Fusion Of Lysosomes Research Articles

YAP Dependent Induction of CD47-Enriched Extracellular Vesicles Inhibit Dendritic Cells Activation and Ameliorate Hepatic Ischemia-Reperfusion Injury

Hepatic ischemia-reperfusion injury (IRI) is a major cause of liver damage during liver transplantation and hepatectomy, and its amelioration is an urgent unmet clinical need. Excess inflammatory response and oxidative stress contributes to the injury and Yes-associated protein (YAP) in the Hippo signaling pathway has been proved participating in it. In this study, using hepatocyte-specific YAP knockout (YAP-HKO) by crossing Yapflox/flox mice with Alb-Cre mice, we surprisingly noticed in the serum of these mice, the amount of serum extracellular vesicles (EVs) decreased after IRI. Then we found that YAP from hepatocytes regulate EVs secretion through F-actin by increasing membrane formation, as well as inhibiting the fusion of multivesicular body (MVB) and lysosomes. We further explored the essential element of YAP-induced EVs by mass spectrometry, and noticed CD47 was one of the target that highly expressed on hepatocyte-derived EVs in YAP-dependent manner. In addition, we found that CD47+ EVs can inhibit dendritic cells (DCs) activation and inflammatory responses via binding to CD172α on the surface of DCs. Finally, we isolated CD47+ EVs in IRI model and found IRI being ameliorated after treatment, suggesting that CD47+ EVs could be a potential therapeutic strategy for the treating hepatic IRI. Our data showed that CD47-enriched EVs were released in a YAP dependent manner by hepatocytes, which could inhibit DCs activation and contribute to the amelioration of hepatic IRI. Funding Statement: National Key RD the China Postdoctoral Science Foundation, Grant/Award Number: 2020M672980; the National 13th Five-Year Science and Technology Plan Major Projects of China, Grant/Award Number: 2017ZX10203205-006-001; National Natural Science Foundation of China, Grant/Award Number: 81702393, 81770648, 81670601, 81570593, 81802897, 81802897, 81900886; Key Scientific and Technological Projects of Guangdong Province, China, Grant/Award Number: 2015B020226004, 2015A030312013, 2017A030311034; Guangdong Natural Science Foundation, China, Grant/Award Number: 2017A030310373; Science and Technology Planning Project of Guangdong Province, China, Grant/Award Number:2017B030314027, 2017B020209004, 20169013, 2017A020215178; Science and Technology Planning Project of Guangzhou, China, Grant/Award Number: 2014Y2-00200, 2014Y2-00544, 201607010024, 201604020001, 201508020262, 201400000001-3 2017. Declaration of Interests: The authors have declared no conflict of interest. Ethics Approval Statement: The study protocol was approved by the Clinical Ethics Review Board of the Third Affiliated Hospital of Sun Yat- sen University. Informed consent was obtained according to the Declaration of Helsinki. All animal studies were approved and monitored by the animal experimental ethics committee of the Third Affiliated hospital of Sun-Yat Sen University.

Bisphenol A increases TLR4-mediated inflammatory response by up-regulation of autophagy-related protein in lung of adolescent mice

In previous studies we found that bisphenol A (BPA) aggravated OVA-induced lung inflammation. The aim of this research was to determine whether BPA exposure alone also induced inflammatory response in the lungs, which mechanism was associated with TLR4/NF-κB signaling pathway and the activation of mTOR-mediated autophagy. Female C57BL/6 mice aged 4 weeks were randomly divided into three groups (10/group): control group, 0.1 and 0.2 μg mL−1 BPA groups. BPA induced the pathological changes in the lung and increased the levels of cytokines and inflammatory cells, as well as affected autophagy related proteins expression. In addition, the RAW264.7 cell culture experiment was conducted in order to confirm the role of autophagy. We found that BPA can enhance autophagy flux by enhancing autophagosome formation. It was further confirmed the details of the mechanism of action with chloroquine (CQ, a compound that inhibits the fusion of autophagosomes and lysosomes) intervention. The inhibition of autophagy led to down-regulation of expression levels associated with inflammation. This research results indicated that BPA induced inflammatory response in vitro and in vivo, and its mechanism may be related to TLR4/NF-κB signaling pathway and the activation of mTOR-mediated autophagy. After autophagy was suppressed, the inflammatory response also weakened. Our findings provide a new perspective into the mechanisms underlying inflammatory responses induced by the environmental exposure. These findings indicate that therapeutic strategies targeting autophagy may provide a new method for the treatment of inflammatory diseases.

