bims-lymeca 2022-03-06 papers

bims-lymeca

Biomed News

on Lysosome metabolism in cancer

Issue of 2022‒03‒06
sixteen papers selected by
Harilaos Filippakis
Harvard University

Protocols to monitor TFEB activation following lysosomal damage in cultured cells using microscopy and immunoblotting.
Lysosomes and lysosome-related organelles in immune responses.
The FACT complex facilitates expression of lysosomal and antioxidant genes through binding to TFEB and TFE3.
Progranulin Insufficiency Affects Lysosomal Homeostasis in Retinal Pigment Epithelium.
Chloroquine treatment induces secretion of autophagy-related proteins and inclusion of Atg8-family proteins in distinct extracellular vesicle populations.
Compartmentation and functions of sphingolipids.
O-GlcNAcylation regulates epidermal growth factor receptor intracellular trafficking and signaling.
Gα13 loss in Kras/Tp53 mouse model of pancreatic tumorigenesis promotes tumors susceptible to rapamycin.
P2X7 Receptor Triggers Lysosomal Leakage Through Calcium Mobilization in a Mechanism Dependent on Pannexin-1 Hemichannels.
Exploiting senescence for the treatment of cancer.
Targeting ULK1 in cancer stem cells: insight from chronic myeloid leukemia.
Estrogen-Induced Extracellular Calcium Influx Promotes Endometrial Cancer Progress by Regulating Lysosomal Activity and Mitochondrial ROS.
The Microscopy-Based Assay to Study and Analyze the Recycling Endosomes using SNARE Trafficking.
Approach to lysosomal diseases.
Calcium Enabled Remote Loading of a Weak Acid Into pH-sensitive Liposomes and Augmented Cytosolic Delivery to Cancer Cells via the Proton Sponge Effect.
Endolysosomal cation channels point the way towards precision medicine of cancer and infectious diseases.

STAR Protoc. 2022 Mar 18. 3(1): 101018

Protocols to monitor TFEB activation following lysosomal damage in cultured cells using microscopy and immunoblotting.

Monami Ogura, Takayuki Shima, Tamotsu Yoshimori, Shuhei Nakamura.

  Following lysosomal damage, activation and nuclear translocation of transcription factor EB (TFEB) is the key event to maintain lysosomal homeostasis. Here, we describe steps to induce lysosomal damage in HeLa cells. This can be followed by monitoring the changes in TFEB localization using widefield fluorescence microscopy. As a complementary approach, we describe the use of immunoblotting to follow the activation and localization of TFEB in cell lysates. These protocols enable quantitative analysis of TFEB. For complete details on the use and execution of this protocol, please refer to Nakamura et al. (2020).

Keywords:  Antibody; Cell Biology; Cell culture; Cell separation/fractionation; Cell-based Assays; Microscopy

DOI:  https://doi.org/10.1016/j.xpro.2021.101018
FEBS Open Bio. 2022 Feb 26.

Lysosomes and lysosome-related organelles in immune responses.

Colin Watts.

  The perception of lysosomes has undergone a remarkable change in recent years. Formerly viewed as end-stage compartments dedicated to macromolecule catabolism enabled by a cohort of hydrolytic enzymes, which if missing or mutated could lead to lysosomal storage diseases (LSDs), they now occupy a prominent position in many other aspects of cellular and organismal physiology. In particular, lysosomes have emerged as key signalling platforms, as regulators of transcription, as agents of plasma membrane repair, in regulated cell death and in autophagy. These exciting developments have been extensively reviewed elsewhere (1-3). Here we confine the discussion to the key roles that lysosomes and lysosome related organelles (LROs) play in immunity. Even with this restricted view their functions are broad. They play a key role in pathogen detection and signalling, in processing and presentation of antigens to T lymphocytes and are closely integrated into the different life stages of dendritic cells (DC) which are key cells that link innate and adaptive immunity (4-6). Some leukocytes assemble specialised lysosome-related organelles (LROs) which can be discharged at the cell surface to achieve distinct, mostly toxic, effector functions. Proteolytic enzymes, most with an acidic pH optimum, are important for many lysosomal functions in immunity. LROs accumulate distinct proteases and other toxic agents and we will see that potential toxicity from these organelles is countered by expression of members of the cystatin and serpin families of protease inhibitors. The term endo-lysosomes rather than lysosomes is often used to refer broadly to later stage endocytic pathway organelles.

