RESUMEN

Rationale: Neovascular ocular diseases (NODs) represent the leading cause of visual impairment globally. Despite significant advances in anti-angiogenic therapies targeting vascular endothelial growth factor (VEGF), persistent challenges remain prevalent. As a proof-of-concept study, we herein demonstrate the effectiveness of targeted degradation of VEGF with bispecific aptamer-based lysosome-targeting chimeras (referred to as VED-LYTACs). Methods: VED-LYTACs were constructed with three distinct modules: a mannose-6-phosphate receptor (M6PR)-binding motif containing an M6PR aptamer, a VEGF-binding module with an aptamer targeting VEGF, and a linker essential for bridging and stabilizing the two-aptamer structure. The degradation efficiency of VED-LYTACs via the autophagy-lysosome system was examined using an enzyme-linked immunosorbent assay (ELISA) and immunofluorescence staining. Subsequently, the anti-angiogenic effects of VED-LYTACs were evaluated using in vitro wound healing assay, tube formation assay, three-dimensional sprouting assay, and ex vivo aortic ring sprouting assay. Finally, the potential therapeutic effects of VED-LYTACs on pathological retinal neovascularization and vascular leakage were tested by employing mouse models of NODs. Results: The engineered VED-LYTACs promote the interaction between M6PR and VEGF, consequently facilitating the translocation and degradation of VEGF through the lysosome. Our data show that treatment with VED-LYTACs significantly suppresses VEGF-induced angiogenic activities both in vitro and ex vivo. In addition, intravitreal injection of VED-LYTACs remarkably ameliorates abnormal vascular proliferation and leakage in mouse models of NODs. Conclusion: Our findings present a novel strategy for targeting VEGF degradation with an aptamer-based LYTAC system, effectively ameliorating pathological retinal angiogenesis. These results suggest that VED-LYTACs have potential as therapeutic agents for managing NODs.

Asunto(s)

Aptámeros de Nucleótidos , Lisosomas , Neovascularización Retiniana , Factor A de Crecimiento Endotelial Vascular , Animales , Aptámeros de Nucleótidos/farmacología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Ratones , Neovascularización Retiniana/tratamiento farmacológico , Neovascularización Retiniana/metabolismo , Humanos , Lisosomas/metabolismo , Lisosomas/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana , Ratones Endogámicos C57BL , Modelos Animales de Enfermedad , Inhibidores de la Angiogénesis/farmacología , Angiogénesis

RESUMEN

Influenza virus-induced viral pneumonia is a major threat to human health, and specific therapeutic agents for viral pneumonia are still lacking. MoringaA (MA) is an anti-influenza virus active compound isolated from Moringa seeds, which can inhibit influenza virus by activating the TFEB-autophagic lysosomal pathway in host cells. In this study, we obtained exosomes from M2-type macrophages and encapsulated and delivered MA (MA-Exos), and we investigated the efficacy of MA-Exos in antiviral and viral pneumonia in vivo and in vitro, respectively. In addition, we provided insights into the mechanism by which MA-Exos regulates TFEB-lysosomal autophagy by RNA sequencing. The MA-Exos showed broad-spectrum inhibition of IAV, and significant promotion of the autophagic lysosomal pathway. Meanwhile, we found that GCN5 gene and protein were significantly down-regulated in IAV-infected cells after MA-Exos intervention, indicating its blocking the acetylation of TFEB by GCN5. In addition, MA-Exos also significantly promoted autophagy in lung tissue cells of mice with viral pneumonia. MA-Exos can inhibit and clear influenza virus by mediating the TFEB-autophagy lysosomal pathway by a mechanism related to the down-regulation of histone acetyltransferase GCN5. Our study provides a strategy for targeting MA-Exos for the treatment of viral pneumonia from both antiviral and virus-induced inflammation inhibition pathways.

Asunto(s)

Antivirales , Autofagia , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Exosomas , Virus de la Influenza A , Lisosomas , Animales , Ratones , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Lisosomas/metabolismo , Lisosomas/efectos de los fármacos , Lisosomas/virología , Exosomas/metabolismo , Antivirales/farmacología , Autofagia/efectos de los fármacos , Humanos , Virus de la Influenza A/efectos de los fármacos , Virus de la Influenza A/fisiología , Infecciones por Orthomyxoviridae/virología , Infecciones por Orthomyxoviridae/tratamiento farmacológico , Macrófagos/virología , Macrófagos/efectos de los fármacos , Pulmón/virología

RESUMEN

Cytopathology induced by methamphetamine (METH) is reminiscent of degenerative disorders such as Parkinson's disease, and it is characterized by membrane organelles arranged in tubulo-vesicular structures. These areas, appearing as clusters of vesicles, have never been defined concerning the presence of specific organelles. Therefore, the present study aimed to identify the relative and absolute area of specific membrane-bound organelles following a moderate dose (100 µM) of METH administered to catecholamine-containing PC12 cells. Organelles and antigens were detected by immunofluorescence, and they were further quantified by plain electron microscopy and in situ stoichiometry. This analysis indicated an increase in autophagosomes and damaged mitochondria along with a decrease in lysosomes and healthy mitochondria. Following METH, a severe dissipation of hallmark proteins from their own vesicles was measured. In fact, the amounts of LC3 and p62 were reduced within autophagy vacuoles compared with the whole cytosol. Similarly, LAMP1 and Cathepsin-D within lysosomes were reduced. These findings suggest a loss of compartmentalization and confirm a decrease in the competence of cell clearing organelles during catecholamine degeneration. Such cell entropy is consistent with a loss of energy stores, which routinely govern appropriate subcellular compartmentalization.