Making peace with the beast within

Immunology One way that immune cells such as neutrophils and macrophages contribute to host defense is by engulfing microbes. This process, known as phagocytosis, typically entails the formation of an organelle around the microbes called the phagosome, which then fuses with lysosomes to facilitate degradation. However, some pathogens can survive and even thrive within phagosomes. Westman et al. investigated the process by which phagosomes sequester and control the rapidly growing fungus Candida albicans after phagocytosis. The macrophage phagosome expands to match the fungus' growth through the calcium-dependent insertion of lysosomes. If lysosomal fusion is inhibited, the phagosomes rupture, leading to NLRP3 inflammasome activation, host cell death, and the enhanced growth and escape of C. albicans . However, by tolerating the burgeoning fungus, lysosomal fusion appears to contain infection and give the host precious time to mount its defenses. Cell Host Microbe 10.1016/j.chom.2020.09.004 (2020).

Cardiac aging is regulated by autophagic disorder mediated by chronic inflammation via the Rubicon/NOX4 pathway

Abstract Background and introduction Aging is known to one of the primary causes of heart failure, in particular in the case of heart failure with preserved left-ventricular ejection fraction (HFpEF). Interestingly, recent evidences demonstrated that DNA damage occurred in aging causes autophagic disorder that contributes to aging phenotype via Rubicon (Run domain Beclin-1 interacting and cysteine-rich containing protein) in non-cardiac tissues. Purpose To elucidate whether aging-related autophagic disorder may lead to myocardial remodeling via chronic inflammation by Rubicon activation. Methods As an aging model with DNA injury, we employed mice model of progeria (mouse model of Werner syndrome that was generated by amino acid mutation exhibiting the functional deletion of DNA helixase; WRN-K577M). Results Cardiac aging markers (PARP-1, p53 and γ H2AX), and apoptosis (TUNEL) were augmented in WRN-K577M (male 18 week-old) as compared to the wild type counterpart. Consistently, cardiac oxidative stress that was measured by DHE was elevated in WRN-K577M with significant increase in oxidative stress enhancer NOX4. WRN-K577M exhibited cardiomegaly and diastolic left-ventricular (LV) dysfunction with preserved systolic LV function. Histological analysis revealed that WRN-K577M exhibited enhanced cardiac fibrosis and cardiomyocyte hypertrophy. Consistently, DNA microarray revealed significant upregulation of sixteen genes, of which ontology was hypertrophy, fibrosis, inflammation. Changes in autophagic activity was assessed by use of LC3 turnover assay and autophagic flux was evaluated by application of pharmacological inhibitor of autolysosome fusion (chloroquine) and fluorescence indicators for monitoring turnover of autophagosome (DAP green) and autolysosome (DAL green) to specify the essential step(s) of the aging-induced changes in the autophagic flux of heart. The LC3 turnover assay revealed that autophagic turnover was pathologically increased in myocardium of WRN-K577M at baseline and chloroquine (50 microg/g body weight) had no effect in WRN-K577M. Furthermore, DAL-positive spots were decreased in cardiomyocytes of WRN-K577M at rest, indicating that the impairment of autophagic flux particularly via impaired lysosome fusion may be responsible for the augmented autophagic turnover in WRN-K577M. Furthermore, we tested the impact of Rubicon. Rubicon was upregulated in heart of WRN-K577M and, more interestingly, Rubicon was found to be co-localized specifically with NOX4 in heart. Using CRISPER-CAS9 system, we generated Rubicon-KD H9C2 and Rubicon-activated HEK293. Upregulation of Rubicon revealed augmented LC3II, p62 and Beclin1 similar to WRN-K577M and augmented oxidative stress and, in contrast, downregulation of Rubicon had no effect on autophagy markers. Conclusion(s) Rubicon, the dual regulator of autophagy and inflammation is essential for autophagic disorder occurred in cardiac aging and the related oxidative stress via NOX4. Funding Acknowledgement Type of funding source: None

Stbd1 promotes glycogen clustering during endoplasmic reticulum stress and supports survival of mouse myoblasts.

