Lysosomal Membrane Permeabilization Research Articles

Copper Chelation Therapy Attenuates Periodontitis Inflammation through the Cuproptosis/Autophagy/Lysosome Axis

Periodontitis development arises from the intricate interplay between bacterial biofilms and the host’s immune response, where macrophages serve pivotal roles in defense and tissue homeostasis. Here, we uncover the mitigative effect of copper chelator Tetrathiomolybdate (TTM) on periodontitis through inhibiting cuproptosis, a newly identified form of cell death which is dependent on copper. Our study reveals concurrent cuproptosis and a macrophage marker within murine models. In response to lipopolysaccharide (LPS) stimulation, macrophages exhibit elevated cuproptosis-associated markers, which are mitigated by the administration of TTM. TTM treatment enhances autophagosome expression and mitophagy-related gene expression, countering the LPS-induced inhibition of autophagy flux. TTM also attenuates the LPS-induced fusion of autophagosomes and lysosomes, the degradation of lysosomal acidic environments, lysosomal membrane permeability increase, and cathepsin B secretion. In mice with periodontitis, TTM reduces cuproptosis, enhances autophagy flux, and decreases Ctsb levels. Our findings underscore the crucial role of copper-chelating agent TTM in regulating the cuproptosis/mitophagy/lysosome pathway during periodontitis inflammation, suggesting TTM as a promising approach to alleviate macrophage dysfunction. Modulating cuproptosis through TTM treatment holds potential for periodontitis intervention.

ROS-mediated lysosomal membrane permeabilization and autophagy inhibition regulate bleomycin-induced cellular senescence

ABSTRACT Bleomycin exhibits effective chemotherapeutic activity against multiple types of tumors, and also induces various side effects, such as pulmonary fibrosis and neuronal defects, which limit the clinical application of this drug. Macroautophagy/autophagy has been recently reported to be involved in the functions of bleomycin, and yet the mechanisms of their crosstalk remain insufficiently understood. Here, we demonstrated that reactive oxygen species (ROS) produced during bleomycin activation hampered autophagy flux by inducing lysosomal membrane permeabilization (LMP) and obstructing lysosomal degradation. Exhaustion of ROS with N-acetylcysteine relieved LMP and autophagy defects. Notably, we observed that LMP and autophagy blockage preceded the emergence of cellular senescence during bleomycin treatment. In addition, promoting or inhibiting autophagy-lysosome degradation alleviated or exacerbated the phenotypes of senescence, respectively. This suggests the alternation of autophagy activity is more a regulatory mechanism than a consequence of bleomycin-induced cellular senescence. Taken together, we reveal a specific role of bleomycin-induced ROS in mediating defects of autophagic degradation and further regulating cellular senescence in vitro and in vivo. Our findings, conversely, indicate the autophagy-lysosome degradation pathway as a target for modulating the functions of bleomycin. These provide a new perspective for optimizing bleomycin as a clinically applicable chemotherapeutics devoid of severe side-effects. Abbreviations: AT2 cells: type II alveolar epithelial cells; ATG7: autophagy related 7; bEnd.3: mouse brain microvascular endothelial cells; BNIP3L: BCL2/adenovirus E1B interacting protein 3-like; CCL2: C-C motif chemokine ligand 2; CDKN1A: cyclin dependent kinase inhibitor 1A; CDKN2A: cyclin dependent kinase inhibitor 2A; FTH1: ferritin heavy polypeptide 1; γ-H2AX: phosphorylated H2A.X variant histone; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; HUVEC: human umbilical vein endothelial cells; HT22: hippocampal neuronal cell lines; Il: interleukin; LAMP: lysosomal-associated membrane protein; LMP: lysosome membrane permeabilization; MTORC1: mechanistic target of rapamycin kinase complex 1; NAC: N-acetylcysteine; NCOA4: nuclear receptor coactivator 4; PI3K: phosphoinositide 3-kinase; ROS: reactive oxygen species; RPS6KB/S6K: ribosomal protein S6 kinase; SA-GLB1/β-gal: senescence-associated galactosidase, beta 1; SAHF: senescence-associated heterochromatic foci; SASP: senescence-associated secretory phenotype; SEC62: SEC62 homolog, preprotein translocation; SEP: superecliptic pHluorin; SQSTM1/p62: sequestosome 1; TFEB: transcription factor EB

Fluoride Induces Neurocytotoxicity by Disrupting Lysosomal Iron Metabolism and Membrane Permeability.

