Search Results (131)

Adipose tissue macrophages (ATMs) are key mediators of obesity-induced inflammation and insulin resistance. However, the contribution of lysosomal dysfunction to ATM inflammatory activation remains poorly defined. Here, we characterized lysosomal structural and functional alterations in ATMs during obesity and examined whether pharmacological restoration of lysosomal function using 2-hydroxypropyl-β-cyclodextrin (HPβCD) ameliorates metabolic inflammation. In diet-induced obese C57BL/6J male mice, adipose tissue exhibited increased lysosomal abundance, accompanied by reduced cathepsin L+V expression, modestly increased lysosomal acid lipase levels, and decreased expression of transcription factor EB (TFEB), a master regulator of lysosomal biogenesis. Despite expanded lysosomal content, ATMs displayed impaired lysosomal acidification, indicating functional lysosomal dysfunction. Intraperitoneal administration of HPβCD for two weeks significantly improved glucose tolerance and insulin sensitivity without affecting body weight. Flow cytometric analysis revealed reduced pro-inflammatory M1 ATMs and CD8⁺ T lymphocytes in visceral adipose tissue, whereas immune cell populations in subcutaneous adipose tissue, blood, and spleen remained unchanged. In vitro, HPβCD suppressed pro-inflammatory gene expression in both classically and metabolically activated macrophages and attenuated inflammatory responses induced by lysosomal stressors, including bafilomycin A1 and chloroquine, while restoring TFEB expression. Collectively, these findings demonstrate that obesity is associated with lysosomal dysfunction in ATMs and that restoration of lysosomal function alleviates adipose tissue inflammation and metabolic dysfunction, highlighting lysosomal regulation in ATMs as a potential therapeutic target for obesity-associated metabolic diseases. Full article

African swine fever virus (ASFV) is a highly contagious and lethal double-stranded DNA virus that relies on host cellular translation machinery for replication and immune evasion. The multigene family 110 (MGF110) contains several members with incompletely defined functions. Here, the role of MGF110-7L in host translation regulation was investigated in HEK-293T and PK15 cells. Ribopuromycylation assays demonstrated that MGF110-7L expression resulted in potent, dose- and time-dependent inhibition of nascent polypeptide synthesis. Western blotting revealed a selective reduction in eIF4G1 protein abundance, with no significant changes in eIF4G2, eIF4E, and eIF4A, while eIF4G1 mRNA levels remained unaffected, indicating post-transcriptional regulation. Overexpression of eIF4G1 partially rescued translation suppression. MGF110-7L also decreased eIF4B phosphorylation and activated the PERK/eIF2α pathway, consistent with the induction of endoplasmic reticulum (ER) stress. ER stress promoted stress granule (SG) formation and enhanced eIF4G1 association with the SG marker G3BP1. The inhibitor assays demonstrated that the suppression of eIF2α phosphorylation by ISRIB restored the abundance of eIF4G1 protein. In addition, the downregulation of eIF4G1 was reversed by the inhibition of autophagy using bafilomycin A1, indicating an SG-linked autophagy–lysosome degradation pathway. Co-immunoprecipitation assays confirmed increased eIF4G1-G3BP1 interaction, but no direct binding between MGF110-7L and eIF4G1. This work provides the first experimental evidence that an ASFV protein, MGF110-7L, suppresses cap-dependent translation through SG-mediated autophagic degradation of eIF4G1, thereby revealing a previously unrecognized mechanism of ASFV translational control. These findings not only extend current understanding of ASFV–host interactions but also suggest potential molecular targets for antiviral strategies and rational vaccine design. Full article

