Accumulation Of LC3-II Research Articles

Unveiling the 4-aminoquinoline derivatives as potent agents against pancreatic ductal adenocarcinoma (PDAC) cell lines

Common antimalarials such as artemisinins, chloroquine and their derivatives also possess potent anti-inflamantory, antiviral and anticancer properties. In the search for new therapeutics to combat difficult-to-treat pancreatic carcinomas, we unveiled that 4-aminoquinoline derivatives, with significant antiplasmodial properties and a great safety profile in vivo, have remarkable anticancer activity against pancreatic ductal adenocarcinoma (PDAC) and considerable efficacy in the xenograft model in vivo. The aim of the present study was to further investigate anticancer properties of these compounds in a drug-repurposing manner. The compounds showed profound cytotoxic effects at nanomolar to low micromolar concentration in 2D cultured cells (in vitro) and in the zebrafish PDAC xenograft model (in vivo). A deeper insight into their mechanisms of cytotoxic action showed these compounds induce apoptosis while increasing reactive oxygen species levels along with autophagy inhibition. Additional investigation of the autophagy modulation proved that tested quinoline derivatives cause P62 and LC3-II accumulation in PDAC cells alongside lysosomal alkalinization. Further, in vivo toxicity studies in the zebrafish model showed low toxicity without developmental side effects of the investigated 4-aminoquinolines, while the applied compounds effectively inhibited tumor growth and prevented the metastasis of xenografted pancreatic cells. Taken together, these results highlight the 4-aminoquinolines as privileged structures that ought to be investigated further for potential application in pancreatic carcinoma treatment.

Abstract We123: Cardiomyocyte Specific Sustained Atg7 Overexpression Activates Mitochondrial Autophagy in the Heart

Introduction: Accumulating evidence indicates that autophagy-related protein 7 (Atg7) is essential in cellular autophagy. Our earlier studies showed that Atg7 is required to promote macro-autophagy in the heart. However, it is unknown if the activation of Atg7 has a role in the heart to regulate selective mitochondrial autophagy (mitophagy). Objectives: We employed in vitro and in vivo approaches to delineate the molecular function and mechanisms of Atg7-mediated activation of mitophagy in the heart. Methods and Results: Subcellular fractionation and quantum dot-labeled electron microscopy revealed Atg7’s localization on the mitochondrial fraction in the heart. Atg7 co-localization to Mitotracker-labeled mitochondria was confirmed using isolated heart mitochondria. To quantitate the mitophagy, we overexpressed Atg7 in cultured primary cardiomyocytes, treated with cyanide 3-chlorophenylhydrazone (CCCP) to induce mitophagy, and stained with pH-sensitive fluorescent mitophagy dye to enumerate mitophagy dots by microscopy. We found significantly increased mitophagy dots in Atg7 overexpressed cardiomyocytes than in β-gal expressing control. To assess the activation of mitophagy in vivo , we treated the cardiac-specific Atg7 overexpressing transgenic (αMHC-Atg7 Tg) and their littermate non-transgenic (Ntg) mice with CCCP. We found an increased accumulation of mature autophagolysosomal marker protein LC3-II in isolated mitochondria from Atg7 Tg mice hearts post-CCCP treatment. To assess the extent of mitochondrial lysosomal delivery in Atg7 overexpressing hearts, we crossed αMHC-Atg7 Tg mice with cardiac-specific Mito-Keima transgenic (Mt-Keima Tg) mice. Mt-Keima Tg mice bear mitochondria-targeted pH-dependent fluorescent lysosomal protease-resistance Keima protein, enabling the measurement of mitophagy activation in vivo . We observed increased Mt-Keima high red-to-green areas in αMHC-Atg7xMt-Keima Tg mice hearts compared to Mt-Keima mice. Conclusions: Our biochemical and genetic approaches revealed that Atg7 localizes to the mitochondria, leading to the recruitment and initiation of mitophagy machinery. Our data further confirm that genetic activation of Atg7 leads to increased mitophagy activity in the heart.

