Lysosomal pH Research Articles

Chemo-drugs in cell microparticles reset antitumor activity of macrophages by activating lysosomal P450 and nuclear hnRNPA2B1

Macrophages in tumors (tumor-associated macrophages, TAMs), a major population within most tumors, play key homeostatic functions by stimulating angiogenesis, enhancing tumor cell growth, and suppressing antitumor immunity. Resetting TAMs by simple, efficacious and safe approach(s) is highly desirable to enhance antitumor immunity and attenuate tumor cell malignancy. Previously, we used tumor cell-derived microparticles to package chemotherapeutic drugs (drug-MPs), which resulted in a significant treatment outcome in human malignant pleural effusions via neutrophil recruitments, implicating that drug-MPs might reset TAMs, considering the inhibitory effects of M2 macrophages on neutrophil recruitment and activation. Here, we show that drug-MPs can function as an antitumor immunomodulator by resetting TAMs with M1 phenotype and IFN-β release. Mechanistically, drug molecules in tumor MPs activate macrophage lysosomal P450 monooxygenases, resulting in superoxide anion formation, which further amplifies lysosomal ROS production and pH value by activating lysosomal NOX2. Consequently, lysosomal Ca2+ signaling is activated, thus polarizing macrophages towards M1. Meanwhile, the drug molecules are delivered from lysosomes into the nucleus where they activate DNA sensor hnRNPA2B1 for IFN-β production. This lysosomal-nuclear machinery fully arouses the antitumor activity of macrophages by targeting both lysosomal pH and the nuclear innate immunity. These findings highlight that drug-MPs can act as a new immunotherapeutic approach by revitalizing antitumor activity of macrophages. This mechanistic elucidation can be translated to treat malignant ascites by drug-MPs combined with PD-1 blockade.

Impact of Enniatin B and Beauvericin on Lysosomal Cathepsin B Secretion and Apoptosis Induction.

Enniatin B (ENN B) and Beauvericin (BEA) are cyclohexadepsipeptides that can be isolated from Fusarium and Beauveria bassiana, respectively. Both compounds are cytotoxic and ionophoric. In the present study, the mechanism of cell death induced by these compounds was investigated. Epidermal carcinoma-derived cell line KB-3-1 cells were treated with different concentrations of these compounds. The extracellular secretion of cathepsin B increased in a concentration-dependent manner, and the lysosomal staining by lysotracker red was reduced upon the treatment with any of the compounds. However, the extracellular secretion of cathepsin L and cathepsin D were not affected. Inhibition of cathepsin B with specific inhibitor CA074 significantly reduced the cytotoxic effect of both compounds, while inhibition of cathepsin D or cathepsin L did not influence the cytotoxic activities of both compounds. In vitro labelling of lysosomal cysteine cathepsins with Ethyl (2S, 3S)-epoxysuccinate-Leu-Tyr-Acp-Lys (Biotin)-NH2 (DCG04) was not affected in case of cathepsin L upon the treatment with both compounds, while it was significantly reduced in case of cathepsin B. In conclusion, ENN B and BEA increase lysosomal Ph, which inhibits delivery of cathepsin B from Golgi to lysosomes, thereby inducing cathepsin B release in cytosol, which activates caspases and hence the apoptotic pathway.

A ratiometric fluorescent probe based on spiropyran in situ switching for tracking dynamic changes of lysosomal autophagy and anticounterfeiting

Autophagy is the controlled breakdown of cellular components that dysfunctional or nonessential, and the decomposition products are further recycled and synthesized for the normal physiological activities of cells. Lysosomal autophagy has been implicated in cancer, neurological disorders, Parkinson's disease, etc. Therefore, it is necessary to develop a fluorescent probe that can clearly describe the process of lysosomal autophagy. However, there are currently limited fluorescent probes for ratiometric monitoring of the autophagic process in dual channels. To solve this problem, a fluorescent probe based on spiropyran with lysosomal targeting and pH response for ratiometric monitoring the autophagy process of lysosomes were designed. The sensitive response of the probe to pH in vitro was verified by UV and fluorescence spectrum tests. Meanwhile, the probe demonstrated the ability to monitor the intracellular pH fluctuations. In addition, the application of Lyso-SD in the field of anti-counterfeiting has been proposed based on the obvious photoluminescence ability of Lyso-SD under UV irradiation.