Asunto(s)

Autofagosomas , Lisosomas , Metanfetamina , Metanfetamina/farmacología , Animales , Células PC12 , Ratas , Lisosomas/metabolismo , Lisosomas/efectos de los fármacos , Autofagosomas/metabolismo , Autofagosomas/efectos de los fármacos , Autofagia/efectos de los fármacos , Mitocondrias/metabolismo , Mitocondrias/efectos de los fármacos , Catepsina D/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo

RESUMEN

Ferroptosis is a promising strategy for cancer therapy, with numerous inhibitors of its braking axes under investigation as potential drugs. However, few studies have explored the potential of activating the driving axes to induce ferroptosis. Herein, phosphatidylcholine peroxide decorating liposomes (LIPPCPO) are synthesized to induce ferroptosis by targeting divalent metal transporter 1 (DMT1). LIPPCPO is found to boost lysosomal Fe2+ efflux by inducing cysteinylation of lysosomal DMT1, resulting in glutathione peroxidase 4 (GPX4) suppression, glutathione depletion and ferroptosis in breast cancer cells and xenografts. Importantly, LIPPCPO induced ferroptotic cell death is independent of acquired resistance to radiation, chemotherapy, or targeted agents in 11 cancer cell lines. Furthermore, a strong synergistic ferroptosis effect is observed between LIPPCPO and an FDA-approved drug, artesunate, as well as X rays. The formula of LIPPCPO encapsulating artesunate significantly inhibits tumor growth and metastasis and improves the survival rate of breast cancer-bearing female mice. These findings provide a distinct strategy for inducing ferroptosis and highlight the potential of LIPPCPO as a vector to synergize the therapeutic effects of conventional ferroptosis inducers.

Asunto(s)

Neoplasias de la Mama , Ferroptosis , Liposomas , Fosfolípido Hidroperóxido Glutatión Peroxidasa , Ferroptosis/efectos de los fármacos , Animales , Humanos , Femenino , Línea Celular Tumoral , Ratones , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Neoplasias de la Mama/genética , Neoplasias de la Mama/tratamiento farmacológico , Liposomas/metabolismo , Fosfolípido Hidroperóxido Glutatión Peroxidasa/metabolismo , Fosfolípido Hidroperóxido Glutatión Peroxidasa/genética , Artesunato/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto , Fosfatidilcolinas/metabolismo , Fosfatidilcolinas/química , Hierro/metabolismo , Lisosomas/metabolismo , Lisosomas/efectos de los fármacos , Ratones Desnudos , Glutatión/metabolismo , Ratones Endogámicos BALB C

RESUMEN

Metformin is a widely employed drug in type 2 diabetes. In addition to warranting good short- and long-term glycemic control, metformin displays many intriguing properties as protection against cardiovascular and neurodegenerative diseases, anti-tumorigenic and longevity promotion. In addition to being a low-cost drug, metformin is generally well tolerated. However, despite the enthusiastic drive to aliment these novel studies, many contradictory results suggest the importance of better elucidating the complexity of metformin action in different tissues/cells to establish its possible employment in neurodegenerative diseases. This review summarises recent data identifying lysosomal-dependent processes and lysosomal targets, such as endosomal Na+/H+ exchangers, presenilin enhancer 2 (PEN2), the lysosomal pathway leading to AMP-activated protein kinase (AMPK) activation, and the transcription factor EB (TFEB), modulated by metformin. Lysosomal dysfunctions resulting in autophagic and lysosomal acidification and biogenesis impairment appear to be hallmarks of many inherited and acquired neurodegenerative diseases. Lysosomes are not yet seen as a sort of cellular dump but are crucial in determining key signalling paths and processes involved in the clearance of aggregated proteins. Thus, the possibility of pharmacologically modulating them deserves great interest. Despite the potentiality of metformin in this context, many additional important issues, such as dosing, should be addressed in the future.

Asunto(s)

Reposicionamiento de Medicamentos , Lisosomas , Metformina , Enfermedades Neurodegenerativas , Metformina/farmacología , Metformina/uso terapéutico , Humanos , Lisosomas/metabolismo , Lisosomas/efectos de los fármacos , Enfermedades Neurodegenerativas/tratamiento farmacológico , Enfermedades Neurodegenerativas/metabolismo , Reposicionamiento de Medicamentos/métodos , Animales , Hipoglucemiantes/farmacología , Hipoglucemiantes/uso terapéutico , Autofagia/efectos de los fármacos

RESUMEN

Specific cancer therapy remains a problem to be solved. Breast and colorectal cancer are among the cancers with the highest prevalence and mortality rates. Although there are some therapeutic options, there are still few effective agents for those cancers, which constitutes a clinical problem that requires further research efforts. Lysosomes play an important role in cancer cells' survival, and targeting lysosomes has gained increased interest. In recent years, our team has been synthetizing and testing novel benzo[a]phenoxazine derivatives, as they have been shown to possess potent pharmacological activities. Here, we investigated the anticancer activity of three of the most potent derivatives from our library, C9, A36, and A42, on colorectal- and breast-cancer-derived cell lines, and compared this with the effect on non-neoplastic cell lines. We observed that the three compounds were selective for the cancer cells, namely the RKO colorectal cancer cell line and the MCF7 breast cancer cell line. In both models, the compounds reduced cell proliferation, cell survival, and cell migration, accumulated on the lysosome, and induced cell death accompanied by lysosomal membrane permeabilization (LMP), increasing the intracellular pH and ROS accumulation. Our results demonstrated that these compounds specifically target lysosomes from cancer cells, making them promising candidates as LMP inducers for cancer therapy.