Keywords:  antigen processing; endocytosis; immune response; lysosome-related organelles; lysosomes; proteases

DOI:  https://doi.org/10.1002/2211-5463.13388
Autophagy. 2022 Mar 01. 1-17

The FACT complex facilitates expression of lysosomal and antioxidant genes through binding to TFEB and TFE3.

Eutteum Jeong, José A Martina, Pablo S Contreras, Juhyung Lee, Rosa Puertollano.

  TFEB (transcription factor EB) and TFE3 (transcription factor binding to IGHM enhancer 3) orchestrate the cellular response to a variety of stressors, including nutrient deprivation, oxidative stress and pathogens. Here we describe a novel interaction of TFEB and TFE3 with the FAcilitates Chromatin Transcription (FACT) complex, a heterodimeric histone chaperone consisting of SSRP1 and SUPT16H that mediates nucleosome disassembly and assembly, thus facilitating transcription. Extracellular stimuli, such as nutrient deprivation or oxidative stress, induce nuclear translocation and activation of TFEB and TFE3, which then associate with the FACT complex to regulate stress-induced gene transcription. Depletion of FACT does not affect TFEB activation, stability, or binding to the promoter of target genes. In contrast, reduction of FACT levels by siRNA or treatment with the FACT inhibitor curaxin, severely impairs induction of numerous antioxidant and lysosomal genes, revealing a crucial role of FACT as a regulator of cellular homeostasis. Furthermore, upregulation of antioxidant genes induced by TFEB over-expression is significantly reduced by curaxin, consistent with a role of FACT as a TFEB transcriptional activator. Together, our data show that chromatin remodeling at the promoter of stress-responsive genes by FACT is important for efficient expression of TFEB and TFE3 targets, thus providing a link between environmental changes, chromatin modifications and transcriptional regulation.Abbreviations: ADNP2, ADNP homeobox 2; ATP6V0D1, ATPase H+ transporting V0 subunit d1; ATP6V1A, ATPase H+ transporting V1 subunit A; ATP6V1C1, ATPase H+ transporting V1 subunit C1; CSNK2/CK2, casein kinase 2; CLCN7, chloride voltage-gated channel 7; CTSD, cathepsin D; CTSZ, cathepsin Z; EBSS, earle's balanced salt solution; FACT complex, facilitates chromatin transcription complex; FOXO3, forkhead box O3; HEXA, hexosaminidase subunit alpha; HIF1A, hypoxia inducible factor 1 subunit alpha; HMOX1, heme oxygenase 1; LAMP1, lysosomal associated membrane protein 1; MAFF, MAF bZIP transcription factor F; MAFG, MAF bZIP transcription factor G; MCOLN1, mucolipin TRP cation channel 1; MTORC1, mechanistic target of rapamycin kinase complex 1; NaAsO2, sodium arsenite; POLR2, RNA polymerase II; PPARGC1A, PPARG coactivator 1 alpha; PYROXD1, pyridine nucleotide-disulfide oxidoreductase domain 1; RRAGC, Ras related GTP binding C; SEC13, SEC13 homolog, nuclear pore and COPII coat complex component; SLC38A9, solute carrier family 38 member 9; SSRP1, structure specific recognition protein 1; SUPT16H, SPT16 homolog, facilitates chromatin remodeling subunit; TFEB, transcription factor EB; TFE3, transcription factor binding to IGHM enhancer 3; TXNRD1, thioredoxin reductase 1; UVRAG, UV radiation resistance associated; WDR59, WD repeat domain 59.