The detrimental effects of fluoride on neurotoxicity have been widely recorded, yet the detailed mechanisms underlying these effects remain unclear. This study explores lysosomal iron metabolism in fluoride-related neurotoxicity, with a focus on the Steap3/TRPML1 axis. Utilizing sodium fluoride (NaF)-treated human neuroblastoma (SH-SY5Y) and mouse hippocampal neuron (HT22) cell lines, our research demonstrates that NaF enhances the accumulation of ferrous ions (Fe2+) in these cells, disrupting lysosomal iron metabolism through the Steap3/TRPML1 axis. Notably, NaF exposure upregulated ACSL4 and downregulated GPX4, accompanied by reduced glutathione (GSH) levels and superoxide dismutase (SOD) activity and increased malondialdehyde (MDA) levels. These changes indicate increased vulnerability to ferroptosis within neuronal cells. The iron chelator deferoxamine (DFO) mitigates this disruption. DFO binds to lysosomal Fe2+ and inhibits the Steap3/TRPML1 axis, restoring normal lysosomal iron metabolism, preventing lysosomal membrane permeabilization (LMP), and reducing neuronal cell ferroptosis. Our findings suggest that interference in lysosomal iron metabolism may mitigate fluoride-induced neurotoxicity, underscoring the critical role of the Steap3/TRPML1 axis in this pathological process.

Increased Prolylcarboxypeptidase Expression Can Serve as a Biomarker of Senescence in Culture.

Prolylcarboxypeptidase (PRCP, PCP, Lysosomal Pro-X-carboxypeptidase, Angiotensinase C) controls angiotensin- and kinin-induced cell signaling. Elevation of PRCP appears to be activated in chronic inflammatory diseases [cardiovascular disease (CVD), diabetes] in proportion to severity. Vascular endothelial cell senescence and mitochondrial dysfunction have consistently been shown in models of CVD in aging. Cellular senescence, a driver of age-related dysfunction, can differentially alter the expression of lysosomal enzymes due to lysosomal membrane permeability. There is a lack of data demonstrating the effect of age-related dysfunction on the expression and function of PRCP. To explore the changes in PRCP, the PRCP-dependent prekallikrein (PK) pathway was characterized in early- and late-passage human pulmonary artery endothelial cells (HPAECs). Detailed kinetic analysis of cells treated with high molecular weight kininogen (HK), a precursor of bradykinin (BK), and PK revealed a mechanism by which senescent HPAECs activate the generation of kallikrein upon the assembly of the HK-PK complex on HPAECs in parallel with an upregulation of PRCP and endothelial nitric oxide (NO) synthase (eNOS) and NO formation. The NO production and expression of both PRCP and eNOS increased in early-passage HPAECs and decreased in late-passage HPAECs. Low activity of PRCP in late-passage HPAECs was associated with rapid decreased telomerase reverse transcriptase mRNA levels. We also found that, with an increase in the passage number of HPAECs, reduced PRCP altered the respiration rate. These results indicated that aging dysregulates PRCP protein expression, and further studies will shed light into the complexity of the PRCP-dependent signaling pathway in aging.

Targeting lysosomal quality control as a therapeutic strategy against aging and diseases.

Previously, lysosomes were primarily referred to as the digestive organelles and recycling centers within cells. Recent discoveries have expanded the lysosomal functional scope and revealed their critical roles in nutrient sensing, epigenetic regulation, plasma membrane repair, lipid transport, ion homeostasis, and cellular stress response. Lysosomal dysfunction is also found to be associated with aging and several diseases. Therefore, function of macroautophagy, a lysosome-dependent intracellular degradation system, has been identified as one of the updated twelve hallmarks of aging. In this review, we begin by introducing the concept of lysosomal quality control (LQC), which is a cellular machinery that maintains the number, morphology, and function of lysosomes through different processes such as lysosomal biogenesis, reformation, fission, fusion, turnover, lysophagy, exocytosis, and membrane permeabilization and repair. Next, we summarize the results from studies reporting the association between LQC dysregulation and aging/various disorders. Subsequently, we explore the emerging therapeutic strategies that target distinct aspects of LQC for treating diseases and combatting aging. Lastly, we underscore the existing knowledge gap and propose potential avenues for future research.

Phenazine Cations as Anticancer Theranostics†.