Asunto(s)

Antineoplásicos , Proliferación Celular , Lisosomas , Oxazinas , Humanos , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Oxazinas/farmacología , Oxazinas/uso terapéutico , Antineoplásicos/farmacología , Proliferación Celular/efectos de los fármacos , Línea Celular Tumoral , Especies Reactivas de Oxígeno/metabolismo , Células MCF-7 , Supervivencia Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos

RESUMEN

Lysosomal degradation pathways coordinate the clearance of superfluous and damaged cellular components. Compromised lysosomal degradation is a hallmark of many degenerative diseases, including lysosomal storage diseases (LSDs), which are caused by loss-of-function mutations within both alleles of a lysosomal hydrolase, leading to lysosomal substrate accumulation. Gaucher's disease, characterized by <15% of normal glucocerebrosidase function, is the most common LSD and is a prominent risk factor for developing Parkinson's disease. Here, we show that either of two structurally distinct small molecules that modulate PIKfyve activity, identified in a high-throughput cellular lipid droplet clearance screen, can improve glucocerebrosidase function in Gaucher patient-derived fibroblasts through an MiT/TFE transcription factor that promotes lysosomal gene translation. An integrated stress response (ISR) antagonist used in combination with a PIKfyve modulator further improves cellular glucocerebrosidase activity, likely because ISR signaling appears to also be slightly activated by treatment by either small molecule at the higher doses employed. This strategy of combining a PIKfyve modulator with an ISR inhibitor improves mutant lysosomal hydrolase function in cellular models of additional LSD.

Asunto(s)

Fibroblastos , Glucosilceramidasa , Enfermedades por Almacenamiento Lisosomal , Lisosomas , Fosfatidilinositol 3-Quinasas , Humanos , Fosfatidilinositol 3-Quinasas/metabolismo , Lisosomas/metabolismo , Lisosomas/efectos de los fármacos , Glucosilceramidasa/metabolismo , Glucosilceramidasa/genética , Fibroblastos/metabolismo , Fibroblastos/efectos de los fármacos , Enfermedades por Almacenamiento Lisosomal/tratamiento farmacológico , Enfermedades por Almacenamiento Lisosomal/genética , Enfermedades por Almacenamiento Lisosomal/metabolismo , Enfermedad de Gaucher/tratamiento farmacológico , Enfermedad de Gaucher/genética , Enfermedad de Gaucher/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3/farmacología

RESUMEN

BACKGROUND: Non-small cell lung cancer (NSCLC) accounts for 85 % of lung cancer, becoming the most mortality of all cancers globally. Blockage of autophagy in NSCLC represents a promising therapeutic strategy that inhibits angiogenesis and overcomes drug resistance. Natural ingredients in anti-tumor adjuvants are increasingly reported to promote cell death with less side effects and the potential to increase chemotherapeutic drugs sensitivity. Baicalin, a Scutellaria baicalensis-extracted flavonoid glycoside, is reported to induce death of NSCLC cells, however, its effects on autophagy in NSCLC cells remain unclear. PURPOSE: This study aimed to investigate the effect of baicalin on autophagic flux in NSCLC cells, unraveling the underlying mechanism including potential target and its role in cell death of NSCLC cells. METHODS: In vitro anti-cancer effects of baicalin were verified by evaluating proliferation, clone formation, cell cycle, and cell migration in three NSCLC cell lines (A549, H1299, and PC-9). In vivo anti-tumor efficacies of baicalin were evaluated in subcutaneous xenograft tumor model in nude mice. Autophagy characterization in NSCLC cells included autophagic marker detection by western blot and immunofluorescence staining, subcellular structure observation by TEM, lysosomal function by RNA-seq and fluorescence staining (LysoTracker®, LysoSensor®, and acridine orange). Based on RNA-seq and molecular biological verification using apoptotic, autophagic, and lysosomal inhibitors, potential target molecule of baicalin was verified via Ca2+ flux assay, MCOLN3 knockdown by shRNA, and virtual molecular docking. RESULTS: Baicalin inhibited NSCLC cell proliferation and migration, and suppressed tumor growth in vivo. Baicalin blocked the autophagic flux via activating the membranal cation channel MCOLN3 of lysosome, which disrupted its Ca2+ balance and induced lysosome dysfunction, leading to failure of autolysosome degradation. The cytoplasmic Ca2+ imbalance further resulted in depolarization of mitochondrial membrane potentials and ROS accumulation in NSCLC cells, mediating autophagy-related apoptosis. CONCLUSION: This study demonstrated that baicalin inhibited autolysosome degradation by activating MCOLN3, leading to dysfunction in lysosomal pH elevation, thereby inhibiting autophagy in NSCLC, leading to apoptotic death of NSCLC cells. These findings enriched the existing theories of cancer therapy based on autophagy inhibition and underlying mechanisms of flavonoids as antitumor agents, paving the way for their clinical application in future.