Keywords:  Autophagy; FACT; TFE3; TFEB; chaperone; histone; lysosomes

DOI:  https://doi.org/10.1080/15548627.2022.2029671
In Vivo. 2022 Mar-Apr;36(2):36(2): 610-617

Progranulin Insufficiency Affects Lysosomal Homeostasis in Retinal Pigment Epithelium.

Kei Takahashi, Shinsuke Nakamura, Masamitsu Shimazawa, Hideaki Hara.

  BACKGROUND: Homozygous loss-of-function progranulin gene (GRN) mutation carriers develop adult-onset neuronal ceroid lipofuscinosis (NCL), a lysosomal storage disease. Clinically, NCL patients display retinal degeneration and visual dysfunction. However, there is little information about the effects of progranulin dysfunction on lysosomal function of the retinal pigment epithelium (RPE).MATERIALS AND METHODS: We performed RNA interference knock down of progranulin in primary human RPE (hRPE) cells and observed RPE function and lysosomal activity.
RESULTS: Progranulin localized to the lysosome in RPE cells. Loss of progranulin did not affect the biogenesis of lysosomes in RPE cells, while it was necessary for the activation of lysosomal proteases. Furthermore, progranulin deficiency decreased cell viability and disrupted the cell-cell junctions.
CONCLUSION: Our results demonstrate that progranulin insufficiency disturbs lysosomal activity and physiological functions in RPE cells.

Keywords:  Progranulin; Retinal pigment epithelium; lysosome

DOI:  https://doi.org/10.21873/invivo.12744
Autophagy. 2022 Feb 28. 1-14

Chloroquine treatment induces secretion of autophagy-related proteins and inclusion of Atg8-family proteins in distinct extracellular vesicle populations.

Jing Xu, Kevin C Yang, Nancy Erro Go, Shane Colborne, Cally J Ho, Elham Hosseini-Beheshti, Alf H Lystad, Anne Simonsen, Emma Tomlinson Guns, Gregg B Morin, Sharon M Gorski.

  Chloroquine (CQ), a lysosomotropic agent, is commonly used to inhibit lysosomal degradation and macroautophagy/autophagy. Here we investigated the cell-extrinsic effects of CQ on secretion. We showed that lysosomal and autophagy inhibition by CQ altered the secretome, and induced the release of Atg8 orthologs and autophagy receptors. Atg8-family proteins, in particular, were secreted inside small extracellular vesicles (sEVs) in a lipidation-dependent manner. CQ treatment enhanced the release of Atg8-family proteins inside sEVs. Using full-length ATG16L1 and an ATG16L1 mutant that enables Atg8-family protein lipidation on double but not on single membranes, we demonstrated that LC3B is released in two distinct sEV populations: one enriched with SDCBP/Syntenin-1, CD63, and endosomal lipidated LC3B, and another that contains LC3B but is not enriched with SDCBP/Syntenin-1 or CD63, and which our data supports as originating from a double-membrane source. Our findings underscore the context-dependency of sEV heterogeneity and composition, and illustrate the integration of autophagy and sEV composition in response to lysosomal inhibition.Abbreviations: ACTB: actin beta; ANOVA: analysis of variance; ATG4B: autophagy related 4B cysteine peptidase; Atg8: autophagy related 8; ATG16L1: autophagy related 16 like 1; ATP5F1A/ATP5a: ATP synthase F1 subunit alpha; CALCOCO2: calcium binding and coiled-coil domain 2; CASP3: caspase 3; CASP7: caspase 7; CQ: chloroquine; CD9: CD9 molecule; CD63: CD63 molecule; DAPI: 4',6-diamidino-2-phenylindole; DQ-BSA: dye quenched-bovine serum albumin; ER: endoplasmic reticulum; ERN1/IRE1a: endoplasmic reticulum to nucleus signaling 1; EV: extracellular vesicles; FBS: fetal bovine serum; FDR: false discovery rate; GABARAP: GABA type A receptor-associated protein; GABARAPL2: GABA type A receptor associated protein like 2; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; GO: gene ontology; HCQ: hydroxychloroquine; HSP90AA1: heat shock protein 90 alpha family class A member 1; IP: immunoprecipitation; KO: knockout; LAMP2: lysosomal associated membrane protein 2; LIR: LC3-interacting region; LMNA: lamin A/C; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MS: mass spectrometry; NBR1: NBR1 autophagy cargo receptor; NCOA4: nuclear receptor coactivator 4; NTA: nanoparticle tracking analysis; PE: phosphatidylethanolamine; PECA: probe-level expression change averaging; SDCBP/syntenin-1: syndecan binding protein; SD: standard deviation; SE: secreted; sEV: small extracellular vesicles; SQSTM1/p62: sequestosome 1; TAX1BP1: Tax1 binding protein 1; TEM: transmission electron microscopy; TMT: tandem-mass tag; TSG101: tumor susceptibility 101; ULK1: unc-51 like autophagy activating kinase 1; WC: whole cell.