Lysosomal inhibition by different agents like chloroquine and bafilomycin A is known to sensitize some tumor cells to chemotherapeutic drugs. The mechanism and signaling pathways are still under investigation. We showed that chloroquine sensitized tumor cells (MCF7, SKBR3, HCT116) to drugs (carboplatin, cisplatin) treatment. Treatment with the combination of platinum drugs and chloroquine resulted in the increased rate of apoptosis compared with single agent treatment. Moreover, we demonstrated the inhibition of the resumption of cell proliferation after cell cycle arrest induced by drugs treatment. Cells treated with the combination of carboplatin (or cisplatin) and chloroquine demonstrated the significant increase in Chk1 protein phosphorylation (Ser345), which together with S-phase increase indicated the induction of replication stress compared to cells treated with carboplatin (or cisplatin) alone. The rescue experiment performed by supplementation the combination of carboplatin and chloroquine with deoxyribonucleotides (dNTPs) demonstrated the reverse of inhibition of cells’ re-proliferation after cell cycle arrest caused by this combination of drugs. Treatment with carboplatin and ATR inhibitor (ceralasertib) greatly increased the level of phospho-Chk1 and induced the replication stress, which is consistent with previous studies. Supplementation of the above drug combination with chloroquine further increased Chk1 phosphorylation and decreased the number of cells able to re-proliferate after the induced stress. Here, we also demonstrated that dNTPs’ supplementation reversed the effect of chloroquine. Similar results were obtained with the combination of carboplatin and Chk1 inhibitor (prexasertib). It was also demonstrated that chloroquine could potentiate the effect of single agent treatment of tumor cells with ATRi/Chk1i in MCF7 cells. Here, we proposed a novel explanation for the chloroquine ability to potentiate the effect of chemotherapy. The results clearly demonstrated that stress induced by chloroquine is due to its ability to increase the replication stress and to reduce the availability of nucleotides. Full article

Background/Objectives: Alzheimer’s disease (AD) is characterized by accumulation of abnormal intracellular substances and autophagy–lysosomal pathway (ALP) dysfunction. While current diagnostic methods rely on cerebrospinal fluid biomarkers and neuroimaging, minimally invasive peripheral biomarkers are needed. Dermal fibroblasts could serve as accessible reporters of AD-related molecular changes. This exploratory pilot study investigated whether ALP-associated proteins in patient-derived fibroblasts could serve as potential peripheral biomarkers for AD diagnosis. Methods: We analyzed dermal fibroblasts from 9 AD patients (amyloid Positron emission tomography (PET)-positive) and 9 age-matched controls (amyloid PET-negative). Comprehensive immunoblot analysis assessed expression profiles of 16 AD- and ALP-associated proteins. Autophagic flux and lysosomal function were evaluated using bafilomycin A1 treatment and LysoTracker staining. Diagnostic performance was assessed through receiver operating characteristic (ROC) curve analysis and multivariable logistic regression. Results: AD fibroblasts showed significantly reduced Beta-site APP cleaving enzyme 1 (BACE1) (p = 0.022) and elevated Tax1-binding protein 1 (TAX1BP1) (p = 0.035) expression. BCL2-associated athanogene proteins 2 (BAG2) and OPTN demonstrated consistent directional changes across patients. Preliminary ROC analysis showed promising performance for protein combinations, with BAG2 + OPTN achieving Area under the curve (AUC) = 0.963 (sensitivity 77.8%, specificity 88.9%). Integration with Apolipoprotein E4 (APOE4) status further enhanced diagnostic accuracy (APOE4 + BACE1: AUC = 0.914). Notably, baseline autophagic flux and lysosomal acidification were preserved, suggesting pathway-specific rather than systemic ALP dysfunction. Conclusions: This exploratory study provides preliminary evidence that dermal fibroblast-derived ALP proteins show disease-associated alterations in AD and may represent potential peripheral biomarkers. However, given the small sample size (n = 18) and lack of independent validation, these findings require confirmation in larger multi-center cohorts before clinical translation. Full article