Asunto(s)

Autofagia , Carcinoma de Pulmón de Células no Pequeñas , Flavonoides , Neoplasias Pulmonares , Lisosomas , Ratones Desnudos , Flavonoides/farmacología , Humanos , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Neoplasias Pulmonares/tratamiento farmacológico , Animales , Autofagia/efectos de los fármacos , Línea Celular Tumoral , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Ratones , Antineoplásicos Fitogénicos/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto , Células A549 , Scutellaria baicalensis/química , Proliferación Celular/efectos de los fármacos , Ratones Endogámicos BALB C , Movimiento Celular/efectos de los fármacos

RESUMEN

Reactive oxygen species (ROS) hold great potential in tumor pyroptosis therapy, yet they are still limited by short species lifespan and limited diffusion distance. Inducing cells into a metastable state and then applying external energy can effectively trigger pyroptosis, but systemic sensitization still faces challenges, such as limited ROS content, rapid decay, and short treatment windows. Herein, a nanohybrid-based redox homeostasis-perturbator system was designed that synergistically induce early lysosomal escape, autophagy inhibition, and redox perturbation functions to effectively sensitize cells to address these challenges. Specifically, weakly alkaline layered double hydroxide nanosheets (LDH NSs) with pH-responsive degradation properties enabled early lysosomal escape within 4 h, releasing poly(L-dopa) nanoparticles for inducing catechol-quinone redox cycling in the cytoplasm. The intracellular ROS levels were systematically rebounded by 3-4 times in tumor cells and lasted for over 4 h. Subsequently induced lysosomal stress and Ca2+ signaling activation resulted in severe mitochondrial dysfunction, as well as a perilous metastable state. Thereby, sequential near-infrared light was applied to trigger amplified stress through a local photothermal conversion. This led to sufficiently high levels of cleaved caspase-1 and GSDMD activation (2.5-2.8-fold increment) and subsequent pyroptosis response. In addition, OH- released by LDH elevated pH to alleviate the limitation of glutathione depletion by quinones at acidic pH and inhibit protective autophagy. Largely secreted inflammatory factors (2.5-5.6-fold increment), efficient maturation of dendritic cells, and further immune stimulation were boosted for tumor inhibition as a consequence. This study offers a new paradigm and insights into the synergy of internal systematic cellular sensitization and sequential external energy treatment to achieve tumor suppression through pyroptosis.

Asunto(s)

Homeostasis , Lisosomas , Oxidación-Reducción , Piroptosis , Piroptosis/efectos de los fármacos , Lisosomas/metabolismo , Lisosomas/efectos de los fármacos , Humanos , Animales , Ratones , Homeostasis/efectos de los fármacos , Homeostasis/efectos de la radiación , Nanopartículas/química , Especies Reactivas de Oxígeno/metabolismo , Línea Celular Tumoral , Antineoplásicos/farmacología , Antineoplásicos/química , Terapia Fototérmica , Hidróxidos/química , Hidróxidos/farmacología , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Neoplasias/terapia , Neoplasias/metabolismo

RESUMEN

In infantile nephropathic cystinosis, variants of the CTNS gene cause accumulation of cystine in lysosomes, causing progressive damage to most organs. Patients usually present before 1 year of age with signs of renal Fanconi syndrome. Cysteamine therapy allows cystine clearance from lysosomes and delays kidney damage but does not prevent progression to end-stage kidney disease, suggesting that pathways unrelated to cystine accumulation are also involved. Among these, impaired autophagy, altered endolysosomal trafficking, and increased apoptosis have emerged in recent years as potential targets for new therapies. We previously showed that luteolin, a flavonoid compound, improves these abnormal pathways in cystinotic cells and in zebrafish models of the disease. Herein, we have investigated if prolonged luteolin treatment ameliorates kidney damage in a murine model of cystinosis. To this end, we have treated Ctns-/- mice from 2 to 8 months with 150 mg/kg/day of luteolin. No significant side effects were observed. Compared to untreated animals, analyses of kidney cortex samples obtained after sacrifice showed that luteolin decreased p62/SQSTM1 levels (p <0.001), improved the number, size, and distribution of LAMP1-positive structures (p <0.02), and decreased tissue expression of cleaved caspase 3 (p <0.001). However, we did not observe improvements in renal Fanconi syndrome and kidney inflammation. Kidney function remained normal during the time of the study. These results indicate that luteolin has positive effects on the apoptosis and endo-lysosomal defects of cystinotic proximal tubular cells. However, these beneficial effects did not translate into improvement of renal Fanconi syndrome.