The biological properties of two water-soluble organic cations based on polypyridyl structures commonly used as ligands for photoactive transition metal complexes designed to interact with biomolecules are investigated. A cytotoxicity screen employing a small panel of cell lines reveals that both cations show cytotoxicity toward cancer cells but show reduced cytotoxicity to noncancerous HEK293 cells with the more extended system being notably more active. Although it is not a singlet oxygen sensitizer, the more active cation also displayed enhanced potency on irradiation with visible light, making it active at nanomolar concentrations. Using the intrinsic luminescence of the cations, their cellular uptake was investigated in more detail, revealing that the active compound is more readily internalized than its less lipophilic analogue. Colocalization studies with established cell probes reveal that the active cation predominantly localizes within lysosomes and that irradiation leads to the disruption of mitochondrial structure and function. Stimulated emission depletion (STED) nanoscopy and transmission electron microscopy (TEM) imaging reveal that treatment results in distinct lysosomal swelling and extensive cellular vacuolization. Further imaging-based studies confirm that treatment with the active cation induces lysosomal membrane permeabilization, which triggers lysosome-dependent cell-death due to both necrosis and caspase-dependent apoptosis. A preliminary toxicity screen in the Galleria melonella animal model was carried out on both cations and revealed no detectable toxicity up to concentrations of 80 mg/kg. Taken together, these studies indicate that this class of synthetically easy-to-access photoactive compounds offers potential as novel therapeutic leads.

VCP Inhibition Augments NLRP3 Inflammasome Activation.

Lysosomal membrane permeabilization caused either via phagocytosis of particulates or the uptake of protein aggregates can trigger the activation of NLRP3 inflammasome- an intense inflammatory response that drives the release of the pro-inflammatory cytokine IL-1β by regulating the activity of CASPASE 1. The maintenance of lysosomal homeostasis and lysosomal membrane integrity is facilitated by the AAA+ ATPase, VCP/p97 (VCP). However, the relationship between VCP and NLRP3 inflammasome activity remains unexplored. Here, we demonstrate that the VCP inhibitors, DBeQ and ML240 elicit the activation of NLRP3 inflammasome in bone marrow-derived macrophages (BMDMs) when used as activation stimuli. Moreover, genetic inhibition of VCP or VCP chemical inhibition enhances lysosomal membrane damage and augments LLoME-associated NLRP3 inflammasome activation in BMDMs. Similarly, VCP inactivation also augments NLRP3 inflammasome activation mediated by aggregated alpha-synuclein fibrils and lysosomal damage. These data suggest that VCP is a participant in the complex regulation of NLRP3 inflammasome activation.

DNA damage-regulated autophagy modulator 1 prevents glioblastoma cells proliferation by regulating lysosomal function and autophagic flux stability

Glioblastoma (GBM) is the most aggressive and life-threatening brain tumor, characterized by its highly malignant and recurrent nature. DNA damage-regulated autophagy modulator 1 (DRAM-1) is a p53 target gene encoding a lysosomal protein that induces macro-autophagy and damage-induced programmed cell death in tumor growth. However, the precise mechanisms underlying how DRAM-1 affects tumor cell proliferation through regulation of lysosomal function and autophagic flux stability remain incompletely understood. We found that DRAM-1 expressions were evidently down-regulated in high-grade glioma and recurrent GBM tissues. The upregulation of DRAM-1 could increase mortality of primary cultured GBM cells. TEM analysis revealed an augmented accumulation of aberrant lysosomes in DRAM-1-overexpressing GBM cells. The assay for lysosomal pH and stability also demonstrated decreasing lysosomal membrane permeabilization (LMP) and impaired lysosomal acidity. Further research revealed the detrimental impact of lysosomal dysfunction, which impaired the autophagic flux stability and ultimately led to GBM cell death. Moreover, downregulation of mTOR phosphorylation was observed in GBM cells following upregulation of DRAM-1. In vivo and in vitro experiments additionally illustrated that the mTOR inhibitor rapamycin increased GBM cell mortality and exhibited an enhanced antitumor effect.

Bioorthogonal In Situ Polymerization of Dendritic Agents for Hijacking Lysosomes and Enhancing Antigen Presentation in Cancer Cells.

A low-generation lysine dendrimer, SPr-G2, responds to intracellular glutathione to initiate bioorthogonal in situ polymerization, resulting in the formation of large assemblies in mouse breast cancer cells. The intracellular large assemblies of SPr-G2 can interact with lysosomes to induce lysosome expansion and enhance lysosomal membrane permeabilization, leading to major histocompatibility complex class I upregulation on tumor cell surfaces and ultimately tumor cell death. Moreover, the use of the SPr-G2 dendrimer to conjugate the chemotherapeutic drug, camptothecin (CPT), can boost the therapeutic potency of CPT. Excellent antitumor effects in vitro and in vivo are obtained from the combinational treatment of the SPr-G2 dendrimer and CPT. This combinational effect also enhances antitumor immunity through promoting activation of cytotoxic T cells in tumor tissues and maturation of dendritic cells. This study can shed new light on the development of peptide dendritic agents for cancer therapy.