Interleukin-1–mediated hyperinflammation in XIAP deficiency is associated with defective autophagy

AbstractDeficiency of X-linked inhibitor of apoptosis protein (XIAP) is a rare genetic condition that can present with recurrent episodes of hemophagocytic lymphohistiocytosis (HLH), though the exact mechanisms leading to this hyperinflammatory disorder are unclear. Understanding its biology is critical to developing targeted therapies for this potentially fatal disease. Here, we report on a novel multiexonic intragenic duplication leading to XIAP deficiency with recurrent HLH that demonstrated complete response to interleukin (IL)-1β blockade. We further demonstrate using both primary patient cells and genetically modified THP-1 monocyte cell lines that, contrary to what has previously been shown in mouse cells, XIAP-deficient human macrophages do not produce excess IL-1β when stimulated under standard conditions. Instead, nucleotide-binding oligomerization domain–like receptor family pyrin domain containing 3 (NLRP3) inflammasome–mediated hyperproduction of IL-1β is observed only when the XIAP-deficient cells are stimulated under autophagy-promoting conditions and this correlates with defective autophagic flux as measured by decreased accumulation of the early autophagy marker LC3-II. This work, therefore, highlights IL-1β blockade as a therapeutic option for patients with XIAP deficiency experiencing recurrent HLH and identifies a critical role for XIAP in promoting autophagy as a means of limiting IL-1β–mediated hyperinflammation during periods of cellular stress.

Hernandezine promotes cancer cell apoptosis and disrupts the lysosomal acidic environment and cathepsin D maturation

Hernandezine (Her), a bisbenzylisoquinoline alkaloid extracted from Thalictrum flavum, is recognized for its range of biological activities inherent to this herbal medicine. Despite its notable properties, the anti-cancer effects of Her have remained largely unexplored. In this study, we elucidated that Her significantly induced cytotoxicity in cancer cells through the activation of apoptosis and necroptosis mechanisms. Furthermore, Her triggered autophagosome formation by activating the AMPK and ATG5 conjugation systems, leading to LC3 lipidation. Our findings revealed that Her caused damage to the mitochondrial membrane, with the damaged mitochondria undergoing mitophagy, as evidenced by the elevated expression of mitophagy markers. Conversely, Her disrupted autophagic flux, demonstrated by the upregulation of p62 and accumulation of autolysosomes, as observed in the RFP-GFP-LC3 reporter assay. Initially, we determined that Her did not prevent the fusion of autophagosomes and lysosomes. However, it inhibited the maturation of cathepsin D and increased lysosomal pH, indicating an impairment of lysosomal function. The use of the early-stage autophagy inhibitor, 3-methyladenine (3-MA), did not suppress LC3II, suggesting that Her also induces noncanonical autophagy in autophagosome formation. The application of Bafilomycin A1, an inhibitor of noncanonical autophagy, diminished the recruitment of ATG16L1 and the accumulation of LC3II by Her, thereby augmenting Her-induced cell death. These observations imply that while autophagy initially plays a protective role, the disruption of the autophagic process by Her promotes programmed cell death. This study provides the first evidence of Her’s dual role in inducing apoptosis and necroptosis while also initiating and subsequently impairing autophagy to promote apoptotic cell death. These insights contribute to a deeper understanding of the mechanisms underlying programmed cell death, offering potential avenues for enhancing cancer prevention and therapeutic strategies.

Nano implant surface triggers autophagy through membrane curvature distortion to regulate the osteogenic differentiation

Anodized titania nanotubes have been considered as an effective coating for bone implants due to their ability to induce osteogenesis, whereas the osteogenic mechanism is not fully understood. Our previous study has revealed the potential role of autophagy in osteogenic regulation of nanotubular surface, whereas how the autophagy is activated remains unknown. In this study, we focused on the cell membrane curvature-sensing protein Bif-1 and its effect on the regulation of autophagy. Both autophagosomes formation and autophagic flux were enhanced on the nanotubular surface, as indicated by LC3-II accumulation and p62 degradation. In the meanwhile, the Bif-1 was significantly upregulated, which contributed to autophagy activation and osteogenic differentiation through Beclin-1/PIK3C3 signaling pathway. In conclusion, these findings have bridged the gap between extracellular physical nanotopography and intracellular autophagy activation, which may provide a deeper insight into the signaling transition from mechanical to biological across the cell membrane.

Missing WD40 Repeats in ATG16L1 Delays Canonical Autophagy and Inhibits Noncanonical Autophagy.

Canonical autophagy is an evolutionarily conserved process that forms double-membrane structures and mediates the degradation of long-lived proteins (LLPs). Noncanonical autophagy (NCA) is an important alternative pathway involving the formation of microtubule-associated protein 1 light chain 3 (LC3)-positive structures that are independent of partial core autophagy proteins. NCA has been defined by the conjugation of ATG8s to single membranes (CASM). During canonical autophagy and NCA/CASM, LC3 undergoes a lipidation modification, and ATG16L1 is a crucial protein in this process. Previous studies have reported that the WDR domain of ATG16L1 is not necessary for canonical autophagy. However, our study found that WDR domain deficiency significantly impaired LLP degradation in basal conditions and slowed down LC3-II accumulation in canonical autophagy. We further demonstrated that the observed effect was due to a reduced interaction between ATG16L1 and FIP200/WIPI2, without affecting lysosome function or fusion. Furthermore, we also found that the WDR domain of ATG16L1 is crucial for chemical-induced NCA/CASM. The results showed that removing the WDR domain or introducing the K490A mutation in ATG16L1 significantly inhibited the NCA/CASM, which interrupted the V-ATPase-ATG16L1 axis. In conclusion, this study highlights the significance of the WDR domain of ATG16L1 for both canonical autophagy and NCA functions, improving our understanding of its role in autophagy.