Keywords:  ATG16L1; Atg8; CD63; MAP1LC3B; SDCBP/syntenin-1; autophagy; chloroquine; endosome; extracellular vesicle; lysosome

DOI:  https://doi.org/10.1080/15548627.2022.2039535
Curr Opin Cell Biol. 2022 Feb 25. pii: S0955-0674(22)00007-2. [Epub ahead of print]74 104-111

Compartmentation and functions of sphingolipids.

Carolin Körner, Florian Fröhlich.

Sphingolipids (SLs) are one of the three major lipid classes in all eukaryotic cells. They function as structural molecules of membranes and can also act as highly active signaling molecules. SL biosynthesis is mainly occurring at the endoplasmic reticulum and the Golgi apparatus. However, SL intermediates are also generated at other organelles such as the plasma membrane and the lysosome. SL biosynthesis is therefore highly compartmentalized. Maintaining SL levels is necessary for the function of multiple trafficking pathways. One major challenge is to decipher the complex regulatory networks controlling SL biosynthesis, the coordination of vesicular and non-vesicular SL transport as well as their role in trafficking. Recent investigations have shed new light on the regulation of SL biosynthesis. Here, we review how SL biosynthesis is coordinated, how SLs are transported and how their levels affect trafficking pathways. Finally, we discuss recently developed methods to study SL metabolism with spatio-temporal resolution.

DOI: https://doi.org/10.1016/j.ceb.2022.01.006
Proc Natl Acad Sci U S A. 2022 Mar 08. 119(10): e2107453119

O-GlcNAcylation regulates epidermal growth factor receptor intracellular trafficking and signaling.

Liming Wu, Yaxian Cheng, Didi Geng, Zhiya Fan, Bingyi Lin, Qiang Zhu, Jingchao Li, Weijie Qin, Wen Yi.

  SignificanceEpidermal growth factor receptor (EGFR) is one of the most important membrane receptors that transduce growth signals into cells to sustain cell growth, proliferation, and survival. EGFR signal termination is initiated by EGFR internalization, followed by trafficking through endosomes, and degradation in lysosomes. How this process is regulated is still poorly understood. Here, we show that hepatocyte growth factor regulated tyrosine kinase substrate (HGS), a key protein in the EGFR trafficking pathway, is dynamically modified by a single sugar N-acetylglucosamine. This modification inhibits EGFR trafficking from endosomes to lysosomes, leading to the accumulation of EGFR and prolonged signaling. This study provides an important insight into diseases with aberrant growth factor signaling, such as cancer, obesity, and diabetes.

Keywords:  EGFR; O-GlcNAcylation; endosomal sorting; membrane receptors

DOI:  https://doi.org/10.1073/pnas.2107453119
Cell Rep. 2022 Mar 01. pii: S2211-1247(22)00168-1. [Epub ahead of print]38(9): 110441

Gα13 loss in Kras/Tp53 mouse model of pancreatic tumorigenesis promotes tumors susceptible to rapamycin.

Mario A Shields, Christina Spaulding, Anastasia E Metropulos, Mahmoud G Khalafalla, Thao N D Pham, Hidayatullah G Munshi.