Abstract 2870: Analysis of cancer cells response to radiotherapy activated NBTXR3 nanoparticles

Abstract In 2011, NBTXR3 emerged as the first-in-class radioenhancer used in clinical practice for treating patients with locally advanced soft tissue sarcoma (LA-STS). NBTXR3 became the first radioenhancer to receive CE mark approval after successfully completing a phase II/III clinical study for LA-STS with a doubling of the pCR rate in the NBTXR3 arm compared with the control arm. NBTXR3 nanoparticles consist of a functionalized core of hafnium oxide, a high atomic number element, specifically designed to amplify the effects of radiotherapy (RT) within cells without additional side effects on healthy tissues. Leveraging this physical mechanism, RT-activated NBTXR3 (NBTXR3+RT) has demonstrated superior efficacy in destroying tumor cells and controlling tumor growth in numerous preclinical models and in human, surpassing the effects of RT alone, while maintaining a favorable safety profile. Preclinical studies have revealed that the benefits of NBTXR3 extend beyond mere radioenhancement and improved cancer cell destruction. Specifically, NBTXR3 has been reported to possess immunomodulatory properties through 1) improved induction of DNA damage, leading to better activation of the cGAS-STING pathway, 2) promotion of immunogenic cell death, 3) enhancement of immunopeptidome presentation, 4) generation of an antitumor immune response leading to the production of an abscopal effect mediated by CD8+ cytotoxic lymphocytes. Notably, a series of recent studies using a two-tumor mouse model of anti-PD1-resistant lung cancer that the addition of NBTXR3 significantly improved the efficacy of various treatment regimens (RT+anti-PD1 and combinations with other check-point inhibitors) in terms of tumor growth, abscopal effect, and survival. It has been demonstrated that same benefits can be achieved when NBTXR3 was combined with proton therapy. Moreover, all these studies (X-ray and proton) reported a robust activation of the antitumor immune response, restoration of the effectiveness of anti-PD1 therapy, and the induction of memory response in cured mice. However, our understanding of the biological events leading to these effects remains limited. To address this question, we conducted a series of in vitro studies that employed cytometry, microscopy, and RNA sequencing on various cancer models, both human and murine. We reported that NBTXR3 nanoparticles primarily accumulate within lysosomes following their uptake by tumor cells, leading to the activation of genes associated with the biogenesis, structure, and contents of these organelles. Upon activation by RT, NBTXR3 within lysosomes induces lysosomal membrane permeabilization (LMP), resulting in an increase in lipid peroxidation, inducing regulation of fatty acid oxidation genes, and impacting mitochondrial genes expression, such as respirasome. These findings provide a deeper insight into the early biological effects of NBTXR3+RT in comparison to RT alone. Citation Format: Jordan Da Silva, Celia Bienassis, Sebastien Paris. Analysis of cancer cells response to radiotherapy activated NBTXR3 nanoparticles [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2870.

Abstract 3271: The casein kinase 2/IKZF1 axis attenuates ceramide accumulation through up-regulation of acid ceramidase: Implications for use of CK2 inhibitors in high-risk pediatric B-cell acute lymphoid leukemia

