RESUMEN
Glioblastoma (GBM), one of the most malignant brain tumors in the world, has limited treatment options and a dismal survival rate. Effective and safe disease-modifying drugs for glioblastoma are urgently needed. Here, we identified a small molecule, Molephantin (EM-5), effectively penetrated the blood-brain barrier (BBB) and demonstrated notable antitumor effects against GBM with good safety profiles both in vitro and in vivo. Mechanistically, EM-5 not only inhibits the proliferation and invasion of GBM cells but also induces cell apoptosis through the reactive oxygen species (ROS)-mediated PI3K/Akt/mTOR pathway. Furthermore, EM-5 causes mitochondrial dysfunction and blocks mitophagy flux by impeding the fusion of mitophagosomes with lysosomes. It is noteworthy that EM-5 does not interfere with the initiation of autophagosome formation or lysosomal function. Additionally, the mitophagy flux blockage caused by EM-5 was driven by the accumulation of intracellular ROS. In vivo, EM-5 exhibited significant efficacy in suppressing tumor growth in a xenograft model. Collectively, our findings not only identified EM-5 as a promising, effective, and safe lead compound for treating GBM but also uncovered its underlying mechanisms from the perspective of apoptosis and mitophagy.
Asunto(s)
Apoptosis , Neoplasias Encefálicas , Proliferación Celular , Glioblastoma , Mitofagia , Especies Reactivas de Oxígeno , Ensayos Antitumor por Modelo de Xenoinjerto , Glioblastoma/tratamiento farmacológico , Glioblastoma/patología , Glioblastoma/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Humanos , Mitofagia/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/metabolismo , Ratones , Proliferación Celular/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Ratones Desnudos , Serina-Treonina Quinasas TOR/metabolismo , Barrera Hematoencefálica/metabolismo , Barrera Hematoencefálica/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismoRESUMEN
Melittin, a naturally occurring polypeptide found in bee venom, has been recognized for its potential anti-tumor effects, particularly in the context of lung cancer. Our previous study focused on its impact on human lung adenocarcinoma cells A549, revealing that melittin induces intracellular reactive oxygen species (ROS) burst and oxidative damage, resulting in cell death. Considering the significant role of mitochondria in maintaining intracellular redox levels and ROS, we further examined the involvement of mitochondrial damage in melittin-induced apoptosis in lung cancer cells. Our findings demonstrated that melittin caused changes in mitochondrial membrane potential (MMP), triggered mitochondrial ROS burst (Figure 1), and activated the mitochondria-related apoptosis pathway Bax/Bcl-2 by directly targeting mitochondria in A549 cells (Figure 2). Further, we infected A549 cells using a lentivirus that can express melittin-Myc and confirmed that melittin can directly target binding to mitochondria, causing the biological effects described above (Figure 2). Notably, melittin induced mitochondrial damage while inhibiting autophagy, resulting in abnormal degradation of damaged mitochondria (Figure 5). To summarize, our study unveils that melittin targets mitochondria, causing mitochondrial damage, and inhibits the autophagy-lysosomal degradation pathway. This process triggers mitoROS burst and ultimately activates the mitochondria-associated Bax/Bcl-2 apoptotic signaling pathways in A549 cells.