Asunto(s)

Cistinosis , Modelos Animales de Enfermedad , Luteolina , Animales , Luteolina/farmacología , Luteolina/uso terapéutico , Cistinosis/tratamiento farmacológico , Ratones , Ratones Noqueados , Apoptosis/efectos de los fármacos , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Sistemas de Transporte de Aminoácidos Neutros/genética , Ratones Endogámicos C57BL , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Masculino , Factores de Tiempo , Riñón/efectos de los fármacos , Riñón/patología , Riñón/metabolismo

RESUMEN

Potential exposure to cobalt nanoparticles (CoNPs) occurs in various fields, including hard alloy industrial production, the increasing use of new energy lithium-ion batteries, and millions of patients with metal-on-metal joint prostheses. Evidence from human, animal, and in vitro experiments suggests a close relationship between CoNPs and neurotoxicity. However, a systematic assessment of central nervous system (CNS) impairment due to CoNPs exposure and the underlying molecular mechanisms is lacking. In this study, we found that CoNPs induced neurodegenerative damage both in vivo and in vitro, including cognitive impairment, ß-amyloid deposition and Tau hyperphosphorylation. CoNPs promoted the formation of autophagosomes and impeding autophagosomal-lysosomal fusion in vivo and in vitro, leading to toxic protein accumulation. Moreover, CoNPs exposure reduced the level of transcription factor EB (TFEB) and the abundance of lysosome, causing a blockage in autophagosomal-lysosomal fusion. Interestingly, overexpression of long noncoding RNA NR_030777 mitigated CoNPs-induced neurodegenerative damage in both in vivo and in vitro models. Fluorescence in situ hybridization assay revealed that NR_030777 directly binds and stabilizes TFEB mRNA, alleviating the blockage of autophagosomal-lysosomal fusion and ultimately restoring neurodegeneration induced by CoNPs in vivo and in vitro. In summary, our study demonstrates that autophagic dysfunction is the main toxic mechanism of neurodegeneration upon CoNPs exposure and NR_030777 plays a crucial role in CoNPs-induced autophagic dysfunction. Additionally, the proposed adverse outcome pathway contributes to a better understanding of CNS toxicity assessment of CoNPs.

Asunto(s)

Autofagosomas , Cobalto , Lisosomas , Nanopartículas del Metal , ARN Largo no Codificante , Lisosomas/metabolismo , Lisosomas/efectos de los fármacos , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Autofagosomas/metabolismo , Autofagosomas/efectos de los fármacos , Cobalto/química , Cobalto/farmacología , Animales , Nanopartículas del Metal/química , Humanos , Ratones , Masculino , Autofagia/efectos de los fármacos , Ratones Endogámicos C57BL , Enfermedades Neurodegenerativas/metabolismo , Enfermedades Neurodegenerativas/patología , Enfermedades Neurodegenerativas/inducido químicamente

RESUMEN

Phagocytosis is an essential physiological mechanism; its impairment is associated with many diseases. A highly smart particle is required for understanding detailed sequential cellular events in phagocytosis. Recently, we identified an Indian traditional medicine named Godanti Bhasma (GB), a bioactive calcium sulfate particle prepared by thermo-transformation ofgypsum. Thermal processing of the gypsum transforms its native physicochemical properties by removing water molecules into the anhydrous GB, which was confirmed by Raman and FT-IR spectroscopy. GB particle showed a 0.5-5 µm size range and a neutral surface charge. Exposure of mammalian cells to GB particles showed a rapid cellular uptake through phagocytosis and induced massive cytoplasmic vacuolation in cells. Interestingly, no cellular uptake and cytoplasmic vacuolation were observed with the parent gypsum particle. The presence of the GB particles in intra-vacuolar space was confirmed using FESEM coupled with EDX. Flow cytometry analysis and live tracking of GB-treated cells showed particle internalization, vacuole formation, particle dissolution, and later vacuolar turnover. Quantification of GB-induced vacuolation was done using neutral red uptake assay in cells. Treatment of lysosomal inhibitors (BFA1 or CQ) with GB could not induce vacuolation, suggesting the requirement of an acidic environment for the vacuolation. In the mimicking experiment, GB particle dissolution in acidic cell-free solution suggested that degradation of GB occurs by acidic pH inside the cell vacuole. Vacuole formation generally accompanies with cell death, whereas GB-induced massive vacuolation does not cause cell death. Moreover, the cell divides and proliferates with the vacuolar process, intra-vacuolar cargo degradation, and eventually vacuolar turnover. Taken together, the sequential cellular events in this study suggest that GB can be used as a smart particle for phagocytosis assay development in animal cells.

Asunto(s)

Fagocitosis , Vacuolas , Fagocitosis/efectos de los fármacos , Vacuolas/efectos de los fármacos , Vacuolas/metabolismo , Animales , Humanos , Ratones , Citoplasma/metabolismo , Citoplasma/efectos de los fármacos , Lisosomas/metabolismo , Lisosomas/efectos de los fármacos

RESUMEN

Multiple myeloma (MM) is the second most common hematological tumor in adults. Immunomodulatory drugs (IMiDs), such as thalidomide and lenalidomide (Len), are effective drugs for the treatment of multiple myeloma. Len can recruit IKZF1 and IKZF3 to cereblon (CRBN), a substrate receptor of the cullin 4-RING E3 ligase (CRL4), promote their ubiquitination and degradation, and finally inhibit the proliferation of myeloma cells. However, MM patients develop resistance to IMiDs over time, leading to disease recurrence and deterioration. To explore the possible approaches that may enhance the sensitivity of IMiDs to MM, in this study, we used the proximity labeling technique TurboID and quantitative proteomics to identify Lys-63-specific deubiquitinase BRCC36 as a CRBN-interacting protein. Biochemical experiments demonstrated that BRCC36 in the BRISC complex protects CRBN from lysosomal degradation by specifically cleaving the K63-linked polyubiquitin chain on CRBN. Further studies found that a small-molecule compound SHIN1, which binds to BRISC complex subunit SHMT2, can upregulate CRBN by elevating BRCC36. The combination of SHIN1 and Len can further increase the sensitivity of MM cells to IMiDs. Therefore, this study provides the basis for the exploration of a possible strategy for the SHIN1 and Len combination treatment for MM.