Abstract Casein Kinase II (CK2), a growth-promoting non-oncogene, is frequently over-expressed in pediatric high-risk B-cell acute lymphoblastic leukemia (B-ALL) leading to proliferation and survival. CK2 phosphorylates the tumor suppressor IKZF1 (IKAROS) resulting in a loss-of DNA-binding activity. Loss of IKZF1 function is associated with poor prognosis in high-risk B-ALL. As such, inhibition of CK2 using silmitasertib (CX-4945), to restore IKZF1 function, is being therapeutically investigated in high-risk B-ALL. CK2 also phosphorylates/activates the mammalian ceramide synthases to increase production of the pro-apoptotic sphingolipid, ceramide (Cer). Therefore, we hypothesized that inhibition of CK2 with CX-4945 would exert both pro-death (restoration of IKAROS function) and pro-survival (decreased Cer production) effects on B-ALL cells. Moreover, this suggests that targeting the sphingolipid metabolic pathway in tandem with CX-4945 could be a novel strategy for treatment of high-risk B-ALL. To this end, we examined the effects of CX-4945 on sphingolipid levels and the enzymes of the sphingolipid metabolic pathway. Unexpectedly, we determined that CX-4945 dose-dependently increased expression of ceramide synthase 1 (CERS1) and acid ceramidase (AC) proteins, in an IKZF1 dependent manner. Increased CERS1 expression/production of C18:0 Cer has been linked to enhanced mitophagy (i.e., lysosomal clearance of damaged mitochondria) in head and neck squamous cell carcinoma. Induction of mitophagy by CX-4945 was confirmed by western blot analysis of mitofusin-1 and Drp-1 levels. Up-regulation of AC, a lysosomal protein, metabolizes C18:0 Cer to sphingosine for recycling and prevents Cer-induced lysosomal membrane permeabilization. Thus, induction of mitophagy and metabolic breakdown of Cer could potentially reduce the apoptotic efficacy of CX-4945 in B-ALL cell lines. We, therefore, tested whether inhibition of AC would synergize with CX-4945 to enhance B-ALL cell death. Indeed, synergy was observed with the AC inhibitor, SACLAC, in multiple cell lines. Consistent with the observation of synergy between CX-4945 and SACLAC, the combination also enhanced cleavage of PARP indicating an enhancement of cell death. We are currently testing the efficacy of a combination of CX-4945 and AC inhibitors in cell-line derived xenograft models of human B-ALL. Together, these findings indicate that simultaneous targeting of CK2 and AC represents a rationally selected combinatorial therapeutic strategy that overcomes the ability of cancer cells to adapt to single agent targeted therapies by altering pro- and/or anti-apoptotic pathways. Citation Format: Jeremy A. Hengst, Diwakar Bastihalli Tukaramrao, Todd Fox, Arati Sharma, Dhimant Desai, Sinisa Dovat. The casein kinase 2/IKZF1 axis attenuates ceramide accumulation through up-regulation of acid ceramidase: Implications for use of CK2 inhibitors in high-risk pediatric B-cell acute lymphoid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3271.

Rutin mitigates fluoride-induced nephrotoxicity by inhibiting ROS-mediated lysosomal membrane permeabilization and the GSDME-HMGB1 axis involved in pyroptosis and inflammation

Fluoride is known to induce nephrotoxicity; however, the underlying mechanisms remain incompletely understood. Therefore, this study aims to explore the roles and mechanisms of lysosomal membrane permeabilization (LMP) and the GSDME/HMGB1 axis in fluoride-induced nephrotoxicity and the protective effects of rutin. Rutin, a naturally occurring flavonoid compound known for its antioxidative and anti-inflammatory properties, is primarily mediated by inhibiting oxidative stress and reducing proinflammatory markers. To that end, we established in vivo and in vitro models. In the in vivo study, rats were exposed to sodium fluoride (NaF) throughout pregnancy and up until 2 months after birth. In parallel, we employed in vitro models using HK-2 cells treated with NaF, n-acetyl-L-cysteine (NAC), or rutin. We assessed lysosomal permeability through immunofluorescence and analyzed relevant protein expression via western blotting. Our findings showed that NaF exposure increased ROS levels, resulting in enhanced LMP and increased cathepsin B (CTSB) and D (CTSD) expression. Furthermore, the exposure to NaF resulted in the upregulation of cleaved PARP1, cleaved caspase-3, GSDME-N, and HMGB1 expressions, indicating cell death and inflammation-induced renal damage. Rutin mitigates fluoride-induced nephrotoxicity by suppressing ROS-mediated LMP and the GSDME/HMGB1 axis, ultimately preventing fluoride-induced renal toxicity occurrence and development. In conclusion, our findings suggest that NaF induces renal damage through ROS-mediated activation of LMP and the GSDME/HMGB1 axis, leading to pyroptosis and inflammation. Rutin, a natural antioxidative and anti-inflammatory dietary supplement, offers a novel approach to prevent and treat fluoride-induced nephrotoxicity.

Impact of Nanoheater Subcellular Localization on the Antitumor Immune Efficacy of Magnetic Hyperthermia