Role of Sphingosine Kinase 1 in Glucolipotoxicity-Induced Early Activation of Autophagy in INS-1 Pancreatic β Cells.

Insulin-producing pancreatic β cells play a crucial role in the regulation of glucose homeostasis, and their failure is a key event for diabetes development. Prolonged exposure to palmitate in the presence of elevated glucose levels, termed gluco-lipotoxicity, is known to induce β cell apoptosis. Autophagy has been proposed to be regulated by gluco-lipotoxicity in order to favor β cell survival. However, the role of palmitate metabolism in gluco-lipotoxcity-induced autophagy is presently unknown. We therefore treated INS-1 cells for 6 and 24 h with palmitate in the presence of low and high glucose concentrations and then monitored autophagy. Gluco-lipotoxicity induces accumulation of LC3-II levels in INS-1 at 6 h which returns to basal levels at 24 h. Using the RFP-GFP-LC3 probe, gluco-lipotoxicity increased both autophagosomes and autolysosmes structures, reflecting early stimulation of an autophagy flux. Triacsin C, a potent inhibitor of the long fatty acid acetyl-coA synthase, completely prevents LC3-II formation and recruitment to autophagosomes, suggesting that autophagic response requires palmitate metabolism. In contrast, etomoxir and bromo-palmitate, inhibitors of fatty acid mitochondrial β-oxidation, are unable to prevent gluco-lipotoxicity-induced LC3-II accumulation and recruitment to autophagosomes. Moreover, bromo-palmitate and etomoxir potentiate palmitate autophagic response. Even if gluco-lipotoxicity raised ceramide levels in INS-1 cells, ceramide synthase 4 overexpression does not potentiate LC3-II accumulation. Gluco-lipotoxicity also still stimulates an autophagic flux in the presence of an ER stress repressor. Finally, selective inhibition of sphingosine kinase 1 (SphK1) activity precludes gluco-lipotoxicity to induce LC3-II accumulation. Moreover, SphK1 overexpression potentiates autophagic flux induced by gluco-lipotxicity. Altogether, our results indicate that early activation of autophagy by gluco-lipotoxicity is mediated by SphK1, which plays a protective role in β cells.

Abstract LB186: Rapid photoinhibition of the autophagy pathway

Abstract Autophagy is a highly regulated and conserved cellular recycling process that is activated under stress conditions. Although cancer cells are known to rely on autophagy for survival, recent studies indicate that when the nutrient recycling process is blocked, a few rare cancer cells still can circumvent autophagy inhibition and maintain their aggressive growth rate. To understand the autophagy inhibition adaptation mechanism, we have developed a novel optogenetic autophagy inhibition tool (ASAP) that can rapidly and reversibly block the autophagy pathway in response to blue light. Our preliminary data shows ASAP releases a mutant autophagy inhibitory protein from the plasma membrane within 5 minutes of blue light exposure, blocks autophagosome-lysosome fusion, and induces LC3-II protein accumulation. In comparison, bafilomycin, a known autophagosome-lysosome fusion inhibitor, takes 4-hours to reproduce similar inhibitory effects as ASAP. Additionally, unlike bafilomycin, ASAP-induced autophagy inhibition is reversible within 2-6 hours. As light-induced autophagy inhibition has not been previously tested, our system brings a novelty to the conventional genetic and pharmacological approaches of autophagy inhibition. We will utilize the novel ASAP tool to understand the rapid changes in cellular proteomics and signaling events that dictate autophagy inhibition adaptation in cancer cells. We will perform tandem mass tag (TMT) based total proteomics and phospho-proteomics screen to visualize the temporal kinetics of protein turnover rates and changes in cell-signaling pathways following rapid autophagy inhibition. Additionally, our optogenetic tool will be applied in vivo in C. elegans to rapidly block autophagy and answer key questions of the spatial and temporal roles of autophagy in aging. Citation Format: Payel Mondal, Amanda Cyril, Christina Towers. Rapid photoinhibition of the autophagy pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB186.