ABSTRACTImbalances in endoplasmic reticulum (ER) homeostasis provoke a condition known as ER stress and activate the unfolded protein response (UPR) pathway, an evolutionarily conserved cell survival mechanism. Here, we show that mouse myoblasts respond to UPR activation by stimulating glycogenesis and the formation of α-amylase-degradable, glycogen-containing ER structures. We demonstrate that the glycogen-binding protein Stbd1 is markedly upregulated through the PERK signalling branch of the UPR pathway and is required for the build-up of glycogen structures in response to ER stress activation. In the absence of ER stress, Stbd1 overexpression is sufficient to induce glycogen clustering but does not stimulate glycogenesis. Glycogen structures induced by ER stress are degraded under conditions of glucose restriction through a process that does not depend on autophagosome–lysosome fusion. Furthermore, we provide evidence that failure to induce glycogen clustering during ER stress is associated with enhanced activation of the apoptotic pathway. Our results reveal a so far unknown response of mouse myoblasts to ER stress and uncover a novel specific function of Stbd1 in this process, which may have physiological implications during myogenic differentiation.This article has an associated First Person interview with the first author of the paper.

Lysosome Fusion Maintains Phagosome Integrity during Fungal Infection

Phagosomes must maintain membrane integrity to exert their microbicidal function. Some microorganisms, however, survive and grow within phagosomes. In such instances, phagosomes must expand to avoid rupture and microbial escape. We studied whether phagosomes regulate their size to preserve integrity during infection with the fungal pathogen Candida albicans. Phagosomes release calcium as C. albicans hyphae elongate, inducing lysosome recruitment and insertion, thereby increasing the phagosomal surface area. As hyphae grow, the expanding phagosome consumes the majority of free lysosomes. Simultaneously, lysosome biosynthesis is stimulated by activation of TFEB, a transcriptional regulator of lysosomal biogenesis. Preventing lysosomal insertion causes phagosomal rupture, NLRP3 inflammasome activation, IL-1β secretion and host-cell death. Whole-genome transcriptomic analysis demonstrate that stress responses elicited in C. albicans upon engulfment are reversed if phagosome expansion is prevented. Our findings reveal a mechanism whereby phagosomes maintain integrity while expanding, ensuring that growing pathogens remain entrapped within this microbicidal compartment.

Amitriptyline interferes with autophagy-mediated clearance of protein aggregates via inhibiting autophagosome maturation in neuronal cells

Amitriptyline is a tricyclic antidepressant commonly prescribed for major depressive disorders, as well as depressive symptoms associated with various neurological disorders. A possible correlation between the use of tricyclic antidepressants and the occurrence of Parkinson’s disease has been reported, but its underlying mechanism remains unknown. The accumulation of misfolded protein aggregates has been suggested to cause cellular toxicity and has been implicated in the common pathogenesis of neurodegenerative diseases. Here, we examined the effect of amitriptyline on protein clearance and its relevant mechanisms in neuronal cells. Amitriptyline exacerbated the accumulation of abnormal aggregates in both in vitro neuronal cells and in vivo mice brain by interfering with the (1) formation of aggresome-like aggregates and (2) autophagy-mediated clearance of aggregates. Amitriptyline upregulated LC3B-II, but LC3B-II levels did not increase further in the presence of NH4Cl, which suggests that amitriptyline inhibited autophagic flux rather than autophagy induction. Amitriptyline interfered with the fusion of autophagosome and lysosome through the activation of PI3K/Akt/mTOR pathway and Beclin 1 acetylation, and regulated lysosome positioning by increasing the interaction between proteins Arl8, SKIP, and kinesin. To the best of our knowledge, we are the first to demonstrate that amitriptyline interferes with autophagic flux by regulating the autophagosome maturation during autophagy in neuronal cells. The present study could provide neurobiological clue for the possible correlation between the amitriptyline use and the risk of developing neurodegenerative diseases.

Unsaturated mannuronate oligosaccharide ameliorates β‐amyloid pathology through autophagy in Alzheimer’s disease cell models

Unsaturated mannuronate oligosaccharide (MOS) is an enzymatic depolymerization product from alginate-derived polymannuronate (PM). In this study, we investigated for the first time the potential therapeutic effect of MOS on Alzheimer’s disease (AD) and its molecular mechanism in N2a-sw cells and 3×Tg-AD primary cortex neurons. Our results showed that MOS ranges from mannuronate dimer to mannuronate undecamer (M2-M11) with an unsaturated nonreducing terminal structure and with a double bond and 1,4-glycosidic linkages. It significantly inhibited the aggregation of amyloid-β (Aβ)1-42 oligomer, decreased expression of Aβ1-42 and reduced levels of amyloid precursor protein (APP) and BACE1. It promoted the autophagy, which involves the inactivation of mTOR signaling pathway and the facilitation of the fusion of autophagosomes and lysosomes. Finally, autophagy inhibitors blocked MOS’ anti-AD actions, confirming the involvement of autophagy. In conclusion, MOS from seaweed alginate might be a promising nutraceutical or natural medicine for AD therapy.