Magnetic nanoparticles (MNPs)-mediated magnetic hyperthermia therapy is garnering attention as a promising modality for tumor remission. This therapy combines localized nano-scale heating and reactive oxygen species-related immunologic effects. A critical aspect in optimizing this therapy involves understanding how nanoheaters’ subcellular localizations influence the therapeutic efficacy and immunological effects of magnetic hyperthermia. Herein, we investigate these effects by focusing on the strategic localization of ferrimagnetic vortex-domain iron oxide (FVIO) nanorings within lysosomes and the cytoplasm of hepatocellular carcinoma (HCC) cells. The comparative analysis revealed that lysosomal-localized magnetic hyperthermia markedly increased apoptosis in Hepa1–6 cells and effectively induced immunogenic cell death (ICD). Mechanistically, lysosomal-localized magnetic hyperthermia led to Bid-associated apoptosis and caspase-1-dependent interleukin-1β (IL-1β) secretion, likely due to enhanced lysosomal membrane permeabilization. Moreover, it reduced calreticulin degradation and increased its presence at the cell membrane, thereby boosting ICD efficiency. In contrast, cytoplasmic-localized magnetic hyperthermia showed comparatively lesser impact than its lysosomal-localized counterpart. In vivo experiments reinforced the exceptional efficacy of lysosomal-localized magnetic hyperthermia, which notably enhanced dendritic cells activation and T cells infiltration in both tumors and lymph nodes, thereby triggering a robust antitumor immune response. Additionally, lysosomal-localized magnetic hyperthermia improved tumor vaccine efficacy and demonstrated the potential for sustaining long-term immune responses in antitumor treatments. This study highlights the crucial role of subcellular positioning of magnetic nanoheaters in determining the efficacy of antitumor immunological responses in magnetic hyperthermia therapy, offering valuable insights for future cancer treatment strategies.

Sequential assembly of DNA nanoparticles inside cells enables lysosome interference and cell behavior regulation

The artificial manipulation of exogenous molecules in living cells is a promising means to regulate cellular behaviors. However, the spatiotemporal control of self-assembly within the complex intracellular environment remains grand challenge. Herein, we report a controlled sequential assembly of DNA nanoparticles in living cells, responding to acidic environment in lysosome, which enables lysosome interference and subsequently regulates a series of cellular behaviors. Two types of ultra-long DNA chains (DNA-chain-1 and 2) synthesized via rolling circle amplification were compressed by magnesium ion to two DNA nanoparticles (DNP-1 and 2), respectively. In the acidic environment of lysosomes, both DNP-1 and DNP-2 dissociated to DNA chains, and the DNA chains were subsequently assembled through base-pairing to form a hydrogel. This sequential assembly process consumed hydrogen ions within the lysosome, decreased lysosomal acidity, and thus altered the hydrolase activity and lysosomal membrane permeability. The interference of sequential assembly on lysosomes promoted cell movement in human breast adenocarcinoma MCF-7 cells, with 81.19% and 36.24% enhancement in cell migration and invasion capacity, respectively; the level of cellular autophagy was increased by 170.00%. Our study provides a new strategy for the sequential assembly of exogenous DNA materials in living cells.

Lysosomes in Cancer-At the Crossroad of Good and Evil.

Although it has been known for decades that lysosomes are central for degradation and recycling in the cell, their pivotal role as nutrient sensing signaling hubs has recently become of central interest. Since lysosomes are highly dynamic and in constant change regarding content and intracellular position, fusion/fission events allow communication between organelles in the cell, as well as cell-to-cell communication via exocytosis of lysosomal content and release of extracellular vesicles. Lysosomes also mediate different forms of regulated cell death by permeabilization of the lysosomal membrane and release of their content to the cytosol. In cancer cells, lysosomal biogenesis and autophagy are increased to support the increased metabolism and allow growth even under nutrient- and oxygen-poor conditions. Tumor cells also induce exocytosis of lysosomal content to the extracellular space to promote invasion and metastasis. However, due to the enhanced lysosomal function, cancer cells are often more susceptible to lysosomal membrane permeabilization, providing an alternative strategy to induce cell death. This review summarizes the current knowledge of cancer-associated alterations in lysosomal structure and function and illustrates how lysosomal exocytosis and release of extracellular vesicles affect disease progression. We focus on functional differences depending on lysosomal localization and the regulation of intracellular transport, and lastly provide insight how new therapeutic strategies can exploit the power of the lysosome and improve cancer treatment.

Adiponectin attenuates H2O2-induced apoptosis in chicken skeletal myoblasts through the lysosomal-mitochondrial axis.