The in vitro and in vivo depigmentation activity of coenzyme Q0, a major quinone derivative from Antrodia camphorata, through autophagy induction in human melanocytes and keratinocytes

BackgroundCoenzyme Q0 (CoQ0), a novel quinone derivative of Antrodia camphorata, has been utilized as a therapeutic agent (including antioxidant, anti-inflammatory, antiangiogenic, antiatherosclerotic, and anticancer agents); however, its depigmenting efficiency has yet to be studied.MethodsWe resolved the depigmenting efficiency of CoQ0 through autophagy induction in melanoma (B16F10) and melanin-feeding keratinocyte (HaCaT) cells and in vivo Zebrafish model. Then, MPLC/HPLC analysis, MTT assay, Western blotting, immunofluorescence staining, LC3 transfection, melanin formation, GFP-LC3 puncta, AVO formation, tyrosinase activity, and TEM were used.ResultsCoQ0-induced autophagy in B16F10 cells was shown by enhanced LC3-II accumulation, ATG7 expression, autophagosome GFP-LC3 puncta, and AVOs formation, and ATG4B downregulation, and Beclin-1/Bcl-2 dysregulation. In α‐MSH-stimulated B16F10 cells, CoQ0 induced antimelanogenesis by suppressing CREB-MITF pathway, tyrosinase expression/activity, and melanin formation via autophagy. TEM data disclosed that CoQ0 increased melanosome-engulfing autophagosomes and autolysosomes in α‐MSH-stimulated B16F10 cells. CoQ0-inhibited melanogenesis in α‐MSH-stimulated B16F10 cells was reversed by pretreatment with the autophagy inhibitor 3-MA or silencing of LC3. Additionally, CoQ0-induced autophagy in HaCaT cells was revealed by enhanced LC3-II accumulation, autophagosome GFP-LC3 puncta and AVO formation, ATG4B downregulation, ATG5/ATG7 expression, and Beclin-1/Bcl-2 dysregulation. In melanin-feeding HaCaT cells, CoQ0 induced melanin degradation by suppressing melanosome gp100 and melanin formation via autophagy. TEM confirmed that CoQ0 increased melanosome-engulfing autophagosomes and autolysosomes in melanin-feeding HaCaT cells. Treatment with 3-MA reversed CoQ0-mediated melanin degradation in melanin-feeding HaCaT cells. In vivo study showed that CoQ0 suppressed endogenous body pigmentation by antimelanogenesis and melanin degradation through autophagy induction in a zebrafish model.ConclusionsOur results showed that CoQ0 exerted antimelanogenesis and melanin degradation by inducing autophagy. CoQ0 could be used in skin-whitening formulations as a topical cosmetic application.

Apoptotic and Autophagic Cell Death Effects of the Hexane Extract of Tropical Marine Algae Halymenia durvillei against Human Glioblastoma Cells: In vitro and in silico Studies

Glioblastoma (GBM) considered as aggressive brain cancer with high mortality rate in patients even after surgical resection. Resistant to chemotherapy is the major problem in GBM therapy. Discovery of novel bioactive compounds from algae is being investigated as alternative sources for potential treatment as well as prevention in glioblastoma. This study revealed the effects of marine red algae extract from hexane solvent fraction of Halymenia durvillei (HDHE) on proliferation and cell death in A172 human GBM cells. HDHE decreased proliferation and promoted cell cycle arrest at G2/M phase. HDHE induced apoptotic cell death in A172 cells through mitochondrial membrane dysfunction, the decrease of anti-apoptotic Bcl-2 protein expression, and activation of caspase 3/7. Moreover, HDHE increased intracellular reactive oxygen species (ROS) production and accumulation of LC3-II, an autophagic marker. The docked conformation of palmitic acid, a major component of HDHE, showed a high affinity binding to TP53 and Beclin-1 as cell death-related target molecules. This research conclusively demonstrated that HDHE might serve as a potent anticancer agent against glioblastoma by promoting apoptotic and autophagic cell death in A172 human GBM cells. HIGHLIGHTS The palmitic acid-enriched extract of red alga Halymenia durvillei (HDHE) could inhibit proliferation of A172 human glioblastoma cells by arresting cell cycle at G2/M phase HDHE could induce both caspase-dependent apoptotic and autophagic death of A172 cells Palmitic acid, a major component of HDHE, showed a high affinity binding to TP53 and Beclin-1 The extract could be beneficial to develop as chemopreventive agents or food supplements against glioblastoma GRAPHICAL ABSTRACT

Erbin accelerates TFEB-mediated lysosome biogenesis and autophagy and alleviates sepsis-induced inflammatory responses and organ injuries