Induction of STK11-dependent cytoprotective autophagy in breast cancer cells upon honokiol treatment

Cancer cells hijack autophagy pathway to evade anti-cancer therapeutics. Many molecular signaling pathways associated with drug-resistance converge on autophagy induction. Honokiol (HNK), a natural phenolic compound purified from Magnolia grandiflora, has recently been shown to impede breast tumorigenesis and, in the present study, we investigated whether breast cancer cells evoke autophagy to modulate therapeutic efficacy and functional networks of HNK. Indeed, breast cancer cells exhibit increased autophagosomes-accumulation, MAP1LC3B-II/LC3B-II-conversion, expression of ATG proteins as well as elevated fusion of autophagosomes and lysosomes upon HNK treatment. Breast cancer cells treated with HNK demonstrate significant growth inhibition and apoptotic induction, and these biological processes are blunted by macroautophagy/autophagy. Consequently, inhibiting autophagosome formation, abrogating autophagosome-lysosome fusion or genetic-knockout of BECN1 and ATG7 effectively increase HNK-mediated apoptotic induction and growth inhibition. Next, we explored the functional impact of tumor suppressor STK11 in autophagy induction in HNK-treated cells. STK11-silencing abrogates LC3B-II-conversion, and blocks autophagosome/lysosome fusion and lysosomal activity as illustrated by LC3B-Rab7 co-staining and DQ-BSA assay. Our results exemplify the cytoprotective nature of autophagy invoked in HNK-treated breast cancer cells and put forth the notion that a combined strategy of autophagy inhibition with HNK would be more effective. Indeed, HNK and chloroquine (CQ) show synergistic inhibition of breast cancer cells and HNK-CQ combination treatment effectively inhibits breast tumorigenesis and metastatic progression. Tumor-dissociated cells from HNK-CQ treated tumors exhibit abrogated invasion and migration potential. Together, these results implicate that breast cancer cells undergo cytoprotective autophagy to circumvent HNK and a combined treatment with HNK and CQ can be a promising therapeutic strategy for breast cancer.

LAMP-2 Is Involved in Surface Expression of RANKL of Osteoblasts In Vitro.

Lysosome associated membrane proteins (LAMPs) are involved in several processes, among which is fusion of lysosomes with phagosomes. For the formation of multinucleated osteoclasts, the interaction between receptor activator of nuclear kappa β (RANK) and its ligand RANKL is essential. Osteoclast precursors express RANK on their membrane and RANKL is expressed by cells of the osteoblast lineage. Recently it has been suggested that the transport of RANKL to the plasma membrane is mediated by lysosomal organelles. We wondered whether LAMP-2 might play a role in transportation of RANKL to the plasma membrane of osteoblasts. To elucidate the possible function of LAMP-2 herein and in the formation of osteoclasts, we analyzed these processes in vivo and in vitro using LAMP-2-deficient mice. We found that, in the presence of macrophage colony stimulating factor (M-CSF) and RANKL, active osteoclasts were formed using bone marrow cells from calvaria and long bone mouse bone marrow. Surprisingly, an almost complete absence of osteoclast formation was found when osteoclast precursors were co-cultured with LAMP-2 deficient osteoblasts. Fluorescence-activated cell sorting FACS analysis revealed that plasma membrane-bound RANKL was strongly decreased on LAMP-2 deficient osteoblasts. These results suggest that osteoblastic LAMP-2 is required for osteoblast-induced osteoclast formation in vitro.