It is well-known that EGF binding to EGFR stimulates signal transduction and endocytosis, with the latter leading to lysosomal degradation of EGFR. However, the majority of data on the regulation of endocytosis have been obtained in tumor-derived cells. Here, we perform a comprehensive analysis of the role of endolysosome acidification in the regulation of endocytic pathway in tumor cells and in endometrial MSCs as a model of proliferating, undifferentiated, non-immortalized cells. Using QD-labeled EGF, the dynamics of co-localization of EGF-receptor complexes with endocytic markers in the control and upon inhibition of V-ATPase by Bafilomycin A1 (BafA1) were studied using confocal microscopy. Image analysis showed that in HeLa and A549 cells, BafA1 significantly slowed down EGFR entry into and exit from EEA1-positive early endosomes without disrupting passage through Rab7, CD63, and Lamp1 compartments, but rather shifting it to later times. In enMSCs, only a portion of EGF-containing endosomes entered the degradation pathway, and lysosomal delivery was significantly delayed. Unlike HeLa, in enMSC early endosomes, BafA1 increased the association of EGF-QDs with EEA1, suggesting a lower pH level, which is suboptimal for EEA1-dependent fusions. It is concluded that, unlike HeLa, enMSCs form a population of pH-independent endosomes containing activated EGFR for a long time. Full article

Extracellular vesicles (EVs) secreted by glioblastoma multiforme and other types of cancer cells are key factors contributing to the aggressiveness of the disease and its resistance to therapy. Chloroquine (CHQ), a lysosomal inhibitor, has shown potential as an enhancer of temozolomide (TMZ) cytotoxicity against glioblastoma cells. Since both CHQ and TMZ are known to modulate EV secretion, we sought to investigate their potential interplay in this process. Simultaneous treatment of TMZ-sensitive (U87-MG) and TMZ-resistant (U138-MG) glioblastoma cells with TMZ and CHQ led to a synergistic upregulation of EV secretion. Although CHQ did not enhance the TMZ cytotoxicity in U87-MG cells, it synergized with the latter to upregulate the release of extracellular nucleic acids implicating activation of unconventional secretory pathways. Synergistic upregulation of the autophagy markers LC3B-II and p62 by CHQ and TMZ in both cells and EVs indicates that secretory autophagy is likely involved in the observed unconventional secretion. Moreover, a significant enrichment of caveolin-1 in small EVs highlights their potential role in modulating tumor aggressiveness. The synergy in EV upregulation was not confined to the specific biological activity of TMZ and CHQ; similar effects were observed upon co-treatments with CHQ and etoposide (a topoisomerase inhibitor) and TMZ and Bafilomycin A1 (another lysosomal inhibitor). Heightened EV release was also observed in THP-1 monocytes and macrophages treated with Bafilomycin and TMZ, highlighting a broader, cell-type-independent mechanism. These findings indicate that combined DNA damage and lysosomal inhibition synergistically stimulate secretory autophagy and EV release, potentially impacting the tumor microenvironment and driving disease progression. Full article

Endocytic uptake and lysosomal localization are suggested to be the key mechanisms underlying the toxicity of metal oxide nanoparticles (MONPs), with dissolution in the acidic milieu driving the response. In this study, we aimed to investigate if MONPs of varying solubility are similarly sequestered intracellularly, including in lysosomes and the role of the acidic lysosomal milieu on toxicity induced by copper oxide (CuO) nanoparticles (NPs), nickel oxide (NiO) NPs, aluminum oxide (Al₂O₃) NPs, and titanium dioxide (TiO₂) NPs of varying solubility in FE1 lung epithelial cells. Mitsui-7 multi-walled carbon nanotubes (MWCNTs) served as contrasts against particles. Enhanced darkfield hyperspectral imaging (EDF-HSI) with fluorescence microscopy was used to determine their potential association with lysosomes. The v-ATPase inhibitor Bafilomycin A1 (BaFA1) was used to assess the role of lysosomal acidification on toxicity. The results showed co-localization of all MONPs with lysosomes, with insoluble TiO₂ NPs showing the greatest co-localization. However, only acute toxicity induced by soluble CuO NPs was affected by the presence of BaFA1, showing a 14% improvement in relative survival. In addition, all MONPs were found to be associated with large actin aggregates; however, treatment with insoluble TiO₂ NPs, but not soluble CuO NPs, impaired the organization of F-actin and α-tubulin. These results indicate that MONPs are sequestered similarly intracellularly; however, the nature or magnitude of their toxicity is not similarly impacted by it. Future studies involving a broader variety of NPs are needed to fully understand the role of differential sequestration of NPs on cellular toxicity. Full article