TRPML1-Induced Lysosomal Ca2+ Signals Activate AQP2 Translocation and Water Flux in Renal Collecting Duct Cells.

Lysosomes are acidic Ca2+ storage organelles that actively generate local Ca2+ signaling events to regulate a plethora of cell functions. Here, we characterized lysosomal Ca2+ signals in mouse renal collecting duct (CD) cells and we assessed their putative role in aquaporin 2 (AQP2)-dependent water reabsorption. Bafilomycin A1 and ML-SA1 triggered similar Ca2+ oscillations, in the absence of extracellular Ca2+, by alkalizing the acidic lysosomal pH or activating the lysosomal cation channel mucolipin 1 (TRPML1), respectively. TRPML1-dependent Ca2+ signals were blocked either pharmacologically or by lysosomes' osmotic permeabilization, thus indicating these organelles as primary sources of Ca2+ release. Lysosome-induced Ca2+ oscillations were sustained by endoplasmic reticulum (ER) Ca2+ content, while bafilomycin A1 and ML-SA1 did not directly interfere with ER Ca2+ homeostasis per se. TRPML1 activation strongly increased AQP2 apical expression and depolymerized the actin cytoskeleton, thereby boosting water flux in response to an hypoosmotic stimulus. These effects were strictly dependent on the activation of the Ca2+/calcineurin pathway. Conversely, bafilomycin A1 led to perinuclear accumulation of AQP2 vesicles without affecting water permeability. Overall, lysosomal Ca2+ signaling events can be differently decoded to modulate Ca2+-dependent cellular functions related to the dock/fusion of AQP2-transporting vesicles in principal cells of the CD.

Nucleic acid uptake occurs independent of lysosomal acidification but dependent on ATP consumption during RNautophagy/DNautophagy

RNautophagy/DNautophagy (RDA) is an autophagic process that refers to the direct uptake of nucleic acids by lysosomes for degradation. Autophagy relies on lysosomes and lysosomal acidification is crucial for the degradation of intracellular components. However, whether lysosomal acidification interferes with nucleic acid uptake during RDA is unclear. In this study, we focused on vacuolar H+-ATPase (V-ATPase), the major proton pump responsible for maintaining an acidic pH in lysosomes. Our results show that lysosomes take up nucleic acids independently of the intralysosomal acidic pH during RDA. Isolated lysosomes treated with bafilomycin A1, a potent V-ATPase inhibitor, did not degrade, but took up RNA at similar levels as the control lysosomes. Similarly, the knockdown of Atp6v1a, the gene that encodes V-ATPase catalytic subunit A, did not affect the RNA uptake ability of isolated lysosomes. In addition, we demonstrated that nucleic acid uptake by isolated lysosomes necessitates ATP consumption, although V-ATPase is not required for the uptake process. These results broaden our understanding of the mechanisms underlying nucleic acid degradation via autophagy.

Ratiometric fluorescent probes for pH mapping in cellular organelles.

The intracellular pH (pHi) in organelles, including mitochondria, endoplasmic reticulum, lysosomes, and nuclei, differs from the cytoplasmic pH, and thus maintaining the pH of these organelles is crucial for cellular homeostasis. Alterations in the intracellular pH (ΔpHi) in organelles lead to the disruption of cell proliferation, ion transportation, cellular homeostasis, and even cell death. Hence, accurately mapping the pH of organelles is crucial. Accordingly, the development of fluorescence imaging probes for targeting specific organelles and monitoring their dynamics at the molecular level has become the forefront of research in the last three decades. Among them, ratiometric fluorescent probes minimize the interference from the excitation wavelength of light, auto-fluorescence from probe concentration, environmental fluctuations, and instrument sensitivity through self-correction compared to monochromatic fluorescent probes, which are known as turn-on/off fluorescent probes. Small-molecular ratiometric fluorescent probes for detecting ΔpHi are challenging yet demanding. To date, sixty-two ratiometric pH probes have been reported for monitoring internal pH alterations in cellular organelles. However, a critical review on organelle-specific ratiometric probes for pH mapping is still lacking. Thus, in the present review, we report the most recent advances in ratiometric pH probes and the previous data on the role of mapping the ΔpHi of cellular organelles. The development strategy, including ratiometric fluorescence with one reference signal (RFRS) and ratiometric fluorescence with two reversible signals (RFRvS), is systematically illustrated. Finally, we emphasize the major challenges in developing ratiometric probes that merit further research in the future.