Syntaxin 17 inhibits ischemic neuronal injury by resuming autophagy flux and ameliorating endoplasmic reticulum stress

Previous studies have shown that syntaxin 17 (STX17) is involved in mediating the fusion of autophagosomes and lysosomes. This study aimed to investigate the role and mechanism of STX17 in neuronal injury following cerebral ischemia/reperfusion. The ischemia/reperfusion (I/R) models were established by transient middle cerebral artery occlusion (tMCAO) in mice and oxygen glucose deprivation/reperfusion (O/R) in primary cultured cortical neurons and HT22 cells. Cerebral ischemia/reperfusion significantly up-regulated the expression of STX17 in neurons. Lentivirus mediated knockdown of STX17 in neurons reduced neuronal viability and increased LDH leakage. Injection of AAV9-shSTX17 into the brain of mice then subjected to tMCAO also significantly augmented the infarct area and exacerbated neurobehavioral deficits and mortality. Depletion of STX17 caused accumulation of autophagic marker/substrate LC3 II and p62, blockade of the autophagic flux, and the accumulation of dysfunctional lysosomes. Knockdown of STX17 also aggravated endoplasmic reticulum (ER) stress-dependent neuronal apoptosis induced by ischemia/reperfusion. Importantly, induction of autophagy-lysosomal pathway and alleviation of ER stress partially rescued STX17 knockdown-induced neuronal damage. These results suggest that STX17 may ameliorate ischemia/reperfusion-induced neuronal damage by enhancing autophagy flux and reducing ER stress-dependent neuronal apoptosis.

A novel Diels-Alder adduct of mulberry leaves exerts anticancer effect through autophagy-mediated cell death.

Guangsangon E (GSE) is a novel Diels-Alder adduct isolated from leaves of Morus alba L, a traditional Chinese medicine widely applied in respiratory diseases. It is reported that GSE has cytotoxic effect on cancer cells. In our research, we investigated its anticancer effect on respiratory cancer and revealed that GSE induces autophagy and apoptosis in lung and nasopharyngeal cancer cells. We first observed that GSE inhibits cell proliferation and induces apoptosis in A549 and CNE1 cells. Meanwhile, the upregulation of autophagosome marker LC3 and increased formation of GFP-LC3 puncta demonstrates the induction of autophagy in GSE-treated cells. Moreover, GSE increases the autophagy flux by enhancing lysosomal activity and the fusion of autophagosomes and lysosomes. Next, we investigated that endoplasmic reticulum (ER) stress is involved in autophagy induction by GSE. GSE activates the ER stress through reactive oxygen species (ROS) accumulation, which can be blocked by ROS scavenger NAC. Finally, inhibition of autophagy attenuates GSE-caused cell death, termed as "autophagy-mediated cell death." Taken together, we revealed the molecular mechanism of GSE against respiratory cancer, which demonstrates great potential of GSE in the treatment of representative cancer.

Decoding three distinct states of the Syntaxin17 SNARE motif in mediating autophagosome–lysosome fusion

Syntaxin17, a key autophagosomal N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein, can associate with ATG8 family proteins SNAP29 and VAMP8 to facilitate the membrane fusion process between the double-membraned autophagosome and single-membraned lysosome in mammalian macroautophagy. However, the inherent properties of Syntaxin17 and the mechanistic basis underlying the interactions of Syntaxin17 with its binding proteins remain largely unknown. Here, using biochemical, NMR, and structural approaches, we systemically characterized Syntaxin17 as well as its interactions with ATG8 family proteins, SNAP29 and VAMP8. We discovered that Syntaxin17 alone adopts an autoinhibited conformation mediated by a direct interaction between its Habc domain and the Qa-SNARE motif. In addition, we revealed that the Qa-SNARE region of Syntaxin17 contains one LC3-interacting region (LIR) motif, which preferentially binds to GABARAP subfamily members. Importantly, the GABARAP binding of Syntaxin17 can release its autoinhibited state. The determined crystal structure of the Syntaxin17 LIR-GABARAP complex not only provides mechanistic insights into the interaction between Syntaxin17 and GABARAP but also reveals an unconventional LIR motif with a C-terminally extended 310 helix for selectively binding to ATG8 family proteins. Finally, we also elucidated structural arrangements of the autophagic Syntaxin17-SNAP29-VAMP8 SNARE core complex, and uncovered its conserved biochemical and structural characteristics common to all other SNAREs. In all, our findings reveal three distinct states of Syntaxin17, and provide mechanistic insights into the Syntaxin17-mediated autophagosome-lysosome fusion process.

Trehalose Inhibits Human Immunodeficiency Virus Type 1 Infection in Primary Human Macrophages and CD4+ T Lymphocytes through Two Distinct Mechanisms.