Mounting attention has been focused on defects of the autophagy-lysosomal pathway in sepsis, however, the precise mechanisms governing the autophagy-lysosomal process in sepsis are poorly known. We have previously reported that Erbin deficiency aggravated the inflammatory response and organ injuries caused by sepsis. In the present study, we found that Erbin knockout impaired the autophagy process in both muramyl dipeptide (MDP)-induced bone marrow-derived macrophages (BMDMs) and sepsis mouse liver and lung, as detected by the accumulation of LC3-II and SQSTM1/p62, and autophagosomes. Pretreatment with autophagy inhibitor chloroquine (CQ) further aggravated inflammatory response and organ injuries in vivo and in vitro sepsis model. We also observed that the impaired lysosomal function mediated autophagic blockade, as detected by the decreased expression of ATP6V, cathepsin B (CTSB) and LAMP2 protein. Immunoprecipitation revealed that the C-terminal of Erbin (aa 391–964) interacts with the N-terminal of transcription factor EB (TFEB) (aa 1–247), and affects the stability of TFEB-14-3-3 and TFEB-PPP3CB complexes and the phosphorylation status of TFEB, thereby promote the nucleus translocation of TFEB and the TFEB target genes transcription. Thus, our study suggested that Erbin alleviated sepsis-induced inflammatory responses and organ injuries by rescuing dysfunction of the autophagy-lysosomal pathway through TFEB-14-3-3 and TFEB-PPP3CB pathway.

Abstract 14526: CREG1 Deletion Inhibits Autophagy and Causes Cardiomyocyte Damage Under Nutritional Stress

Introduction: The cellular repressor of E1A-stimulated genes 1 (CREG1) is a small, highly conserved glycoprotein ubiquitously expressed in mammalian tissues. It localizes in the endosomal-lysosomal compartment and promotes lysosome biogenesis and acidification. The goal of the current study was to elucidate the physiological function of CREG1 in the mouse heart. Hypothesis: We hypothesized that deletion of Creg1 would result in pathological cardiac remodeling following nutritional stress. Methods: We generated whole-body and cardiomyocyte-specific Creg1 KO mouse lines via mating Creg1 fl/fl mice with UBC-Cre-ERT2 and Myh6-Cre mice, respectively. Mice were fasted for 16-40 hours, and hearts were evaluated by H&E staining, immunofluorescence, electron microscopy, and biochemical analysis. Knockout mice were compared to within-litter controls. Results: Under ad lib feeding, some whole-body knockout mice exhibited myofibril disarray and irregular nuclei in the heart. After prolonged fasting, both whole-body and cardiomyocyte-specific deletion of Creg1 resulted in significantly increased intracellular vacuoles, pale cytoplasm, and loss of nuclei in the papillary muscles and interventricular septa ( Figure 1 ). Immunofluorescence staining demonstrated significantly increased size and number of LC3 puncta (an autophagosome marker), and Western blot demonstrated accumulation of LC3II in the Creg1 KO hearts. In Creg1 KO cardiomyocytes, electron microscopy revealed condensed and irregular mitochondria, which lost their registry with myofibers. In addition, mitophagy was impaired and sarcomeric Z-discs were either damaged or obliterated. Conclusion: CREG1 protects the heart from nutritional stress-induced injury, likely through the promotion of autophagic flux.

CREBH promotes autophagy to ameliorate NASH by regulating Coro1a

Dysfunctional autophagy aggravates oxidative stress and inflammation in hepatocytes and accelerates the progression of nonalcoholic steatohepatitis (NASH). Here, we demonstrated that cAMP-responsive element-binding protein H (CREBH) is a transcriptional regulator of hepatic autophagy in response to diet-induced NASH. The results showed that the upregulation of CREBH in lipid-overloaded hepatocytes improves cell damage, dysfunction of autophagic flux and associated abnormal accumulation of the autophagosome marker LC3-II and autophagic substrate p62. CREBH deficiency aggravated the dysfunctional autophagy and liver injury and even caused NASH-associated liver fibrosis. In addition, the changing trend of autolysosomes and lysosome membrane-associated protein (LAMP1) was consistent with the expression level of CREBH. This result indicated that CREBH might promote autophagic degradation by restoring the formation of autolysosomes, thereby improving the blocked autophagic flux. Moreover, we observed that CREBH inhibited the expression of Coronin 1a (Coro1a), an autophagosome-lysosome fusion-related gene, through transcriptional regulation. The overexpression of Coro1a in LO2 liver cells inhibited autophagic flux and elevated inflammatory cytokine levels upon palmitic acid (PA) stimulation. Overall, our findings provide new insights into the regulatory role of CREBH in the progression of NASH and reveal that Coro1a is a novel target gene of CREBH based on the autophagy pathway.