Monitoring Lysosome Function in Ferroptosis.

Ferroptosis is a type of regulated cell death that occurs due to iron-induced membrane lipid peroxidation. Lysosomes, which are acidic, membrane-bound organelles containing various hydrolases, play a vital role in ferroptosis. They not only aid in the degradation of autophagic substrates, but also serve as signaling hubs in cell death. Specifically, lysosomes are involved in the induction and execution of ferroptosis through autophagy-mediated degradation of anti-ferroptotic proteins, lysosomal membrane permeability-mediated release of cathepsins, and iron-induced lysosomal membrane lipid peroxidation. Therefore, it is essential to have reliable methods for monitoring lysosomal functions, including lysosomal activity, pH, and membrane integrity, as well as iron accumulation and lipid peroxidation, to understand ferroptosis. This chapter introduces several protocols, such as western blotting, immunofluorescence, lysosomal probes, and lipid peroxidation assay kits, for monitoring the process of lysosome-related ferroptosis.

A water-soluble fluorescent pH probe and its application for monitoring lysosomal pH changes in living cells.

Intracellular pH plays a crucial role in many cellular processes, and abnormal intracellular pH has been linked to common diseases such as cancer and Alzheimer's. To address this issue, a water-soluble fluorescent pH probe was designed based on the protonation/deprotonation of the 4-methylpiperazin-1-yl group, using dicyanoisophorone as the fluorophore. In the neutral form of the probe, fluorescence is quenched due to charge transfer from the 4-methylpiperazin-1-yl group to the fluorophore upon excitation. Under acidic conditions, protonation of the 4-methylpiperazin-1-yl group inhibits the photoinduced electron transfer process, leading to an increase in fluorescence intensity. Density-functional theory calculations also verified the fluorescence OFF-ON mechanism. The probe exhibits high selectivity, photostability, fast response to pH changes, and low cytotoxicity to cells. Additionally, the probe selectively accumulates in lysosomes, with a high Pearson coefficient (0.95) using LysoTracker Green DND-26 as a reference. Notably, the probe can monitor lysosomal pH changes in living cells and track pH changes stimulated by chloroquine. We anticipate that the probe has potential for diagnosing pH-related diseases.

Nanodrug regulates ROS homeostasis via enhancing fatty acid oxidation and inhibiting autophagy to overcome tumor drug resistance.

The treatment of drug-resistant tumors poses a significant challenge in the field of tumor therapy. Disrupting the homeostasis of reactive oxygen species (ROS) within tumor cells may represent a pivotal strategy for overcoming the prevalent issue of drug resistance. However, the restricted sustainability of ROS generation and the increased autophagy capacity exhibited by tumor cells hinder the application of ROS-based therapies. In this study, we developed liposome nanoparticles (Ato/CQ@L) for co-encapsulation of atorvastatin (Ato), an activator of AMP-activated protein kinase (AMPK), and chloroquine (CQ), an autophagy inhibitor. Upon internalization by tumor cells, Ato upregulated carnitine palmitoyltransferase 1(CPT1) concentration and promoted fatty acid oxidation (FAO) within the tumor cells. The process of FAO coupled with an abundance of fatty acid substrates, facilitates a sustained generation of ROS production. Concurrently, a positive feedback loop is established between escalated concentration of ROS and AMPK protein levels, resulting in a persistent elevation in ROS levels. In addition, CQ disrupted lysosomes, leading to an increased lysosomal pH and reducing autophagy in tumor cells. In both in vivo and in vitro experiments, the Ato/CQ@L treatment group exhibited a considerable enhancement in tumor cell apoptosis, validating the efficacy of this combined therapy. In summary, the combined therapy involving Ato and CQ addresses the inherent limitations of conventional ROS therapy, which include insufficient ROS production and increased autophagy. This approach holds significant potential as a treatment strategy for drug-resistant triple-negative breast cancer.

Macro-microporous ZIF-8 MOF complexed with lysosomal pH-adjusting hexadecylsulfonylfluoride as tumor vaccine delivery systems for improving anti-tumor cellular immunity.