  Gα13 transduces signals from G-protein-coupled receptors. While Gα13 functions as a tumor suppressor in lymphomas, it is not known whether Gα13 is pro-tumorigenic or tumor suppressive in genetically engineered mouse (GEM) models of epithelial cancers. Here, we show that loss of Gα13 in the Kras/Tp53 (KPC) GEM model promotes well-differentiated tumors and reduces survival. Mechanistically, tumors developing in KPC mice with Gα13 loss exhibit increased E-cadherin expression and mTOR signaling. Importantly, human pancreatic ductal adenocarcinoma (PDAC) tumors with low Gα13 expression also exhibit increased E-cadherin expression and mTOR signaling. Treatment with the mTOR inhibitor rapamycin decreases the growth of syngeneic KPC tumors with Gα13 loss by promoting cell death. This work establishes a tumor-suppressive role of Gα13 in pancreatic tumorigenesis in the KPC GEM model and suggests targeting mTOR in human PDAC tumors with Gα13 loss.

Keywords:  E-cadherin; Gα13; KC mouse model; KPC mouse model; human PDAC tumors; mTOR; rapamycin

DOI:  https://doi.org/10.1016/j.celrep.2022.110441
Front Immunol. 2022 ;13 752105

P2X7 Receptor Triggers Lysosomal Leakage Through Calcium Mobilization in a Mechanism Dependent on Pannexin-1 Hemichannels.

Stephanie Alexia Cristina Silva Santos, Pedro Muanis Persechini, Bianca Monteiro Henriques-Santos, Victória Gabriela Bello-Santos, Newton G Castro, Júlia Costa de Sousa, Fernando Ariel Genta, Marcelo Felippe Santiago, Robson Coutinho-Silva, Luiz Eduardo Baggio Savio, Eleonora Kurtenbach.

  The P2X7 receptor is a critical purinergic receptor in immune cells. Its activation was associated with cathepsin release into macrophage cytosol, suggesting its involvement in lysosomal membrane permeabilization (LMP) and leakage. Nevertheless, the mechanisms by which P2X7 receptor activation induces LMP and leakage are unclear. This study investigated cellular mechanisms associated with endosomal and lysosomal leakage triggered by P2X7 receptor activation. We found that ATP at 500 μM and 5 mM (but not 50 μM) induced LMP in non-stimulated peritoneal macrophages. This effect was not observed in P2X7-deficient or A740003-pretreated macrophages. We found that the P2X7 receptor and pannexin-1 channels mediate calcium influx that might be important for activating specific ion channels (TRPM2 and two-pore channels) on the membranes of late endosomes and lysosomes leading to LMP leakage and consequent cathepsin release. These findings suggest the critical role of the P2X7 receptor in inflammatory and infectious diseases via lysosomal dysfunction.

Keywords:  P2 receptor; cathepsins; extracellular ATP; lysosomal permeabilization; lysosomes

DOI:  https://doi.org/10.3389/fimmu.2022.752105
Nat Rev Cancer. 2022 Mar 03.

Exploiting senescence for the treatment of cancer.

Liqin Wang, Lina Lankhorst, René Bernards.

Senescence is a cellular response to a variety of stress signals that is characterized by a stable withdrawal from the cell cycle and major changes in cell morphology and physiology. While most research on senescence has been performed on non-cancer cells, it is evident that cancer cells can also mount a senescence response. In this Review, we discuss how senescence can be induced in cancer cells. We describe the distinctive features of senescent cancer cells and how these changes in cellular physiology might be exploited for the selective eradication of these cells (senolysis). We discuss activation of the host immune system as a particularly attractive way to clear senescent cancer cells. Finally, we consider the challenges and opportunities provided by a 'one-two punch' sequential treatment of cancer with pro-senescence therapy followed by senolytic therapy.

DOI: https://doi.org/10.1038/s41568-022-00450-9
Autophagy. 2022 Mar 01. 1-3

Targeting ULK1 in cancer stem cells: insight from chronic myeloid leukemia.