ROS-mediated mitophagy and necroptosis regulate osteocytes death caused by TCP particles in MLO-Y4 cells

Our previous data have revealed TCP particles caused cell death of osteocytes, comprising over 95 % of all bone cells, which contribute to periprosthetic osteolysis, joint loosening and implant failure, but its mechanisms are not fully understood. Here, we reported that TCP particles inhibited cell viability of osteocytes MLO-Y4, and caused cell death. TCP particles caused mitochondrial impairment and increased expressions of LC-3 II, Parkin and PINK 1, accompanied by the elevation of autophagy flux and intracellular acidic components, the accumulation of LC-3II, PINK1 and Parkin in damaged mitochondria, and p62 reduction. The increased LC-3II expression and cell death extent were significantly enhanced by the autophagy inhibitor Baf A1, compared with Baf A1 (or TCP particles) alone, indicating that TCP particles increase autophagic flux and lead to cell even death of MLO-Y4 cells, closely associated with mitophagy. Furthermore, TCP particles induced propidium iodide (PI) uptake and the phosphorylation of RIP1, RIP3 and MLKL, thereby increasing necroptosis in MLO-Y4 cells. The pro-necroptotic effect was alleviated by the RIP1 inhibitor Nec-1 or the MLKL inhibitor NSA. Additionally, TCP particles promoted the production of intracellular reactive oxygen species (ROS) and mitochondrial ROS (mtROS), and increased TXNIP expression, but decreased protein levels of TRX1, Nrf2, HO-1 and NQO1, leading to oxidative stress. The ROS scavenger NAC remarkably reversed mitophagy and necroptosis caused by TCP particles, suggesting that ROS is responsible for mitophagy and necroptosis. Collectively, ROS-mediated mitophagy and necroptosis regulate osteocytes death caused by TCP particles in MLO-Y4 cells, which enhances osteoclastogenesis and periprosthetic osteolysis.

MAGED2 Depletion Promotes Stress-Induced Autophagy by Impairing the cAMP/PKA Pathway.

Melanoma-associated antigen D2 (MAGED2) plays an essential role in activating the cAMP/PKA pathway under hypoxic conditions, which is crucial for stimulating renal salt reabsorption and thus explaining the transient variant of Bartter's syndrome. The cAMP/PKA pathway is also known to regulate autophagy, a lysosomal degradation process induced by cellular stress. Previous studies showed that two members of the melanoma-associated antigens MAGE-family inhibit autophagy. To explore the potential role of MAGED2 in stress-induced autophagy, specific MAGED2-siRNA were used in HEK293 cells under physical hypoxia and oxidative stress (cobalt chloride, hypoxia mimetic). Depletion of MAGED2 resulted in reduced p62 levels and upregulation of both the autophagy-related genes (ATG5 and ATG12) as well as the autophagosome marker LC3II compared to control siRNA. The increase in the autophagy markers in MAGED2-depleted cells was further confirmed by leupeptin-based assay which concurred with the highest LC3II accumulation. Likewise, under hypoxia, immunofluorescence in HEK293, HeLa and U2OS cell lines demonstrated a pronounced accumulation of LC3B puncta upon MAGED2 depletion. Moreover, LC3B puncta were absent in human fetal control kidneys but markedly expressed in a fetal kidney from a MAGED2-deficient subject. Induction of autophagy with both physical hypoxia and oxidative stress suggests a potentially general role of MAGED2 under stress conditions. Various other cellular stressors (brefeldin A, tunicamycin, 2-deoxy-D-glucose, and camptothecin) were analyzed, which all induced autophagy in the absence of MAGED2. Forskolin (FSK) inhibited, whereas GNAS Knockdown induced autophagy under hypoxia. In contrast to other MAGE proteins, MAGED2 has an inhibitory role on autophagy only under stress conditions. Hence, a prominent role of MAGED2 in the regulation of autophagy under stress conditions is evident, which may also contribute to impaired fetal renal salt reabsorption by promoting autophagy of salt-transporters in patients with MAGED2 mutation.