Major histocompatibility complex class I (MHC-I)-mediated antigen presentation plays a pivotal role in anti-tumor immunity by enabling CD8⁺ T cells to recognize and eliminate malignant cells. In melanoma, modulation of this pathway is critical for improving the efficacy of immunotherapies. Our study demonstrates that the natural compound Cepharanthine (CEP) exhibits notable antitumor activity by enhancing MHC-I-mediated antigen presentation. CEP treatment upregulated MHC-I expression (both membrane-bound and total levels) in melanoma cells in a concentration-dependent manner, thereby improving antigen-presenting capacity. Interestingly, when autophagy was pharmacologically blocked using Bafilomycin A1, co-treatment with CEP did not lead to further elevation of MHC-I expression, suggesting that CEP’s effect is mediated through disruption of the autophagic pathway. Mechanistically, CEP induced autophagosome accumulation, as evidenced by an increase in GFP-LC3 puncta. Fluorescence imaging further confirmed that CEP selectively impaired lysosomal acidification without affecting autophagosome–lysosome fusion, thereby inhibiting late-stage autophagic flux. Furthermore, CEP treatment promoted CD8⁺ T cell infiltration into tumor tissues and enhanced the antitumor efficacy of anti-PD-1 therapy, resulting in greater tumor suppression compared to either treatment alone. The study elucidates how CEP’s selective lysosomal inhibition creates a tumor microenvironment more susceptible to immune surveillance, primarily through preserved MHC-I surface expression and subsequent T cell recognition. This work highlights CEP as a promising immunomodulatory agent and provides a potential strategy for improving the outcomes of immune checkpoint blockade therapy. Full article

The resistance of breast cancer cells to therapeutic antibodies such as anti-HER2 trastuzumab can be overcome by engaging natural killer (NK) cells for killing antibody-binding tumor cells via antibody-dependent cellular cytotoxicity (ADCC). Here, we investigated how autophagy modulation affects trastuzumab-mediated ADCC in HER2-positive JIMT1 breast cancer cells and NK cells. Autophagy inducers (rapamycin and resveratrol) had no significant impact, but the inhibitor bafilomycin nearly abolished ADCC. Protection occurred when either cancer or NK cells were pretreated, indicating dual effects. Bafilomycin reduced phosphatidylserine externalization, the loss of plasma membrane integrity, caspase-3/7 activity, and DNA fragmentation. It downregulated pro-apoptotic BAK1 and BAX without altering BCL-2. Additionally, bafilomycin decreased HER2 surface expression, impairing trastuzumab binding, and modulated immune regulators (STAT1, CD95, and PD-L1) in NK and/or in the cancer cells. Bafilomycin disrupted HER2 trafficking and induced HER2 internalization, leading to its accumulation in cytoplasmic vesicles. These findings show that autophagy inhibition by bafilomycin confers ADCC resistance by altering apoptosis, immune signaling, and HER2 dynamics. The study underscores autophagy’s role in antibody-based cancer therapy efficacy. Full article