Tumor vaccine therapy, which can induce tumor antigen-specific cellular immune responses to directly kill tumor cells, is considered to be one of the most promising tumor immunotherapies. How to elicit effective tumor antigen-specific cellular immunity is the key for the development of tumor vaccines. However, current tumor vaccines with conventional antigen delivery systems mainly induce humoral immunity but not effective cellular immunity. In this study, based on pH-sensitive, ordered macro-microporous zeolitic imidazolate framework-8 (SOM-ZIF-8) and hexadecylsulfonylfluoride (HDSF), an intelligent tumor vaccine delivery system SOM-ZIF-8/HDSF was developed to elicit potent cellular immunity. Results demonstrated that the SOM-ZIF-8 particles could efficiently encapsulate antigen into the macropores, promote antigen uptake by antigen-presenting cells, facilitate lysosomal escape, and enhance antigen cross-presentation and cellular immunity. In addition, the introduction of HDSF could up-regulate the lysosomal pH to protect antigens from acid degradation, which further promoted antigen cross-presentation and cellular immunity. The immunization tests showed that the tumor vaccines based on the delivery system improved antigen-specific cellular immune response. Moreover, the tumor vaccines significantly inhibited tumor growth in B16 melanoma-bearing C57BL/6 mice. These results indicate that SOM-ZIF-8/HDSF as an intelligent vaccine delivery system could be used for the development of novel tumor vaccines.

Acidic pH-Triggered Live-Cell Lysosome Specific Tracking, Ratiometric pH Sensing, and Multicolor Imaging by Visible to NIR Switchable Cy-7 Dyes.

We have demonstrated an efficient synthetic route with crystal structures for the construction of acidic pH-triggered visible-to-NIR interchangeable ratiometric fluorescent pH sensors. This bioresponsive probe exhibits pH-sensitive reversible absorption/emission features, low cytotoxicity, a huge 322 nm bathochromic spectral shift with augmented quantum yield from neutral to acidic pH, high sensitivity and selective targeting ability of live-cell lysosomes with ideal pKa , off-to-on narrow NIR absorption/fluorescence signals with high molar absorption coefficient at acidic lysosomal lumen, and in-situ live-cell pH-activated ratiometric imaging of lysosomal pH. Selective staining and ratiometric pH imaging in human carcinoma live-cell lysosomes were monitored by dual-channel confocal laser scanning microscope using a pH-activatable organic fluorescent dye comprising a morpholine moiety for lysosome targeting and an acidic pH openable oxazolidine ring. Moreover, real-time tracking of lysosomes, 3D, and multicolor live-cell imaging have been achieved using the synthesized pH-activatable probe.

Glutamine Produces Ammonium to Tune Lysosomal pH and Regulate Lysosomal Function.

Glutamine is one of the most abundant amino acids in the cell. In mitochondria, glutaminases 1 and 2 (GLS1/2) hydrolyze glutamine to glutamate, which serves as the precursor of multiple metabolites. Here, we show that ammonium generated during GLS1/2-mediated glutaminolysis regulates lysosomal pH and in turn lysosomal degradation. In primary human skin fibroblasts BJ cells and mouse embryonic fibroblasts, deprivation of total amino acids for 1 h increased lysosomal degradation capacity as shown by the increased turnover of lipidated microtubule-associated proteins 1A/1B light chain 3B (LC3-II), several autophagic receptors, and endocytosed DQ-BSA. Removal of glutamine but not any other amino acids from the culture medium enhanced lysosomal degradation similarly as total amino acid starvation. The presence of glutamine in regular culture media increased lysosomal pH by >0.5 pH unit and the removal of glutamine caused lysosomal acidification. GLS1/2 knockdown, GLS1 antagonist, or ammonium scavengers reduced lysosomal pH in the presence of glutamine. The addition of glutamine or NH4Cl prevented the increase in lysosomal degradation and curtailed the extension of mTORC1 function during the early time period of amino acid starvation. Our findings suggest that glutamine tunes lysosomal pH by producing ammonium, which regulates lysosomal degradation to meet the demands of cellular activities. During the early stage of amino acid starvation, the glutamine-dependent mechanism allows more efficient use of internal reserves and endocytosed proteins to extend mTORC1 activation such that the normal anabolism is not easily interrupted by a brief disruption of the amino acid supply.

Ursolic Acid Impairs Cellular Lipid Homeostasis and Lysosomal Membrane Integrity in Breast Carcinoma Cells.