Asunto(s)

Proteínas Adaptadoras Transductoras de Señales , Lenalidomida , Lisosomas , Mieloma Múltiple , Ubiquitina-Proteína Ligasas , Humanos , Mieloma Múltiple/patología , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/metabolismo , Lenalidomida/farmacología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Lisosomas/metabolismo , Lisosomas/efectos de los fármacos , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Línea Celular Tumoral , Ubiquitinación/efectos de los fármacos , Proteolisis/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Enzimas Desubicuitinizantes/metabolismo , Enzimas Desubicuitinizantes/antagonistas & inhibidores

RESUMEN

Aloperine (ALO), a quinolizidine-type alkaloid isolated from a natural Chinese herb, has shown promising antitumor effects. Nevertheless, its common mechanism of action and specific target remain elusive. Here, it is demonstrated that ALO inhibits the proliferation and migration of non-small cell lung cancer cell lines in vitro and the tumor development in several mouse tumor models in vivo. Mechanistically, ALO inhibits the fusion of autophagosomes with lysosomes and the autophagic flux, leading to the accumulation of sequestosome-1 (SQSTM1) and production of reactive oxygen species (ROS), thereby inducing tumor cell apoptosis and preventing tumor growth. Knockdown of SQSTM1 in cells inhibits ROS production and reverses ALO-induced cell apoptosis. Furthermore, VPS4A is identified as a direct target of ALO, and the amino acids F153 and D263 of VPS4A are confirmed as the binding sites for ALO. Knockout of VPS4A in H1299 cells demonstrates a similar biological effect as ALO treatment. Additionally, ALO enhances the efficacy of the anti-PD-L1/TGF-ß bispecific antibody in inhibiting LLC-derived subcutaneous tumor models. Thus, ALO is first identified as a novel late-stage autophagy inhibitor that triggers tumor cell death by targeting VPS4A.

Asunto(s)

Autofagosomas , Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Lisosomas , Quinolizidinas , Animales , Ratones , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Humanos , Autofagosomas/metabolismo , Autofagosomas/efectos de los fármacos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Lisosomas/metabolismo , Lisosomas/efectos de los fármacos , Línea Celular Tumoral , Quinolizidinas/farmacología , Modelos Animales de Enfermedad , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Transporte Vesicular/genética , Progresión de la Enfermedad , Proliferación Celular/efectos de los fármacos , Autofagia/efectos de los fármacos , Apoptosis/efectos de los fármacos

RESUMEN

Anaplastic thyroid cancer (ATC) is among the most aggressive and metastatic malignancies, often resulting in fatal outcomes due to the lack of effective treatments. Prosapogenin A (PA), a bioactive compound prevalent in traditional Chinese herbs, has shown potential as an antineoplastic agent against various human tumors. However, its effects on ATC and the underlying mechanism remain unclear. Here, we demonstrate that PA exhibits significant anti-ATC activity both in vitro and in vivo by inducing GSDME-dependent pyroptosis in ATC cells. Mechanistically, PA promotes lysosomal membrane permeabilization (LMP), leading to the release of cathepsins that activate caspase 8/3 to cleave GSDME. Remarkably, PA significantly upregulates three key functional subunits of V-ATPase-ATP6V1A, ATP6V1B2, and ATP6V0C-resulting in lysosomal over-acidification. This over-acidification exacerbates LMP and subsequent lysosomal damage. Neutralization of lysosomal lumen acidification or inhibition/knockdown of these V-ATPase subunits attenuates PA-induced lysosomal damage, pyroptosis and growth inhibition of ATC cells, highlighting the critical role for lysosomal acidification and LMP in PA's anticancer effects. In summary, our findings uncover a novel link between PA and lysosomal damage-dependent pyroptosis in cancer cells. PA may act as a V-ATPase agonist targeting lysosomal acidification, presenting a new potential therapeutic option for ATC treatment.

Asunto(s)

Lisosomas , Piroptosis , Carcinoma Anaplásico de Tiroides , ATPasas de Translocación de Protón Vacuolares , Lisosomas/metabolismo , Lisosomas/efectos de los fármacos , Humanos , Piroptosis/efectos de los fármacos , ATPasas de Translocación de Protón Vacuolares/metabolismo , Carcinoma Anaplásico de Tiroides/metabolismo , Carcinoma Anaplásico de Tiroides/patología , Carcinoma Anaplásico de Tiroides/tratamiento farmacológico , Animales , Línea Celular Tumoral , Sapogeninas/farmacología , Ratones , Ratones Desnudos , Neoplasias de la Tiroides/patología , Neoplasias de la Tiroides/metabolismo , Neoplasias de la Tiroides/tratamiento farmacológico , Ensayos Antitumor por Modelo de Xenoinjerto , Ratones Endogámicos BALB C , Gasderminas