Background/Objectives: Metabolic dysfunction-associated fatty liver disease (MAFLD) is closely associated with type 2 diabetes mellitus (T2DM), where T2DM serves as a crucial driving factor for MAFLD progression. While vitamin D (VD) demonstrates protective effects against MAFLD, the underlying mechanisms through which it influences MAFLD-related liver sinusoidal endothelial cell (LSEC) capillarization remain to be elucidated. This study aimed to explore how vitamin D ameliorates LSEC capillarization in T2DM-associated MAFLD. Methods: Culture human liver sinusoidal endothelial cells (HLSECs) according to the established protocol. After 1,25(OH)₂D₃ intervention in high glucose (HG)-induced HLSECs, determine the changes in liver sinusoidal capillarization-related proteins (LN, PLVAP), autophagy and pyroptosis levels. Observe the changes in cell lipid accumulation and fenestration structures. After adding Bafilomycin A1, MCC950, compound C and rapamycin to HLSECs, explore the therapeutic mechanism of 1,25(OH)₂D₃. After supplementing VD to MAFLD model mice, further verify the therapeutic mechanism of VD on MAFLD. Results: HG can induce the capillarization and lipid accumulation of HLSEC, increase the level of pyroptosis, and simultaneously reduce the autophagy level. Vitamin D alleviated high-glucose-induced pyroptosis (by suppressing GSDMD/NLRP3) and autophagic inhibition by activating the AMPK-mTOR axis (upregulating p-AMPK and downregulating mTOR), and improved lipid accumulation and hepatic sinusoidal capillarization. In the mouse model of MAFLD, VD supplementation can induce autophagy, inhibit pyroptosis and capillarization, and improve MAFLD. Conclusions: These results demonstrate, for the first time, that VD mitigates LSEC dysfunction through dual mechanisms: activating AMPK-dependent autophagy and inhibiting pyroptosis, providing a therapeutic rationale for VD in treating MAFLD-related sinusoidal pathology. Full article

Microplastics (MPs) are pervasive environmental pollutants that have raised concerns due to their potential toxic impacts on human health. This study investigates the impact of polyethylene (PE) and polyethylene terephthalate (PET) microplastics on Caco-2 cells, a commonly used in vitro model for the intestinal barrier. Caco-2 cells were exposed to MPs of different sizes (1 µm and 2.6 µm) for 72 h. The results demonstrated a significant decrease in cell viability, accompanied by increased reactive oxygen species (ROS) production, suggesting oxidative-stress-induced cytotoxicity. Flow cytometry and Western blot analyses revealed that the MPs induced apoptosis, as evidenced by an increased Bax/Bcl-2 ratio and caspase-3 activation. Additionally, MPs triggered autophagy, indicated by elevated LC3-II levels and decreased p62 expression. The use of bafilomycin A1 further confirmed the enhancement of autophagic flux. These findings highlight the potential cytotoxic effects of MPs on intestinal epithelial cells, raising concerns about their impact on human health. Full article

Non-alcoholic steatohepatitis (NASH), a progressive liver disease characterized by lipid accumulation and chronic inflammation, lacks effective therapies targeting its multifactorial pathogenesis. This study investigates marine-derived chondroitin sulfate (CS) as a multi-organelle modulator capable of regulating lipid metabolism, oxidative stress, and inflammation in NASH. By employing subcellular imaging and organelle-specific labeling techniques, we demonstrate that CS restores lysosomal acidification in a NASH model, enabling the reduction of lipid droplets via lysosomal–lipid droplet fusion. Concurrently, CS upregulates dynamin-related protein 1 (DRP1), driving mitochondrial terminal fission to spatially isolate reactive oxygen species (ROS) segments for mitophagy, thereby reducing ROS levels. Notably, pharmacological inhibition of lysosomal activity using chloroquine or bafilomycin A1 abolished the therapeutic effects of CS, confirming lysosomal acidification as an essential prerequisite. Collectively, these findings reveal the potential of CS as a therapeutic agent for NASH and provide critical insights into the subcellular mechanisms underlying its protective effects, thus offering a foundation for future research and therapeutic development. Full article