Asunto(s)
Neoplasias Pulmonares , Mitofagia , Humanos , Células A549 , Meliteno/farmacología , Meliteno/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Proteína X Asociada a bcl-2/metabolismo , Proteína X Asociada a bcl-2/farmacología , Mitocondrias/metabolismo , Apoptosis , Potencial de la Membrana Mitocondrial , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/metabolismoRESUMEN
Aims: Acute myocardial infarction (MI), caused by acute coronary artery obstruction, is a common cardiovascular event leading to mortality. Nuclear dot protein 52 (NDP52) is an essential selective autophagy adaptor, although its function in MI is still obscure. This study was designed to examine the function of NDP52 in MI and the associated mechanisms. Results: Our results revealed that MI challenge overtly impaired myocardial geometry and systolic function, along with cardiomyocyte apoptosis, myocardial interstitial fibrosis, and mitochondrial damage, and NDP52 nullified such devastating responses. Further studies showed that the blockade of mitochondrial clearance is related to MI-induced buildup of damaged mitochondria. Mechanistic approaches depicted that 7-day MI induced abnormal mitophagy flux, resulting in poor lysosomal clearance of injured mitochondria. NDP52 promoted mitophagy flux through the recruitment of Ras-associated protein RAB7 (RAB7) and TANK-binding kinase 1 (TBK1). On protein co-localization, TBK1 phosphorylated RAB7, in line with the finding that chloroquine or a TBK1 inhibitor reversed NDP52-dependent beneficial responses. Innovation: This study denoted a novel mechanism that NDP52 promotes cardioprotection against ischemic heart diseases through interaction with TBK1 and RAB7, leading to RAB7 phosphorylation, induction of mitophagy to clear ischemia-induced impaired mitochondria, thus preventing cardiomyocyte apoptosis in MI. Conclusion: Our results indicate that NDP52 promotes autophagic flux and clears damaged mitochondria to diminish reactive oxygen species and cell death in a TBK1/RAB7-dependent manner and thus limits MI-induced injury. Antioxid. Redox Signal. 36, 1119-1135.
Asunto(s)
Infarto del Miocardio , Proteínas del Tejido Nervioso , Proteínas Serina-Treonina Quinasas , Receptores Citoplasmáticos y Nucleares , Proteínas de Unión a GTP rab7 , Animales , Autofagosomas/metabolismo , Autofagia , Lisosomas/metabolismo , Ratones , Mitofagia , Infarto del Miocardio/metabolismo , Miocitos Cardíacos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Proteínas de Unión a GTP rab7/metabolismoRESUMEN
Bladder cancer is one of the most common malignancy in the urinary tract with high recurrence and drug resistance in clinics. Alternative treatments from existing drugs might be a promising strategy. Nitazoxanide (NTZ), an FDA-approved antiprotozoal drug, has got increasingly noticed because of its favorable safety profile and antitumor potential, yet the effects in bladder cancer and underlying mechanisms remain poorly understood. Herein, we find that NTZ induces mitochondrial damage and mitophagy initiation through PINK1-generated phospho-ubiquitin(pS65-Ub) and autophagy receptor-mediated pathway even in the absence of Atg5/Beclin1. Meanwhile, NTZ inhibits lysosomal degradation activity, leading to mitophagy flux impairment at late stage. Mitochondrial reactive oxygen species (ROS) production is critical in this process, as eliminating ROS with N-acetylcysteine (NAC) efficiently inhibits PINK1 signaling-mediated mitophagy initiation and alleviates lysosomal dysfunction. Co-treatment with NTZ and autophagy inhibitor Chloroquine (CQ) to aggravate mitophagy flux impairment promotes NTZ-induced apoptosis, while alleviation of mitophagy flux impairment with ROS scavenger reduces cell death. Moreover, we also discover a similar signaling response in the 3D bladder tumor spheroid after NTZ exposure. In vivo study reveals a significant inhibition of orthotopic bladder tumors with no obvious systemic toxicity. Together, our results uncover the anti-tumor activities of NTZ with the involvement of ROS-mediated mitophagy modulation at different stages and demonstrate it as a potential drug candidate for fighting against bladder tumors.