RESUMEN

Neuropathic lysosomal storage diseases (NLSDs), including ceroid lipofuscinosis neuronal 3 (CLN3) disease and Gaucher disease type 2 (GD2), are typically present in adolescents; however, there are no approved therapies. CLN3 disease is the most common of the 13 types of neuronal ceroid lipofuscinosis, and Gaucher disease is the most common type of lysosomal storage disease. These NLSDs share oxidative stress and lysosomal dysfunction with Parkinson's disease. In this study, we used patient-derived cells (PDCs) and resorcinol to develop a therapeutic agent based on peroxisome proliferator-activated receptor γ (PPARγ) activation. PPARγ is a major regulator of autophagy and reactive oxygen species (ROS). Resorcinol, a polyphenolic compound, has been reported to exhibit PPARγ agonistic potential. Protein levels were analyzed by immunoblotting and immunofluorescence microscopy. Changes in cellular metabolism, including ROS levels, lipid droplet content, and lysosomal activity, were measured by flow cytometry. Resorcinol reduced ROS levels by suppressing hypoxia-inducible factor 1α levels in CLN3-PDCs. Resorcinol upregulated autophagy and reduced lipid accumulation in CLN3-PDCs; however, these effects were abolished by autophagy inhibitors. Resorcinol increased nuclear PPARγ levels in CLN3-PDCs, and PPARγ antagonists abolished the therapeutic effects of resorcinol. Moreover, Resorcinol upregulated nuclear PPARγ levels and lysosomal activity in GD2-PDCs, and reduced lipid accumulation and ROS levels. In summary, resorcinol alleviated the shared pathogenesis of CLN3 disease and GD2 through PPARγ upregulation. These findings suggest that resorcinol is a potential therapeutic candidate for alleviating NLSD progression.

Asunto(s)

PPAR gamma , Especies Reactivas de Oxígeno , Resorcinoles , Regulación hacia Arriba , Especies Reactivas de Oxígeno/metabolismo , Humanos , Resorcinoles/farmacología , PPAR gamma/metabolismo , PPAR gamma/genética , Regulación hacia Arriba/efectos de los fármacos , Metabolismo de los Lípidos/efectos de los fármacos , Lisosomas/metabolismo , Lisosomas/efectos de los fármacos , Autofagia/efectos de los fármacos , Enfermedades por Almacenamiento Lisosomal/metabolismo , Enfermedades por Almacenamiento Lisosomal/tratamiento farmacológico , Enfermedades por Almacenamiento Lisosomal/patología , Enfermedades por Almacenamiento Lisosomal/genética , Lipofuscinosis Ceroideas Neuronales/metabolismo , Lipofuscinosis Ceroideas Neuronales/tratamiento farmacológico , Lipofuscinosis Ceroideas Neuronales/patología , Lipofuscinosis Ceroideas Neuronales/genética

RESUMEN

BACKGROUND: Vascular dementia (VaD) resulting from chronic cerebral hypoperfusion (CCH) induces cognitive impairment and white matter injury (WMI). We previously found that CCH induces dysfunction of the autophagy-lysosomal pathway (ALP) in white matter (WM) of rats. Enhancing oligodendrocyte autophagy to counteract ALP deficiency is beneficial for cognitive recovery. Pseudogenoside-F11 (PF11), a saponin extracted from Panax quinquefolium l., provides neuroprotective benefits in many animal models of cerebral ischemia and dementia. PURPOSE: To investigate how PF11 affects cognitive deterioration in rats with VaD induced by two vessel occlusion (2VO), and to determine if PF11 regulates ALP dysfunction in WM. METHODS: CCH-related VaD was induced in rats using the 2VO method. PF11 (6, 12, 24 mg/kg, intragastric administration) was given continuously for 4 weeks postoperatively. Behavioral tests related to cognitive function were performed on the 28th day following 2VO. Transmission electron microscopy, immunofluorescence, western blotting and Luxol fast blue staining were used to assess the WMI and the mechanism of action of PF11 in 2VO-induced VaD. RESULTS: PF11 (12 mg/kg) ameliorated 2VO-induced cognitive impairment. PF11 also alleviated WMI on the 28th day following 2VO, as characterized by reduction of neuronal axonal demyelination and axonal loss. Furthermore, PF11 prevented mature oligodendrocytes death by attenuating ALP deficiency in WM on the 14th day following 2VO, as manifested by enhancement of mechanistic target of rapamycin-mediated autophagy and lysosomal function, thereby reducing the aberrant accumulation of autophagy substrates and increasing the level of autophagosomes in WM. In addition, PF11 also prevented microglia and astrocytes from activating in WM on the 28th day following 2VO. CONCLUSION: PF11 significantly ameliorates cognitive impairment and WMI, and the mechanism is at least partly related to lessening ALP dysfunction in WM by enhancing autophagy and reducing lysosomal defects in oligodendrocytes.