Cancer is one of the leading causes of death worldwide, thus the search for new cancer therapies is of utmost importance. Ursolic acid is a naturally occurring pentacyclic triterpene with a wide range of pharmacological activities including anti-inflammatory and anti-neoplastic effects. The latter has been assigned to its ability to promote apoptosis and inhibit cancer cell proliferation by poorly defined mechanisms. In this report, we identify lysosomes as the essential targets of the anti-cancer activity of ursolic acid. The treatment of MCF7 breast cancer cells with ursolic acid elevates lysosomal pH, alters the cellular lipid profile, and causes lysosomal membrane permeabilization and leakage of lysosomal enzymes into the cytosol. Lysosomal membrane permeabilization precedes the essential hallmarks of apoptosis placing it as an initial event in the cascade of effects induced by ursolic acid. The disruption of the lysosomal function impairs the autophagic pathway and likely partakes in the mechanism by which ursolic acid kills cancer cells. Furthermore, we find that combining treatment with ursolic acid and cationic amphiphilic drugs can significantly enhance the degree of lysosomal membrane permeabilization and cell death in breast cancer cells.

Excessive Lysosomal Stress Response and Consequently Impaired Autophagy Contribute to Fluoride-Induced Developmental Neurotoxicity.

Fluoride can cause developmental neurotoxicity; however, the precise mechanism has yet to be determined. We aimed to explore the possible role and mechanism of fluoride-induced developmental neurotoxicity, specifically the significance of the lysosomal stress response. As an in vivo model, Sprague Dawley rats were exposed to sodium fluoride (NaF) from embryo to 2months of age. We found that NaF caused autophagic flux blockage and apoptosis in the rat hippocampus. These results were validated in human neuroblastoma (SH-SY5Y) cells in vitro. In addition, in SH-SY5Y cells, NaF hindered autophagosome-lysosome fusion, decreased lysosomal degradation, and elevated lysosomal pH, which is the most prominent hallmark of a lysosomal stress response. Interestingly, rapamycin promoted autophagosome-lysosome fusion, effectively restoring autophagic flux and reducing apoptosis. Notably, bafilomycin A1, a lysosomal lumen alkalizer, unsurprisingly exacerbated the NaF-induced increase in lysosomal pH and decreased lysosomal degradability, as well as enhanced apoptosis of SH-SY5Y cells. In conclusion, our results suggest that NaF exposure initiates excessive lysosomal stress response, resulting in elevated lysosomal pH, decreased lysosomal degradation, and blocked autophagic flux, which leads to neuronal apoptosis. Thus, the lysosomal stress response may be a promising target for the prevention and treatment of fluoride-induced developmental neurotoxicity.

Ginsenoside protopanaxadiol protects adult retinal pigment epithelial-19 cells from chloroquine by modulating autophagy and apoptosis.

Chloroquine often causes serious eye and vision problems, which are mainly mediated by lysosomotropic alteration. In this study, we investigated whether the ginsenoside protopanaxadiol relieves chloroquine-induced retinopathy by restoring lysosomotropic abnormalities in human adult retinal pigment epithelial-19 cells. Cytotoxicity was assessed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Morphological alterations in autophagosomes of adult retinal pigment epithelial-19 cells was detected using confocal microscopy. Apoptosis was examined using flow cytometry, whereas cellular reactive oxygen species levels were determined by measuring the fluorescence intensity of 5-(and-6)-carboxy-2'-7'-dichlorohydrofluorescein diacetate. Lysosomal function was assessed by measuring lysosomal pH and enzyme activity. Immunoprecipitation and western blotting analyses were performed. Adult retinal pigment epithelial-19 cells accumulated autophagosomes with fusion defects in lysosomes and reactive oxygen species formation following exposure to chloroquine. This effect trapped Beclin-1 and B-cell lymphoma 2 interfering with autophagy initiation and autophagosome development. Protopanaxadiol alleviated chloroquine-induced toxicity by modulating the interaction between Beclin-1 and Bcl-2, which was mediated by the AMP-activated protein kinase-mammalian target of rapamycin signal axis. Furthermore, autophagy and apoptosis were simultaneously controlled by protopanaxadiol via upregulation of autophagy flux and decreased reactive oxygen species formation and apoptotic protein expression. These findings suggest that protopanaxadiol is a promising treatment strategy for chloroquine-mediated retinopathy.

Carbon Nanotubes as Carriers in Drug Delivery for Non-Small Cell Lung Cancer, Mechanistic Analysis of Their Carcinogenic Potential, Safety Profiling and Identification of Biomarkers.