Angela Ianniciello, G Vignir Helgason.

  Minimal residual disease (MRD) refers to a low number of cells that persist anti-cancer treatment and is the major cause of relapse in solid cancers and leukemias. In chronic myeloid leukemia (CML), a paradigm for stem cell-driven cancer, MRD is maintained by tyrosine kinase inhibitor (TKI)-insensitive leukemic stem cells (LSCs), which may rely on fundamental metabolic processes to resist drug treatment. Macroautophagy/autophagy is a cytoprotective process that has been highlighted as critical for sustaining LSC survival during TKI treatment in robust experimental models of CML. Our recent study shows that the autophagy-initiating kinase ULK1 is required for maintaining energy and redox balance in CML LSCs. Pharmacological inhibition of ULK1 results in stress-induced differentiation of LSCs, rendering them sensitive to TKI treatment, uncovering a promising strategy for selective eradication of LSCs in CML patients.Abbreviations CML: chronic myeloid leukemia; LSC: leukemic stem cell; MAPK: mitogen-activated protein kinase; MRD: minimal residual disease; TKI: tyrosine kinase inhibitor.

Keywords:  Autophagy; ULK1; cancer; drug resistance; leukemia; leukemic stem cells

DOI:  https://doi.org/10.1080/15548627.2022.2041152
Front Med (Lausanne). 2022 ;9 835700

Estrogen-Induced Extracellular Calcium Influx Promotes Endometrial Cancer Progress by Regulating Lysosomal Activity and Mitochondrial ROS.

Boqiang Shen, Juan Hao, Yanying Lin, Xingchen Li, Xiao Yang, Ting Huang, Jiaqi Wang, Yuanyuan Jia, Jingyi Zhou, Jianliu Wang.

  Objective: Calcium is present in serum mainly in filterable and bound forms, and Ca2+ is a major key to modulate signaling pathways that control oncogenesis and oncochannels associated with several types of cancer. However, the biological significance of serum calcium and its related mechanism with estrogen in endometrial cancer (EC) still remains elusive. This study aims to ascertain the relationship between serum calcium and clinicopathology in EC.Methods: Retrospective assessment of a total of 502 patients diagnosed with EC after surgery in Peking University People's Hospital from 2010 to 2018. Preoperative serum ionized calcium and the albumin corrected calcium was calculated in quartiles for various postoperative clinicopathological characteristics, logistic regression adjusted for potential confounders. Intracellular calcium homeostasis change induced by estrogen was detected by confocal analysis. Downstream pathways were analyzed by transcriptome and proteomics. Mitochondrial Ca2+ and ROS (reactive oxygen species) level was detected by confocal and flow cytometry. Lysosomal morphological and membrane changes were verified by confocal or Western blot assays.
Results: High level of albumin-corrected serum calcium was significantly correlated with EC clinicopathological characteristics progression include lymph vascular space invasion, lymph nodes metastasis, myometrial invasion, and cervical invasion. Calcium homeostasis regulated by estrogen in EC cells derived from extracellular calcium influx but not the release of the endoplasmic reticulum. Proteomic and bioinformatic analysis revealed the calcium influx might be involved in the regulation of autophagy and mitochondrial-related pathways. Mechanistic investigation demonstrated that calcium influx acted on the function of mitochondrial ROS and lysosomal activity.
Conclusion: Our findings revealed that serum calcium level was significantly related to poor outcomes. The extracellular calcium influx induced by estrogen was targeted to mitochondrial ROS and lysosome activity, which should be oriented to improve EC therapeutic strategies.

Keywords:  calcium homeostasis; endometrial cancer (EC); estrogen; prognosis (carcinoma); serum calcium

DOI:  https://doi.org/10.3389/fmed.2022.835700
J Vis Exp. 2022 Feb 12.

The Microscopy-Based Assay to Study and Analyze the Recycling Endosomes using SNARE Trafficking.

Anshul Milap Bhatt, Subba Rao Gangi Setty.