Autophagy is a highly conserved recycling pathway that promotes cell survival during periods of stress. We previously reported that induction of autophagy through the inhibition of the mechanistic target of rapamycin (MTOR) inhibits HIV replication in human macrophages and CD4+ T lymphocytes (T cells). However, the inhibition of MTOR has modulatory effects beyond autophagy that might affect viral replication. Here, we examined the effect on HIV replication of trehalose, a nontoxic, nonreducing disaccharide that induces autophagy through an MTOR-independent mechanism. Treatment of HIV-infected macrophages and T cells with trehalose inhibited infection in a dose-dependent manner. Uninfected and HIV-infected macrophages and T cells treated with trehalose exhibited increased markers of autophagy, including LC3B lipidation with further accumulation following bafilomycin A1 treatment, and increased levels of LAMP1, LAMP2, and RAB7 proteins required for lysosomal biogenesis and fusion. Moreover, the inhibition of HIV by trehalose was significantly reduced by knockdown of ATG5 Additionally, trehalose downregulated the expression of C-C motif chemokine receptor 5 (CCR5) in T cells and CD4 in both T cells and macrophages, which reduced HIV entry in these cells. Our data demonstrate that the naturally occurring sugar trehalose at doses safely achieved in humans inhibits HIV through two mechanisms: (i) decreased entry through the downregulation of CCR5 in T cells and decreased CD4 expression in both T cells and macrophages and (ii) degradation of intracellular HIV through the induction of MTOR-independent autophagy. These findings demonstrate that cellular mechanisms can be modulated to inhibit HIV entry and intracellular replication using a naturally occurring, nontoxic sugar.IMPORTANCE Induction of autophagy through inhibition of MTOR has been shown to inhibit HIV replication. However, inhibition of the mechanistic target of rapamycin (MTOR) has cellular effects that may alter HIV infection through other mechanisms. Here, we examined the HIV-inhibitory effects of the MTOR-independent inducer of autophagy, trehalose. Of note, we identified that in addition to the inhibition of the intracellular replication of HIV by autophagy, trehalose decreased viral entry in human primary macrophages and CD4+ T cells through the downregulation of C-C motif chemokine receptor 5 (CCR5) in T cells and CD4 in both T cells and macrophages. Thus, we showed that trehalose uniquely inhibits HIV replication through inhibition of viral entry and intracellular degradation in the two most important target cells for HIV infection.

Ursolic acid inhibits pigmentation by increasing melanosomal autophagy in B16F1 cells

Melanosomes are specialized membrane-bound organelles that are involved in melanin synthesis. Unlike melanosome biogenesis, the melanosome degradation pathway is poorly understood. Among the cellular processes, autophagy controls degradation of intracellular components by cooperating with lysosomes. In this study, we showed that ursolic acid inhibits skin pigmentation by promoting melanosomal autophagy, or melanophagy, in melanocytes. We found that B16F1 cells treated with ursolic acid suppressed alpha-melanocyte stimulating hormone (α-MSH) stimulated increase in melanin content and activated autophagy. In addition, we found that treatment with ursolic acid promotes melanosomal degradation, and bafilomycin A1 inhibition of autophagosome–lysosome fusion blocked the removal of melanosomes in α-MSH-stimulated B16F1 cells. Furthermore, depletion of the autophagy-related gene 5 (ATG5) resulted in significant suppression of ursolic acid–mediated anti-pigmentation activity and autophagy in α-MSH-treated B16F1 cells. Taken together, our results suggest that ursolic acid inhibits skin pigmentation by increasing melanosomal degradation in melanocytes.

Transcriptome profiling analysis reveals that ATP6V0E2 is involved in the lysosomal activation by anlotinib