Tranexamic acid improves psoriasis-like skin inflammation: Evidence from in vivo and in vitro studies

The chronic disease psoriasis is associated with severe inflammation and abnormal keratinocyte propagation in the skin. Tranexamic acid (TXA), a plasmin inhibitor, is used to cure serious bleeding. We investigated whether TXA ointment mitigated Imiquimod (IMQ)-induced psoriasis-like inflammation. Furthermore, this study investigated the effect of noncytotoxic concentrations of TXA on IL-17-induced human keratinocyte (HaCaT) cells to determine the status of proliferative psoriatic keratinocytes. We found that TXA reduced IMQ-induced psoriasis-like erythema, thickness, scaling, and cumulative scores (erythema plus thickness plus scaling) on the back skin of BALB/c mice. Additionally, TXA decreased ear thickness and suppressed hyperkeratosis, hyperplasia, and inflammation of the ear epidermis in IMQ-induced BALB/c mice. Furthermore, TXA inhibited IMQ-induced splenomegaly in BALB/c mouse models. In IL-17-induced HaCaT cells, TXA inhibited ROS production and IL-8 secretion. Interestingly, TXA suppressed the IL-17-induced NFκB signaling pathway via IKK-mediated IκB degradation. TXA inhibited IL-17-induced activation of the NLRP3 inflammasome through caspase-1 and IL1β expression. TXA inhibited IL-17-induced NLRP3 inflammasome activation by enhancing autophagy, as indicated by LC3-II accumulation, p62/SQSTM1 expression, ATG4B inhibition, and Beclin-1/Bcl-2 dysregulation. Notably, TXA suppressed IL-17-induced Nrf2-mediated keratin 17 expression. N-acetylcysteine pretreatment reversed the effects of TXA on NFκB, NLRP3 inflammasomes, and the Nrf2-mediated keratin 17 pathway in IL-17-induced HaCaT cells. Results further confirmed that in the ear skin of IMQ-induced mice, psoriasis biomarkers such as NLRP3, IL1β, Nrf2, and keratin 17 expression were downregulated by TXA treatment. TXA improves IMQ-induced psoriasis-like inflammation in vivo and psoriatic keratinocytes in vitro. Tranexamic acid is a promising future treatment for psoriasis.

Metformin inhibits methylglyoxal-induced retinal pigment epithelial cell death and retinopathy via AMPK-dependent mechanisms: Reversing mitochondrial dysfunction and upregulating glyoxalase 1

Diabetic retinopathy (DR) is a major cause of blindness in adult, and the accumulation of advanced glycation end products (AGEs) is a major pathologic event in DR. Methylglyoxal (MGO), a highly reactive dicarbonyl compound, is a precursor of AGEs. Although the therapeutic potential of metformin for retinopathy disorders has recently been elucidated, possibly through AMPK activation, it remains unknown how metformin directly affects the MGO-induced stress response in retinal pigment epithelial cells. Therefore, in this study, we compared the effects of metformin and the AMPK activator A769662 on MGO-induced DR in mice, as well as evaluated cytotoxicity, mitochondrial dynamic changes and dysfunction in ARPE-19 cells. We found MGO can induce mitochondrial ROS production and mitochondrial membrane potential loss, but reduce cytosolic ROS level in ARPE-19 cells. Although these effects of MGO can be reversed by both metformin and A769662, we demonstrated that reduction of mitochondrial ROS production rather than restoration of cytosolic ROS level contributes to cell protective effects of metformin and A769662. Moreover, MGO inhibits AMPK activity, reduces LC3II accumulation, and suppresses protein and gene expressions of MFN1, PGC-1α and TFAM, leading to mitochondrial fission, inhibition of mitochondrial biogenesis and autophagy. In contrast, these events of MGO were reversed by metformin in an AMPK-dependent manner as evidenced by the effects of compound C and AMPK silencing. In addition, we observed an AMPK-dependent upregulation of glyoxalase 1, a ubiquitous cellular enzyme that participates in the detoxification of MGO. In intravitreal drug-treated mice, we found that AMPK activators can reverse the MGO-induced cotton wool spots, macular edema and retinal damage. Functional, histological and optical coherence tomography analysis support the protective actions of both agents against MGO-elicited retinal damage. Metformin and A769662 via AMPK activation exert a strong protection against MGO-induced retinal pigment epithelial cell death and retinopathy. Therefore, metformin and AMPK activator can be therapeutic agents for DR.

Autophagy blockage and lysosomal dysfunction are involved in diallyl sulfide-induced inhibition of malignant growth in hepatocellular carcinoma cells.