Non-small cell lung cancer (NSCLC) is a global burden leading to millions of deaths worldwide every year. Nanomedicine refers to the use of materials at the nanoscale for drug delivery and subsequent therapeutic approaches in cancer. Carbon nanotubes (CNTs) are widely used as nanocarriers for therapeutic molecules such as plasmids, siRNAs, antisense agents, aptamers and molecules related to the immunotherapy for several cancers. They are usually functionalized and loaded with standard drug molecules to improve their therapeutic efficiency. Functionalization and drug loading possibly decrease the genotoxic and carcinogenic potential of CNTs. In addition, the targeted cytotoxic properties of the drug improve and undesired toxicity decreases after drug loading and/or conjugation with proteins, including antibodies. For intended drug delivery, a lysosomal pH of 5.5 is more suitable and effective for the slow and extended release of cytotoxic drugs than a physiological of pH 7.4. Remarkably, CNTs possess intrinsic antitumor properties and are usually internalized by endocytosis. After being internalized, several mechanisms are involved in the therapeutic and carcinogenic effects of CNTs. They are generally safe for therapy, and their toxicity profile remains dependent on their physicochemical properties. Moreover, the dose, route, duration of exposure, surface properties and degradative potential determine the toxicity outcomes of CNTs locally or systemically. In summary, the use of CNTs in drug delivery and NSCLC therapy, as well as their genotoxic and carcinogenic potential and the possible mechanisms, has been discussed in this review. The therapeutic index is generally high for NSCLC cells treated with drug-loaded CNTs; therefore, they are effective carriers in implementing targeted therapy for NSCLC.

RAB7A GTPase Is Involved in Mitophagosome Formation and Autophagosome-Lysosome Fusion in N2a Cells Treated with the Prion Protein Fragment 106-126.

Failed communication between mitochondria and lysosomes causes dysfunctional mitochondria, which may induce mitochondria-related neurodegenerative diseases. Here, we show that RAB7A, a small GTPase of the Rab family, mediates the crosstalk between these two important organelles to maintain homeostasis in N2a cells treated with PrP106-126. Specifically, we demonstrate that mitophagy deficiency in N2a cells caused by PrP106-126 is associated with dysregulated RAB7A localization in mitochondria. Cells lacking RAB7A display decreased mitochondrial colocalization with lysosomes and significantly increased mitochondrial protein expression, resulting in inhibited mitophagy. In contrast, overexpression of GTP-bound RAB7A directly induces lysosome colocalization with mitochondria. Further study revealed that GTP-bound RAB7A protects mitochondrial homeostasis by supporting autophagosome biogenesis. Moreover, wesuggest that depletion of RAB7A leads to gross morphological changes in lysosomes, which prevents autophagosome-lysosome fusion and interferes with the breakdown of autophagic cargo within lysosomes. Overexpression of GTP-bound RAB7A can also alleviate PrP106-126-induced morphological damage and dysfunction of mitochondria, reducing neuronal apoptosis. Collectively, our data demonstrate that RAB7A successfully drives mitochondria to the autophagosomal lumen for degradation, suggesting that the communication of proteotoxic stress from mitochondria to lysosomes requires RAB7A, as a signaling molecule, to establish a link between the disturbed mitochondrial network and its remodeling. These findings indicate that small molecules regulating mitophagy have the potential to modulate cellular homeostasis and the clinical course of neurodegenerative diseases. Proposed model of mitophagy regulated by RAB7A. (1) Accumulating PrP106-126 induced mitophagy. (2) RAB7A is recruited to mitochondria. (3) ATG5-12 and ATG9A (5) vesicles are recruited to the autophagosome formation sites in a RAB7A-dependent manner. The ATG5-12 complex recruits and anchors LC3-I to form active LC3-II (4), accelerating mitophagosomal formation. The ATG9A vesicles are thought to be a source of membranes for autophagosome assembly. The recruitment of proteins and lipids induces membrane expansion and subsequent closure to form the mitophagosome. (6) Maintenance of the normal low lysosomal PH depends on active (GTP-bound) RAB7A. (7) RAB7A recruits effector molecules responsible for tight membrane interactions, and directly or indirectly, the subsequent autophagosome merges with the lysosome, and the cargo is completely degraded.

Dieckol Inhibits Autophagic Flux and Induces Apoptotic Cell Death in A375 Human Melanoma Cells via Lysosomal Dysfunction and Mitochondrial Membrane Impairment.