Adiponectin has previously been investigated for exerting its protective effect against myocardial injury through anti-apoptotic and anti-oxidative actions. Therefore, the present study aimed to investigate the nature and mechanism of adiponectin inhibition of H2O2-induced apoptosis in chicken skeletal myoblasts. Skeletal muscle satellite cells were differentiated and assigned into three groups. Group C was on the blank control group, group H was stimulated with the H2O2 (500μmol/L, 4h) alone group, group A + H was pre-treated with adiponectin (10μg/mL, 24h) and stimulated with the H2O2 (500μmol/L, 4h) group. Cytotoxicity inhibited by adiponectin was evaluated by the CCK-8 assay. The degree of apoptosis and oxidative damage was investigated by the TdT-mediated dUTP nick end labeling (TUNEL) and reactive oxygen species (ROS) staining assays. Oxidative stress was assessed by evaluating lipid peroxidation, superoxide dismutase, and reduced glutathione. Acridine orange (AO) staining detected lysosomal membrane permeability. The changes in mitochondrial membrane potential (MMP) were analyzed using 5,5,6,6'-tetrachloro-1,1,3,3-tetraethylimidacarbocyanine iodide (JC-1) dye under a fluorescence microscope. The lysosomal function, mitochondrial function, and apoptosis-related mRNA and protein expression levels were quantified by real-time quantitative PCR and western blot, respectively. The results suggested that adiponectin treatment attenuated H2O2-induced cytotoxicity and oxidative stress in skeletal myoblasts. Compared with H2O2 treatment, TUNEL and ROS staining demonstrated lower apoptosis upon adiponectin treatment. AO staining confirmed the amelioration of lysosomal membrane damage, and JC-1 staining revealed an increase in mitochondrial membrane potential after adiponectin treatment. At the molecular level, adiponectin treatment inhibited the expression of the lysosomal apoptotic factors cathepsin B, chymotrypsin B, and the mitochondrial apoptotic pathway cytochrome-c (cyt-c) and caspase-8; decreased the apoptotic marker gene Bax; and increased the expression of the anti-apoptotic marker gene Bcl-2. Adiponectin treatment attenuated H2O2-induced apoptosis in skeletal myoblasts, possibly by inhibiting oxidative stress and apoptosis through the lysosomal-mitochondrial axis.

Transformable Supramolecular Self-Assembled Peptides for Cascade Self-Enhanced Ferroptosis Primed Cancer Immunotherapy.

Immunotherapy has received widespread attention for its effective and long-term tumor-eliminating ability. However, for immunogenic "cold" tumors, such as prostate cancer (PCa), the low immunogenicity of the tumor itself is a serious obstacle to efficacy. Here, this work reports a strategy to enhance PCa immunogenicity by triggering cascade self-enhanced ferroptosis in tumor cells, turning the tumor from "cold" to "hot". This work develops a transformable self-assembled peptide TEP-FFG-CRApY with alkaline phosphatase (ALP) responsiveness and glutathione peroxidase 4 (GPX4) protein targeting. TEP-FFG-CRApY self-assembles into nanoparticles under aqueous conditions and transforms into nanofibers in response to ALP during endosome/lysosome uptake into tumor cells, promoting lysosomal membrane permeabilization (LMP). On the one hand, the released TEP-FFG-CRAY nanofibers target GPX4 and selectively degrade the GPX4 protein under the light irradiation, inducing ferroptosis; on the other hand, the large amount of leaked Fe2+ further cascade to amplify the ferroptosis through the Fenton reaction. TEP-FFG-CRApY-induced immunogenic ferroptosis improves tumor cell immunogenicity by promoting the maturation of dendritic cells (DCs) and increasing intratumor T-cell infiltration. More importantly, recovered T cells further enhance ferroptosis by secreting large amounts of interferon-gamma (IFN-γ). This work provides a novel strategy for the molecular design of synergistic molecularly targeted therapy for immunogenic "cold" tumors.

The HN protein of Newcastle disease virus induces cell apoptosis through the induction of lysosomal membrane permeabilization.

Lysosomes are acidic organelles that mediate the degradation and recycling of cellular waste materials. Damage to lysosomes can cause lysosomal membrane permeabilization (LMP) and trigger different types of cell death, including apoptosis. Newcastle disease virus (NDV) can naturally infect most birds. Additionally, it serves as a promising oncolytic virus known for its effective infection of tumor cells and induction of intensive apoptotic responses. However, the involvement of lysosomes in NDV-induced apoptosis remains poorly understood. Here, we demonstrate that NDV infection profoundly triggers LMP, leading to the translocation of cathepsin B and D and subsequent mitochondria-dependent apoptosis in various tumor and avian cells. Notably, the released cathepsin B and D exacerbate NDV-induced LMP by inducing the generation of reactive oxygen species. Additionally, we uncover that the viral Hemagglutinin neuraminidase (HN) protein induces the deglycosylation and degradation of lysosome-associated membrane protein 1 (LAMP1) and LAMP2 dependent on its sialidase activity, which finally contributes to NDV-induced LMP and cellular apoptosis. Overall, our findings elucidate the role of LMP in NDV-induced cell apoptosis and provide novel insights into the function of HN during NDV-induced LMP, which provide innovative approaches for the development of NDV-based oncolytic agents.