Mammalian renal intercalated cells are known for their role in acid secretion and maintaining acid–base balance. Herein, we discuss the theoretical reasons behind their development based on published data, focusing on the unique characteristics of renal intercalated cell biology that distinguish them from other mammalian cell types, while simultaneously attempting to explain the persistence of cells similar to intercalated cells throughout evolution. In addition, we traced these characteristics phylogenetically back to the simplest organisms. Intercalated cells have several functions and attributes. First, they contribute to kidney defense mechanisms in response to both infectious and non-infectious kidney damage. Second, intercalated cells are energized by V-ATPases in a manner similar to that of protozoa. Third, they possess T-antigens, which are commonly found in embryonic and cancer cells and which confer invasive abilities to these cells. Fourth, their plasticity enables the regeneration of other epithelial cells. These observations indicate that the origins of renal intercalated cells may be traceable back to amoeboid cells that originated from an evolutionary lineage including protists, or even to the last eukaryote common ancestor. The theoretical framework presented herein supports two predictions: first, that sponge amoebocytes possess membrane V-ATPase and are sensitive to bafilomycin, but not to ouabain; and second, that sponge amoebocytes—along with cells from diploblasts (such as Xenacoelomorpha), cnidarians, worms, fish and mollusk ionocytes, and the entire cell lineage containing V-ATPase, carbonic anhydrase, and anion exchangers (HCO₃⁻/Cl⁻)—have innate immunity, cellular dedifferentiation, and regeneration capabilities. Full article

Background: Chloroquine (CQ) has been extensively validated for its safety as an antimalarial drug. The treatment regimen combining CQ with 5-fluorouracil (5-FU) has demonstrated promising antitumor effects in both in vitro and animal models. However, the clinical application of this combination therapy still faces numerous challenges, primarily due to the unelucidated mechanistic underpinnings. Methods: We validated the synergistic effect of CQ in antitumor therapy using 5-fluorouracil and N-acetylcysteine. Subsequently, we employed lysosomal pH probes and inhibitors (5-BDBD and bafilomycin A1) to verify the mechanism of CQ in synergistic antitumor therapy. Finally, the therapeutic efficacy and underlying mechanisms of CQ were further confirmed through in vivo experiments. Results: Here, we found that CQ can inhibit the ATP-induced activation of mammalian target of rapamycin (mTOR), enhancing the inhibition of 5-FU on the proliferation and survival of tumors. Mechanistically, CQ affects the lysosomal pH value, leading to the inhibition of P2X4 receptor activity. The ATP-P2X4-mTOR axis is consequently disrupted, resulting in the weakened activation of mTOR. Conclusions: Our findings suggest that CQ may inhibit ATP-induced mTOR activation by suppressing P2X4 receptor signaling, thereby altering the apoptosis resistance of tumors. The combination of CQ and 5-FU represents a promising therapeutic strategy, particularly for mTOR-hyperactivated malignancies refractory to conventional chemotherapy. These findings not only advance our understanding of the mechanisms underlying CQ-based combination therapy but also highlight the therapeutic potential of pharmacologically targeting mTOR and its alternative pathways in combination chemotherapy regimens. Full article

Tauroursodeoxycholic acid (TUDCA) has been shown to protect against oxidative damage in retinal pigment epithelial (RPE) cells. However, the mechanisms by which it mediates these protective effects have not been thoroughly investigated in the context of age-related macular degeneration (AMD) disease onset and progression. We measured LC3-II and p62 expression via Western blot and immunohistochemistry in RPE cells treated with H₂O₂, TUDCA, or a combination of both to measure autophagy induction. To determine autophagy flux, we measured the expression of LC3-II/LC3-I in RPE cells in the presence of bafilomycin via Western blot. To determine the mechanistic pathways of TUDCA-induced autophagy, we measured the protein expression of autophagy regulators (Atg5, Beclin-1, S6, AMPK, and Akt) via Western blot. We show that TUDCA-mediated autophagy induction confers protection of RPE cells against oxidative damage via mTORC1/mTORC2 independent pathways but depends on Atg5. Our work adds to the overall understanding of RPE cell homeostasis and highlights the role of TUDCA in maintaining RPE health. Full article