Asunto(s)

Autofagia , Disfunción Cognitiva , Demencia Vascular , Ginsenósidos , Lisosomas , Fármacos Neuroprotectores , Ratas Sprague-Dawley , Sustancia Blanca , Animales , Demencia Vascular/tratamiento farmacológico , Autofagia/efectos de los fármacos , Masculino , Disfunción Cognitiva/tratamiento farmacológico , Sustancia Blanca/efectos de los fármacos , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Ginsenósidos/farmacología , Ratas , Fármacos Neuroprotectores/farmacología , Modelos Animales de Enfermedad , Panax/química

RESUMEN

Photosensitizer-based phototherapies, including photodynamic therapy (PDT) and photothermal therapy (PTT), offer safe treatment modalities for tumor ablation with spatiotemporal precision. After photons are absorbed, PDT creates localized chemical damage by generating reactive oxygen species (ROS), while PTT induces localized thermal damage. However, PDT still faces hypoxic tumor challenges, while PTT encounters issues related to heat resistance and potential overheating. The combination of PDT and PTT shows great potential as an effective anticancer strategy. By targeting lysosomes with carefully designed phototherapeutic reagents for combined phototherapy, rapid dysfunction and cell death in cancer cells can be induced, showing promise for cancer treatment. Herein, two α-α-linked bisBODIPYs with tetraphenylethene (TPE) moieties are designed and synthesized. These TPE-substituted bisBODIPYs expand the absorption into NIR range (λmaxabs/λmaxem ∼ 740/810 nm) and confer aggregation-induced emission (AIE) activity (λmaxem ∼ 912 nm). Moreover, these bisBODIPYs self-assemble with surfactant F-127 into nanoparticles (NPs), which efficiently generate ROS (1O2 and •OH) in both solution and cellular environments and demonstrate superior photothermal conversion efficiencies (η ∼ 68.3%) along with exceptional photothermal stability. More importantly, these NPs showed lysosomal targeting and remarkable tumor ablation in cellular and murine models, indicating their potential in precision tumor therapy.

Asunto(s)

Lisosomas , Nanopartículas , Fármacos Fotosensibilizantes , Lisosomas/metabolismo , Lisosomas/efectos de los fármacos , Humanos , Animales , Ratones , Fármacos Fotosensibilizantes/química , Fármacos Fotosensibilizantes/farmacología , Nanopartículas/química , Rayos Infrarrojos , Fotoquimioterapia , Estilbenos/química , Estilbenos/farmacología , Especies Reactivas de Oxígeno/metabolismo , Fototerapia , Antineoplásicos/química , Antineoplásicos/farmacología , Línea Celular Tumoral , Ratones Endogámicos BALB C , Neoplasias/tratamiento farmacológico , Neoplasias/terapia , Neoplasias/patología , Ratones Desnudos

RESUMEN

Advanced hepatocellular carcinoma (HCC) patients have poor prognosis. As an endogenous antioxidant enzyme involved in a variety of bioprocesses, sulfiredoxin-1 (SRXN1) plays an irreplaceable role in promoting the development of tumors. However, the role and working mechanism of SRXN1 in HCC remain unclear. In this study, we confirmed that SRXN1 promoted the cell proliferation of HCC at genetic and pharmacological level, respectively. Transcriptome sequencing analysis revealed SRXN1 knockdown had a significant effect on the expression of lysosome biogenesis related genes. Further experiments validated that lysosome biogenesis and autophagic flux were enhanced after SRXN1 inhibition and reduced as SRXN1 overexpression. Mechanism study revealed that ROS accumulation induced TFEB nuclear translocation, followed by increased autophagy. Following this rationale, the combination of SRXN1 inhibitor and sorafenib demonstrated noticeable synergistic antitumor effect through the boost of ROS both in vivo and in vitro. Taken together, SRXN1 could be a potential therapeutic target for HCC therapy.

Asunto(s)

Autofagia , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Carcinoma Hepatocelular , Proliferación Celular , Neoplasias Hepáticas , Lisosomas , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/tratamiento farmacológico , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/tratamiento farmacológico , Humanos , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/genética , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/metabolismo , Autofagia/efectos de los fármacos , Autofagia/genética , Proliferación Celular/efectos de los fármacos , Lisosomas/metabolismo , Lisosomas/efectos de los fármacos , Animales , Ratones , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Ratones Desnudos , Especies Reactivas de Oxígeno/metabolismo , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Ratones Endogámicos BALB C , Masculino , Sorafenib/farmacología

RESUMEN

Autophagy, a crucial intracellular degradation process facilitated by lysosomes, plays a pivotal role in maintaining cellular homeostasis. The elucidation of autophagy key genes and signaling pathways has significantly advanced our understanding of this process and has led to the exploration of autophagy as a promising therapeutic approach. This review comprehensively assesses the latest developments in small molecule modulators targeting autophagy. Moreover, the review delves into the most recent strategies for drug discovery, specifically focusing on selective agents that exploit autophagosomes and lysosomes for targeted protein degradation. Additionally, this article highlights the prevailing challenges and outlines potential future advancements in the field. By amalgamating the cutting-edge knowledge in the field, we aim to offer valuable insights and references for the anti-cancer drug development of autophagy-targeted therapies, thus contributing to the advancement of novel therapeutic interventions.

Asunto(s)

Antineoplásicos , Autofagia , Neoplasias , Humanos , Autofagia/efectos de los fármacos , Antineoplásicos/farmacología , Antineoplásicos/química , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Animales , Lisosomas/metabolismo , Lisosomas/efectos de los fármacos , Estructura Molecular , Descubrimiento de Drogas