Asunto(s)
Antineoplásicos/farmacología , Lisosomas/metabolismo , Mitofagia/efectos de los fármacos , Nitrocompuestos/farmacología , Especies Reactivas de Oxígeno/metabolismo , Tiazoles/farmacología , Neoplasias de la Vejiga Urinaria/metabolismo , Animales , Antineoplásicos/uso terapéutico , Antiparasitarios/farmacología , Antiparasitarios/uso terapéutico , Relación Dosis-Respuesta a Droga , Femenino , Células HEK293 , Humanos , Lisosomas/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Mitofagia/fisiología , Nitrocompuestos/uso terapéutico , Especies Reactivas de Oxígeno/antagonistas & inhibidores , Tiazoles/uso terapéutico , Neoplasias de la Vejiga Urinaria/tratamiento farmacológicoRESUMEN
Rationale: Ischemic heart disease remains a primary threat to human health, while its precise etiopathogenesis is still unclear. TBC domain family member 15 (TBC1D15) is a RAB7 GTPase-activating protein participating in the regulation of mitochondrial dynamics. This study was designed to explore the role of TBC1D15 in acute myocardial infarction (MI)-induced cardiac injury and the possible mechanism(s) involved. Methods: Mitochondria-lysosome interaction was evaluated using transmission electron microscopy and live cell time-lapse imaging. Mitophagy flux was measured by fluorescence and western blotting. Adult mice were transfected with adenoviral TBC1D15 through intra-myocardium injection prior to a 3-day MI procedure. Cardiac morphology and function were evaluated at the levels of whole-heart, cardiomyocytes, intracellular organelles and cell signaling transduction. Results: Our results revealed downregulated level of TBC1D15, reduced systolic function, overt infarct area and myocardial interstitial fibrosis, elevated cardiomyocyte apoptosis and mitochondrial damage 3 days after MI. Overexpression of TBC1D15 restored cardiac systolic function, alleviated infarct area and myocardial interstitial fibrosis, reduced cardiomyocyte apoptosis and mitochondrial damage although TBC1D15 itself did not exert any myocardial effect in the absence of MI. Further examination revealed that 3-day MI-induced accumulation of damaged mitochondria was associated with blockade of mitochondrial clearance because of enlarged defective lysosomes and subsequent interrupted mitophagy flux, which were attenuated by TBC1D15 overexpression. Mechanistic studies showed that 3-day MI provoked abnormal mitochondria-lysosome contacts, leading to lysosomal enlargement and subsequently disabled lysosomal clearance of damaged mitochondria. TBC1D15 loosened the abnormal mitochondria-lysosome contacts through both the Fis1 binding and the RAB7 GAPase-activating domain of TBC1D15, as TBC1D15-dependent beneficial responses were reversed by interference with either of these two domains both in vitro and in vivo. Conclusions: Our findings indicated a pivotal role of TBC1D15 in acute MI-induced cardiac anomalies through Fis1/RAB7 regulated mitochondria-lysosome contacts and subsequent lysosome-dependent mitophagy flux activation, which may provide a new target in the clinical treatment of acute MI.
Asunto(s)
Proteínas Activadoras de GTPasa/metabolismo , Infarto del Miocardio/complicaciones , Daño por Reperfusión Miocárdica/patología , Miocardio/patología , Animales , Animales Recién Nacidos , Células Cultivadas , Modelos Animales de Enfermedad , Proteínas Activadoras de GTPasa/genética , Humanos , Lisosomas/patología , Masculino , Ratones , Ratones Transgénicos , Mitocondrias/patología , Dinámicas Mitocondriales , Proteínas Mitocondriales/metabolismo , Mitofagia , Daño por Reperfusión Miocárdica/etiología , Miocardio/citología , Miocitos Cardíacos/citología , Miocitos Cardíacos/patología , Cultivo Primario de Células , Unión Proteica/genética , Dominios Proteicos/genética , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión a GTP rab7RESUMEN