Dieckol is a natural brown algal-derived polyphenol and its cytotoxic potential against various types of cancer cells has been studied. However, the effects of dieckol on autophagy in cancer cells remain unknown. Here, we show that dieckol inhibits the growth of A375 human melanoma cells by inducing apoptotic cell death, which is associated with lysosomal dysfunction and the inhibition of autophagic flux. Dieckol induces autophagosome accumulation by inhibiting autophagosome-lysosome fusion. Moreover, dieckol not only triggers lysosomal membrane permeabilization, followed by an increase in lysosomal pH and the inactivation of cathepsin B and D, but also causes the loss of mitochondrial membrane potential. Importantly, a cathepsin D inhibitor partially relieved dieckol-induced mitochondrial membrane impairment and caspase-mediated apoptosis. Collectively, our findings indicate that dieckol is a novel autophagy inhibitor that induces apoptosis-mediated cell death via lysosomal dysfunction and mitochondrial membrane impairment in A375 human melanoma cells. This suggests the novel potential value of dieckol as a chemotherapeutic drug candidate for melanoma treatment.

A Sensitive Fluorescent Probe with Large Stokes Shift for Real‐Time Tracking Lysosomal pH Changes in Live Cells

AbstractThe lysosome is the typical acidic organelle involved in many biological processes, the fluctuation of its pH influences its normal digestive function and even the fate of the cells. To conveniently detect and visualize lysosomal pH changes during heatstroke, we designed and synthesized a lysosomal‐targeted ratiometric pH‐sensitive fluorescent probe TCF‐LysopH. TCF‐LysopH showed an ideal pKa value (pKa=6.12) in the lysosomal pH range during heatstroke (5.00–7.00), rapid response for the changes of pH in real‐time, reversible pH switching ability, good selectivity, low cytotoxicity, and excellent lysosome‐targeting ability. The large Stokes shift of 110 nm allows us to apply the probe for fluorescence imaging in MCF‐7 cells. The probe has much better photostability (only less than 20 % decrease in intensity for 15 min irradiation) than LysoTracker Green. The fluorescence intensity became higher with the increase of pH, which is consistent with the tendency of the lysosomal pH change during heatstroke. Importantly, TCF‐LysopH was applied to measure the change of lysosomal pH during heatstroke in live cells, the results prove once again that the pH of the lysosome is increased during heatstroke. Thus, TCF‐LysopH was successfully developed as a practical and useful tool to provide a better understanding of the complicated mechanisms between lysosome pH change and heatstroke.

CUR5g, a novel autophagy inhibitor, exhibits potent synergistic anticancer effects with cisplatin against non-small-cell lung cancer

Autophagy, a highly conserved degradation process of eukaryotic cells, has been proven to be closely related to chemoresistance and metastasis of non-small-cell lung cancer (NSCLC). Autophagy inhibitors, such as chloroquine (CQ) and its derivative hydroxychloroquine (HCQ), has been shown to mediate anticancer effects in preclinical models, especially when combined with chemotherapy. However, the vast majority of autophagy inhibitors, including CQ and HCQ, actually disrupt lysosomal or/and possibly non-lysosomal processes other than autophagy. It is therefore of great significance to discover more specific autophagy inhibitors. In this study, after screening a series of curcumin derivatives synthesized in our laboratory, we found that (3E,5E)-1-methyl-3-(4-hydroxybenzylidene)-5-(3-indolymethylene)-piperidine-4-one (CUR5g) selectively inhibited autophagosome degradation in cancer cells by blocking autophagosome-lysosome fusion. CUR5g did not affect the lysosomal pH and proteolytic function, nor did it disturb cytoskeleton. CUR5g blocked the recruitment of STX17, a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein, to autophagosomes via a UVRAG-dependent mechanism, resulting in the inability of autophagosomes to fuse with lysosomes. CUR5g alone did not induce apoptosis and necrosis of A549 cells, but significantly inhibited the mobility and colony formation of A549 cells. More excitingly, CUR5g showed no obvious toxicity to normal HUVECs in vitro or mice in vivo. CUR5g enhances the cisplatin sensitivity of A549 cells and effectively inhibited autophagy in tumor tissues in vivo. Collectively, our study identified a new late-stage autophagy inhibitor and provided a novel option for NSCLC treatment, particular when combined with cisplatin.