Diallyl sulfide (DAS), as a major component of garlic extracts, has been shown to inhibit growth of hepatocellular carcinoma cells (HCC), but the underlying mechanism is still elusive. In this study, we aimed to explore the involvement of autophagy in DAS-induced growth inhibition of HepG2 and Huh7 hepatocellular carcinoma cells. We studied growth of DAS-treated HepG2 and Huh7 cells using the MTS and clonogenic assays. Autophagic flux was examined by immunofluorescence and confocal microscopy. The expression levels of autophagy-related proteins AMPK, mTOR, p62, LC3-II, LAMP1, and cathepsin D in the HepG2 and Huh7 cells treated with DAS as well as the tumors formed by HepG2 cells in the nude mice in the presence or absence of DAS were examined using western blotting and immunohistochemistry analysis. We found that DAS treatment induced activation of AMPK/mTOR, and accumulation of LC3-II and p62 both in vivo and in vitro. DAS inhibited autophagic flux through blocking the fusion of autophagosomes with lysosomes. Furthermore, DAS induced an increase in lysosomal pH and inhibition of Cathepsin D maturation. Co-treatment with an autophagy inhibitor (Chloroquine, CQ) further enhanced the growth inhibitory activity of DAS in HCC cells. Thus, our findings indicate that autophagy is involved in DAS-mediated growth inhibition of HCC cells both in vitro and in vivo.

Intranasal 15d-PGJ2 inhibits the growth of rat lactotroph pituitary neuroendocrine tumors by inducing PPARγ-dependent apoptotic and autophagic cell death.

PPARγ agonists have been reported to induce cell death in pituitary neuroendocrine tumor (PitNET) cell cultures. However, the therapeutic effects of PPARγ agonists in vivo remain unclear. In the present study, we found that intranasal 15d-PGJ2, an endogenous PPARγ agonist, resulted in growth suppression of Fischer 344 rat lactotroph PitNETs induced by subcutaneous implantation with a mini-osmotic pump containing estradiol. Intranasal 15d-PGJ2 reduced the volume and weight of the pituitary gland and the level of serum prolactin (PRL) in rat lactotroph PitNETs. 15d-PGJ2 treatment attenuated pathological changes and significantly decreased the ratio of PRL/pituitary-specific transcription factor 1 (Pit-1) and estrogen receptor α (ERα)/Pit-1 double-positive cells. Moreover, 15d-PGJ2 treatment induced apoptosis in the pituitary gland characterized by an increased ratio of TUNEL-positive cells, cleavage of caspase-3, and elevated activity of caspase-3. 15d-PGJ2 treatment decreased the levels of cytokines, including TNF-α, IL-1β, and IL-6. Furthermore, 15d-PGJ2 treatment markedly increased the protein expression of PPARγ and blocked autophagic flux, as evidenced by the accumulation of LC3-II and SQSTM1/p62 and the decrease in LAMP-1 expression. Importantly, all these effects mediated by 15d-PGJ2 were abolished by cotreatment with the PPARγ antagonist GW9662. In conclusion, intranasal 15d-PGJ2 suppressed the growth of rat lactotroph PitNETs by inducing PPARγ-dependent apoptotic and autophagic cell death. Therefore, 15d-PGJ2 may be a potential new drug for lactotroph PitNETs.

DcR3 suppresses LPS-Induced Aggresome-like induced structures in Macrophages via inhibition of ROS and p38 MAPK

Abstract Decoy receptor 3 (DcR3), also known as tumor necrosis factor receptor (TNFR) superfamily member 6b (TNFRSF6B), is a pleiotropic factor which modulates cell functions via decoy and non-decoy actions. DcR3 has been shown its anti-apoptotic and anti-inflammatory effects on cancers and inflammatory diseases. Toll-like receptors (TLRs), which are the members of pattern recognition receptors (PRRs), trigger inflammatory response against invading pathogens. However, it remains unclear whether DcR3 plays a role in TLR4-induced inflammatory response. In this study, we demonstrated that in bone marrow-derived macrophages (BMDMs) DcR3 did not affect lipopolysaccharide (LPS)-induced COX-2, iNOS, NLRP3 and pro-IL-1b protein expression. It has been reported that aggresome-like induced structures (ALIS) are formed in response to LPS in macrophages. ALIS, which containing ubiquitinated protein aggregates, are stress-induced response involved in MHC class I antigen presentation. Our findings illustrated that DcR3 can decrease LPS-induced ALIS formation via inhibiting cellular ROS production and p38 MAPK phosphorylation. In addition to LPS, ZnPP-, bafilomycin A1- and MG132-induced ubiquitinated protein expression were reduced by DcR3. Confirming the notion that ALIS is controlled by autophagy, we found that rapamycin can decrease LPS-induced ALIS formation, while bafilomycin A1 can increase LC3-II accumulation with similar extent in WT and DcR3 macrophages. Nevertheless, DcR3 expression does not change p62 and HO-1 expression induced by LPS. Taken together, despite the fact that DcR3 does not alter LPS-induced inflammatory response, our data suggest a novel role of DcR3 in suppression of ALIS formation. MOST 111-2320-B-002-028