The maintenance of muscle health with advancing age is dependent on mitochondrial homeostasis. While reductions in mitochondrial biogenesis have been observed with age, less is known regarding organelle degradation. Parkin is an E3 ubiquitin ligase implicated in mitophagy, but few studies have examined Parkin's contribution to mitochondrial turnover in muscle. Wild-type (WT) and Parkin knockout (KO) mice were used to delineate a role for Parkin-mediated mitochondrial degradation in aged muscle, in concurrence with exercise. Aged animals exhibited declines in muscle mass and mitochondrial content, paralleled by a nuclear environment endorsing the transcriptional repression of mitochondrial biogenesis. Mitophagic signaling was enhanced following acute endurance exercise in young WT mice but was abolished in the absence of Parkin. Basal mitophagy flux of the autophagosomal protein lipidated microtubule-associated protein 1A/1B-light chain 3 was augmented in aged animals but did not increase additionally with exercise when compared with young animals. In the absence of Parkin, exercise increased the nuclear localization of Parkin-interacting substrate, corresponding to a decrease in nuclear peroxisome proliferator gamma coactivator-1α. Remarkably, exercise enhanced mitochondrial ubiquitination in both young WT and KO animals. This suggested compensation of alternative ubiquitin ligases that were, however, unable to restore the diminished exercise-induced mitophagy in KO mice. Under basal conditions, we demonstrated that Parkin was required for mitochondrial mitofusin-2 ubiquitination. We also observed an abrogation of exercise-induced mitophagy in aged muscle. Our results demonstrate that acute exercise-induced mitophagy is dependent on Parkin and attenuated with age, which likely contributes to changes in mitochondrial content and quality in aging muscle.
Asunto(s)
Envejecimiento/fisiología , Mitofagia/genética , Músculo Esquelético/crecimiento & desarrollo , Músculo Esquelético/fisiología , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/fisiología , Animales , Complejo IV de Transporte de Electrones/metabolismo , GTP Fosfohidrolasas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias Musculares/genética , Mitocondrias Musculares/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/genética , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Condicionamiento Físico Animal , Proteínas Represoras/metabolismo , Ubiquitinación/genéticaRESUMEN
BACKGROUND: Parkin is a ubiquitin ligase that is involved in the selective removal of dysfunctional mitochondria. This process is termed mitophagy and can assist in mitochondrial quality control. Endurance training can produce adaptations in skeletal muscle toward a more oxidative phenotype, an outcome of enhanced mitochondrial biogenesis. It remains unknown whether Parkin-mediated mitophagy is involved in training-induced increases in mitochondrial content and function. Our purpose was to determine a role for Parkin in maintaining mitochondrial turnover in muscle, and its requirement in mediating mitochondrial biogenesis following endurance exercise training. METHODS: Wild-type and Parkin knockout (KO) mice were trained for 6 weeks and then treated with colchicine or vehicle to evaluate the role of Parkin in mediating changes in mitochondrial content, function and acute exercise-induced mitophagy flux. RESULTS: Our results indicate that Parkin is required for the basal maintenance of mitochondrial function. The absence of Parkin did not significantly alter mitophagy basally; however, acute exercise produced an elevation in mitophagy flux, a response that was Parkin-dependent. Mitochondrial content was increased following training in both genotypes, but this occurred without an induction of PGC-1α signaling in KO animals. Interestingly, the increased muscle mitochondrial content in response to training did not influence basal mitophagy flux, despite an enhanced expression and localization of Parkin to mitochondria in WT animals. Furthermore, exercise-induced mitophagy flux was attenuated with training in WT animals, suggesting a lower rate of mitochondrial degradation resulting from improved organelle quality with training. In contrast, training led to a higher mitochondrial content, but with persistent dysfunction, in KO animals. Thus, the lack of a rescue of mitochondrial dysfunction with training in the absence of Parkin is the likely reason for the impaired training-induced attenuation of mitophagy flux compared to WT animals. CONCLUSIONS: Our study demonstrates that Parkin is required for exercise-induced mitophagy flux. Exercise-induced mitophagy is reduced with training in muscle, likely due to attenuated signaling consequent to increased mitochondrial content and quality. Our data suggest that Parkin is essential for the maintenance of basal mitochondrial function, as well as for the accumulation of normally functioning mitochondria as a result of training adaptations in muscle.