Anlotinib is a receptor tyrosine kinase inhibitor with potential anti-neoplastic and anti-angiogenic activities. It has been approved for the treatment of non-small-cell lung cancer. Lysosomes are acidic organelles and have been implicated in various mechanisms of cancer therapeutics. However, the effect of anlotinib on lysosomal function has not been investigated. In the present study, anlotinib induces apoptosis in human colon cancer cells. Through transcriptome sequencing, we found for the first time that anlotinib treatment upregulates ATP6V0E2 (ATPase H+ Transporting V0 Subunit E2) and other lysosome-related genes expression in human colon cancer. In human colon cancer, we validated that anlotinib activates lysosomal function and enhances the fusion of autophagosomes and lysosomes. Moreover, anlotinib treatment is shown to inhibit mTOR (mammalian target of rapamycin) signaling and the activation of lysosomal function by anlotinib is mTOR dependent. Furthermore, anlotinib treatment activates TFEB, a key nuclear transcription factor that controls lysosome biogenesis and function. We found that anlotinib treatment promotes TFEB nuclear translocation and enhances its transcriptional activity. When TFEB or ATP6V0E2 are knocked down, the enhanced lysosomal function and autophagy by anlotinib are attenuated. Finally, inhibition of lysosomal function enhances anlotinib-induced cell death and tumor suppression, which may be attributed to high levels of ROS (reactive oxygen species). These findings suggest that the activation of lysosomal function protects against anlotinib-mediated cell apoptosis via regulating the cellular redox status. Taken together, our results provide novel insights into the regulatory mechanisms of anlotinib on lysosomes, and this information could facilitate the development of potential novel cancer therapeutic agents that inhibit lysosomal function.

Hederagenin potentiated cisplatin- and paclitaxel-mediated cytotoxicity by impairing autophagy in lung cancer cells

Autophagy inhibition has been demonstrated to increase the efficacy of conventional chemotherapy. In this study, we identified hederagenin, a triterpenoid derived from Hedera helix, as a potent inhibitor of autophagy and then hypothesized that hederagenin might synergize with chemotherapeutic drugs (e.g., cisplatin and paclitaxel) to kill lung cancer cells. Firstly, we observed that hederagenin induced the increased autophagosomes in lung cancer cells concomitantly with the upregulation of LC3-II and p62, which indicated the impairment of autophagic flux. The colocalization assay indicated hederagenin could not block the fusion of lysosomes and autophagosomes, whereas the lysosomal acidification might be inhibited by hederagenin as revealed by the reduced staining of acidity-sensitive reagents (i.e., Lysotracker and acridine orange). The aberrant acidic environment then impaired the function of lysosome, which was evidenced by the decrease of mature cathepsin B and cathepsin D. Lastly, hederagenin, in agree with our hypothesis, promoted pro-apoptotic effect of cisplatin and paclitaxel with the accumulation of reactive oxygen species (ROS); while the synergistic effect could be abolished by the ROS scavenger, N-acetyl-L-cysteine. These data summarily demonstrated hederagenin-induced accumulation of ROS by blocking autophagic flux potentiated the cytotoxicity of cisplatin and paclitaxel in lung cancer cells.

Development of a pH-responsive polymersome inducing endoplasmic reticulum stress and autophagy blockade.

Autophagy is involved in the occurrence and development of tumors. Here, a pH-responsive polymersome codelivering hydroxychloroquine (HCQ) and tunicamycin (Tuni) drugs is developed to simultaneously induce endoplasmic reticulum (ER) stress and autophagic flux blockade for achieving an antitumor effect and inhibiting tumor metastasis. The pH response of poly(β-amino ester) and HCQ synergistically deacidifies the lysosomes, thereby blocking the fusion of autophagosomes and lysosomes and lastly blocking autophagic flux. The function mechanism of regulating autophagy was systematically investigated on orthotopic luciferase gene-transfected, 4T1 tumor-bearing BALB/c mice through Western blot and immunohistochemistry analyses. The Tuni triggers ER stress to regulate the PERK/Akt signaling pathway to increase the autophagic level. The "autophagic stress" generated by triggering ER stress-induced autophagy and blocking autophagic flux is effective against tumors. The reduced expression of matrix metalloproteinase-2 due to ER stress and reduced focal adhesions turnover due to the blockade of autophagic flux synergistically inhibit tumor metastasis.

Palmitic acid reduces the autophagic flux in hypothalamic neurons by impairing autophagosome-lysosome fusion and endolysosomal dynamics

ABSTRACT High-fat diet (HFD)-induced obesity is associated with increased cancer risk. Long-term feeding with HFD increases the concentration of the saturated fatty acid palmitic acid (PA) in the hypothalamus. We previously showed that, in hypothalamic neuronal cells, exposure to PA inhibits the autophagic flux, which is the whole autophagic process from the synthesis of the autophagosomes, up to their lysosomal fusion and degradation. However, the mechanism by which PA impairs autophagy in hypothalamic neurons remains unknown. Here, we show that PA-mediated reduction of the autophagic flux is not caused by lysosomal dysfunction, as PA treatment does not impair lysosomal pH or the activity of cathepsin B.Instead, PA dysregulates autophagy by reducing autophagosome-lysosome fusion, which correlates with the swelling of endolysosomal compartments that show areduction in their dynamics. Finally, because lysosomes undergo constant dynamic regulation by the small Rab7 GTPase, we investigated the effect of PA treatment on its activity. Interestingly, we found PA treatment altered the activity of Rab7. Altogether, these results unveil the cellular process by which PA exposure impairs the autophagic flux. As impaired autophagy in hypothalamic neurons promotes obesity, and balanced autophagy is required to inhibit malignant transformation, this could affect tumor initiation, progression, and/or response to therapy of obesity-related cancers.