Radiotherapy-activated NBTXR3 nanoparticles promote ferroptosis through induction of lysosomal membrane permeabilization

PurposeRadiotherapy-activated NBTXR3 (NBTXR3 + RT) has demonstrated superior efficacy in cancer cell destruction and tumor growth control, compared to radiotherapy (RT), in preclinical and clinical settings. Previous studies highlighted the immunomodulatory properties of NBTXR3 + RT, such as modification of tumor cell immunogenicity/adjuvanticity, producing an effective local tumor control and abscopal effect, related to an enhanced antitumor immune response. Furthermore, NBTXR3 + RT has shown potential in restoring anti-PD1 efficacy in a refractory tumor model. However, the early events leading to these results, such as NBTXR3 endocytosis, intracellular trafficking and primary biological responses induced by NBTXR3 + RT remain poorly understood.MethodsWe analyzed by transmission electron microscopy endocytosis and intracellular localization of NBTXR3 nanoparticles after endocytosis in various cell lines, in vitro and in vivo. A kinetic of NBTXR3 endocytosis and its impact on lysosomes was conducted using LysoTracker staining, and a RNAseq analysis was performed. We investigated the ability of NBTXR3 + RT to induce lysosomal membrane permeabilization (LMP) and ferroptosis by analyzing lipid peroxidation. Additionally, we evaluated the recapture by cancer cells of NBTXR3 released from dead cells.ResultsNBTXR3 nanoparticles were rapidly internalized by cells mainly through macropinocytosis and in a less extend by clathrin-dependent endocytosis. NBTXR3-containing endosomes were then fused with lysosomes. The day following NBTXR3 addition, we measured a significant increase in LysoTracker lysosome labeling intensity, in vitro as in vivo. Following RT, a significant lysosomal membrane permeabilization (LMP) was measured exclusively in cells treated with NBTXR3 + RT, while RT had no effect. The day post-irradiation, a significant increase in lipid peroxidation, a biomarker of ferroptosis, was measured with NBTXR3 + RT compared to RT. Moreover, we demonstrated that NBTXR3 nanoparticles released from dead cells can be recaptured by cancer cells.ConclusionsOur findings provide novel insights into the early and specific biological effects induced by NBTXR3 + RT, especially LMP, not induced by RT in our models. The subsequent significant increase in lipid peroxidation partially explains the enhanced cancer cell killing capacity of NBTXR3 + RT compared to RT, potentially by promoting ferroptosis. This study improves our understanding of the cellular mechanisms underlying NBTXR3 + RT and highlights its potential as an agnostic therapeutic strategy for solid cancers treatment.Graphical

Lysosomal BK channels facilitate silica-induced inflammation in macrophages

Background Lysosomal ion channels are proposed therapeutic targets for a number of diseases, including those driven by NLRP3 inflammasome-mediated inflammation. Here, the specific role of the lysosomal big conductance Ca2+-activated K+ (BK) channel was evaluated in a silica model of inflammation in murine macrophages. A specific-inhibitor of BK channel function, paxilline (PAX), and activators NS11021 and NS1619 were utilized to evaluate the role of lysosomal BK channel activity in silica-induced lysosomal membrane permeabilization (LMP) and NLRP3 inflammasome activation resulting in IL-1β release. Methods Murine macrophages were exposed in vitro to crystalline silica following pretreatment with BK channel inhibitors or activators and LMP, cell death, and IL-1β release were assessed. In addition, the effect of PAX treatment on silica-induced cytosolic K+ decrease was measured. Finally, the effects of BK channel modifiers on lysosomal pH, proteolytic activity, and cholesterol transport were also evaluated. Results PAX pretreatment significantly attenuated silica-induced cell death and IL-1β release. PAX caused an increase in lysosomal pH and decrease in lysosomal proteolytic activity. PAX also caused a significant accumulation of lysosomal cholesterol. BK channel activators NS11021 and NS1619 increased silica-induced cell death and IL-1β release. BK channel activation also caused a decrease in lysosomal pH and increase in lysosomal proteolytic function as well as a decrease in cholesterol accumulation. Conclusion Taken together, these results demonstrate that inhibiting lysosomal BK channel activity with PAX effectively reduced silica-induced cell death and IL-1β release. Blocking cytosolic K+ entry into the lysosome prevented LMP through the decrease of lysosomal acidification and proteolytic function and increase in lysosomal cholesterol.