, which was statistically significantly lower 30 min and 24 h after US in the control athletes compared to the baseline activity determined directly before the procedure (47.5% and 55.7% differences, respectively). Similar tendency was observed after DEMS (p > 0.05). The procedures, especially low-frequency US, decrease lysosomal proteolytic activity and do not significantly disrupt the oxidant-antioxidant and lysosomal equilibriums in the peripheral blood both of healthy and injured athletes. No systemic acute-phase response of AAT was also detected in the athletes after both procedures. This trial is registered with CTRI/2018/01/011344.

Withaferin A inhibits lysosomal activity to block autophagic flux and induces apoptosis via energetic impairment in breast cancer cells.

Withaferin A (WFA), a steroidal lactone, negatively regulates breast cancer growth however, its mechanisms of action remain largely elusive. We found that WFA blocks autophagy flux and lysosomal proteolytic activity in breast cancer cells. WFA increases accumulation of autophagosomes, LC3B-II conversion, expression of autophagy-related proteins and autophagosome/lysosome fusion. Autolysosomes display the characteristics of acidic compartments in WFA-treated cells; however, the protein degradation activity of lysosomes is inhibited. Blockade of autophagic flux reduces the recycling of cellular fuels leading to insufficient substrates for tricarboxylic acid (TCA) cycle and impaired oxidative phosphorylation. WFA decreases expression and phosphorylation of lactate dehydrogenase, the key enzyme that catalyzes pyruvate-to-lactate conversion, reduces adenosine triphosphate levels and increases AMP-activated protein kinase (AMPK) activation. AMPK inhibition abrogates while AMPK activation potentiates WFA's effect. WFA and 2-deoxy-d-glucose combination elicits synergistic inhibition of breast cancer cells. Genetic knockout of BECN1 and ATG7 fails to rescue cells from WFA treatment; in contrast, addition of methyl pyruvate to supplement TCA cycle protects WFA-treated cells. Together, these results implicate that WFA is a potent lysosomal inhibitor; energetic impairment is required for WFA-induced apoptosis and growth inhibition and combining WFA and 2-DG is a promising therapeutic strategy for breast cancer.

OCRL deficiency impairs endolysosomal function in a humanized mouse model for Lowe syndrome and Dent disease.

Mutations in OCRL encoding the inositol polyphosphate 5-phosphatase OCRL (Lowe oculocerebrorenal syndrome protein) disrupt phosphoinositide homeostasis along the endolysosomal pathway causing dysfunction of the cells lining the kidney proximal tubule (PT). The dysfunction can be isolated (Dent disease 2) or associated with congenital cataracts, central hypotonia and intellectual disability (Lowe syndrome). The mechanistic understanding of Dent disease 2/Lowe syndrome remains scarce due to limitations of animal models of OCRL deficiency. Here, we investigate the role of OCRL in Dent disease 2/Lowe syndrome by using OcrlY/− mice, where the lethal deletion of the paralogue Inpp5b was rescued by human INPP5B insertion, and primary culture of proximal tubule cells (mPTCs) derived from OcrlY/− kidneys. The OcrlY/− mice show muscular defects with dysfunctional locomotricity and present massive urinary losses of low-molecular-weight proteins and albumin, caused by selective impairment of receptor-mediated endocytosis in PT cells. The latter was due to accumulation of phosphatidylinositol 4,5–bisphosphate PI(4,5)P2 in endolysosomes, driving local hyper-polymerization of F-actin and impairing trafficking of the endocytic LRP2 receptor, as evidenced in OcrlY/− mPTCs. The OCRL deficiency was also associated with a disruption of the lysosomal dynamic and proteolytic activity. Partial convergence of disease-pathways and renal phenotypes observed in OcrlY/− and Clcn5Y/− mice suggest shared mechanisms in Dent diseases 1 and 2.These studies substantiate the first mouse model of Lowe syndrome and give insights into the role of OCRL in cellular trafficking of multiligand receptors. These insights open new avenues for therapeutic interventions in Lowe syndrome and Dent disease.

Reprogramming Tumor-Associated Macrophages by Nanoparticle-Based Reactive Oxygen Species Photogeneration.

Without coordinated strategies to mitigate the immunosuppressive nature of the tumor microenvironment, cancer immunotherapy generally offers limited clinical benefit for established tumors. Tumor-associated macrophages (TAMs) are the critical driver of this immunosuppressive tumor microenvironment, which also promotes tumor metastasis. Here we successfully reprogrammed TAMs to an antitumor M1 phenotype using precision nanoparticle-based reactive oxygen species photogeneration, which demonstrated superior efficiency and efficacy over lipopolysaccharide stimulation. Meanwhile, antigen presentation and T-cell-priming by TAMs were enhanced by inhibiting lysosomal proton pump and proteolytic activity or by promoting tumor associated antigen release in the cytoplasm. The reprogrammed TAMs orchestrate cytotoxic lymphocyte (CTL) recruitment in the tumor and direct memory T-cells toward tumoricidal responses. This strategy could effectively eradicate tumors, inhibit metastasis, and further prevent their recurrence, which holds tremendous promise to realize potent cancer immunotherapy.

Intracellular Delivery of Rapamycin From FKBP Elastin-Like Polypeptides Is Consistent With Macropinocytosis.

Rapamycin (Rapa) is a highly potent drug; however, its clinical potential is limited by poor solubility, bioavailability, and cytotoxicity. To improve Rapa delivery, our team has fused the cognate protein receptor for Rapa, FKBP12, to high molecular weight elastin-like polypeptides (ELPs). One construct, FAF, includes an FKBP domain at each termini of an ELP. In a recent report, FAF/Rapa outperformed a family of related carriers with higher tumor accumulation and efficacy. Despite apparent efficacy, an explanation for how FAF carries Rapa into cells has not been elucidated. This manuscript explores the intracellular fate of FAF in MDA-MB-468, a triple negative (ER-/PR-/HER2-) breast cancer line. Based on a lack of displacement by excess unlabeled FAF, no evidence was found for the involvement of a receptor in cell-surface binding. Cellular association showed no dose-dependent saturation at concentrations up to 100 μM, which is consistent with uptake through fluid phase endocytosis. FAF does colocalize with dextran, a marker of fluid phase endocytosis. Upon internalization, both FAF and dextran target low pH intracellular compartments similarly. Despite likely exposure to lysosomal pH and proteolytic activity, intracellular FAF is eliminated from cells with a relatively long half-life of 17.7 and 19.0 h by confocal microscopy and SDS-PAGE respectively. A split luciferase reporter assay demonstrated that FAF delays the cytosolic access of Rapa in comparison to free drug by 30 min. A specific macropinocytosis inhibitor, amiloride, completely inhibits the cytosolic delivery of Rapa from FAF. Each of these results are consistent with macropinocytosis as the mechanism of cellular uptake necessary for the hand-off of Rapa from FKBP-based drug carriers like FAF to endogenous FKBP12 in the cytosol.