Human enteroviral infection impairs autophagy in clonal INS(832/13) cells and human pancreatic islet cells

Aim/hypothesisHuman enteroviral infections are suggested to be associated with type 1 diabetes. However, the mechanism by which enteroviruses can trigger disease remains unknown. The present study aims to investigate the impact of enterovirus on autophagy, a cellular process that regulates beta cell homeostasis, using the clonal beta cell line INS(832/13) and human islet cells as in vitro models.MethodsINS(832/13) cells and human islet cells were infected with a strain of echovirus 16 (E16), originally isolated from the stool of a child who developed type 1 diabetes-associated autoantibodies. Virus production and release was determined by 50% cell culture infectious dose (CCID50) assay and FACS analysis. The occurrence of autophagy, autophagosomes, lysosomes and autolysosomes was detected by western blot, baculoviral-mediated expression of microtubule-associated protein light chain 3 (LC3)II-GFP and LysoTracker Red, and quantified by Cellomics ArrayScan. Autophagy was also monitored with a Cyto-ID detection kit. Nutrient deprivation (low glucose [2.8 mmol/l]), amino acid starvation (Earle’s Balanced Salt Solution [EBSS]) and autophagy-modifying agents (rapamycin and chloroquine) were used in control experiments. Insulin secretion and the expression of autophagy-related (Atg) genes and genes involved in autophagosome–lysosome fusion were determined.ResultsE16-infected INS(832/13) cells displayed an accumulation of autophagosomes, compared with non-treated (NT) cells (grown in complete RPMI1640 containing 11.1 mmol/l glucose) (32.1 ± 1.7 vs 21.0 ± 1.2 μm2/cell; p = 0.05). This was accompanied by increased LC3II ratio both in E16-infected cells grown in low glucose (LG) (2.8 mmol/l) (0.42 ± 0.03 vs 0.11 ± 0.04 (arbitrary units [a.u.]); p < 0.0001) and grown in media containing 11.1 mmol/l glucose (0.37 ± 0.016 vs 0.05 ± 0.02 (a.u.); p < 0.0001). Additionally, p62 accumulated in cells after E16 infection when grown in LG (1.23 ± 0.31 vs 0.36 ± 0.12 (a.u.); p = 0.012) and grown in media containing 11.1 mmol/l glucose (1.79 ± 0.39 vs 0.66 ± 0.15 (a.u.); p = 0.0078). mRNA levels of genes involved in autophagosome formation and autophagosome–lysosome fusion remained unchanged in E16-infected cells, except Atg7, which was significantly increased when autophagy was induced by E16 infection, in combination with LG (1.48 ± 0.08-fold; p = 0.02) and at 11.1 mmol/l glucose (1.26 ± 0.2-fold; p = 0.001), compared with NT controls. Moreover, autophagosomes accumulated in E16-infected cells to the same extent as when cells were treated with the lysosomal inhibitor, chloroquine, clearly indicating that autophagosome turnover was blocked. Upon infection, there was an increased viral titre in the cell culture supernatant and a marked reduction in glucose-stimulated insulin secretion (112.9 ± 24.4 vs 209.8 ± 24.4 ng [mg protein]–1 h–1; p = 0.006), compared with uninfected controls, but cellular viability remained unaffected. Importantly, and in agreement with the observations for INS(832/13) cells, E16 infection impaired autophagic flux in primary human islet cells (46.5 ± 1.6 vs 34.4 ± 2.1 μm2/cell; p = 0.01).Conclusions/interpretationEnteroviruses disrupt beta cell autophagy by impairing the later stages of the autophagic pathway, without influencing expression of key genes involved in core autophagy machinery. This results in increased viral replication, non-lytic viral spread and accumulation of autophagic structures, all of which may contribute to beta cell demise and type 1 diabetes.Graphical abstract