Recycling endosomes (REs) are tubular-vesicular organelles generated from early/sorting endosomes in all cell types. These organelles play a key role in the biogenesis of melanosomes, a lysosome-related organelle produced by melanocytes. REs deliver the melanocyte-specific cargo to premature melanosomes during their formation. Blockage in the generation of REs, observed in several mutants of Hermansky-Pudlak syndrome, results in hypopigmentation of skin, hair, and eye. Therefore, studying the dynamics (refer to number and length) of REs is useful to understand the function of these organelles in normal and disease conditions. In this study, we aim to measure the RE dynamics using a resident SNARE STX13.

DOI: https://doi.org/10.3791/63087
Med Clin (Barc). 2022 Feb 28. pii: S0025-7753(22)00059-8. [Epub ahead of print]

Approach to lysosomal diseases.

Miguel Ángel Torralba Cabeza, Sofía Aznárez Nogueras.

DOI: https://doi.org/10.1016/j.medcli.2022.02.001
Pharm Res. 2022 Feb 28.

Calcium Enabled Remote Loading of a Weak Acid Into pH-sensitive Liposomes and Augmented Cytosolic Delivery to Cancer Cells via the Proton Sponge Effect.

Mimi M Yang, Sasi Bhushan Yarragudi, Stephen M F Jamieson, Mingtan Tang, William R Wilson, Zimei Wu.

  While delivery of chemotherapeutics to cancer cells by nanomedicines can improve therapeutic outcomes, many fail due to the low drug loading (DL), poor cellular uptake and endosomal entrapment. This study investigated the potential to overcome these limitations using pH-sensitive liposomes (PSL) empowered by the use of calcium acetate. An acidic dinitrobenzamide mustard prodrug SN25860 was used as a model drug, with non pH-sensitive liposomes (NPSL) as a reference. Calcium acetate as a remote loading agent allowed to engineer PSL- and NPSL-SN25860 with DL of > 31.1% (w/w). The IC50 of PSL-SN25860 was 21- and 141-fold lower than NPSL and free drug, respectively. At 48 h following injection of PSL-SN25860, NPSL-SN25860 and the free drug, drug concentrations in EMT6-nfsB murine breast tumors were 56.3 µg/g, 6.76 µg/g and undetectable (< 0.015 µg/g), respectively (n = 3). Meanwhile, the ex vivo tumor clonogenic assay showed 9.1%, 19.4% and 42.7% cell survival in the respective tumors. Live-cell imaging and co-localization analysis suggested endosomal escape was accomplished by destabilization of PSL followed by release of Ca2+ in endosomes allowing induction of a proton sponge effect. Subsequent endosomal rupture was observed approximately 30 min following endocytosis of PSL containing Ca2+. Additionally, calcium in liposomes promoted internalization of both PSL and NPSL. Taken together, this study demonstrated multifaceted functions of calcium acetate in promoting drug loading into liposomes, cellular uptake, and endosomal escape of PSL for efficient cytoplasmic drug delivery. The results shed light on designing nano-platforms for cytoplasmic delivery of various therapeutics.

Keywords:  Calcium acetate; Co-localization analysis; Cytosolic delivery; Endosomal entrapment; Proton sponge effect; Remote drug loading; pH-sensitive liposomes

DOI:  https://doi.org/10.1007/s11095-022-03206-0
Biomed Pharmacother. 2022 Feb 28. pii: S0753-3322(22)00139-1. [Epub ahead of print]148 112751

Endolysosomal cation channels point the way towards precision medicine of cancer and infectious diseases.

Cheng-Chang Chen, Einar Krogsaeter, Ching-Ying Kuo, Min-Chuan Huang, Sui-Yuan Chang, Martin Biel.

  Infectious diseases and cancer are among the key medical challenges that humankind is facing today. A growing amount of evidence suggests that ion channels in the endolysosomal system play a crucial role in the pathology of both groups of diseases. The development of advanced patch-clamp technologies has allowed us to directly characterize ion fluxes through endolysosomal ion channels in their native environments. Endolysosomes are essential organelles for intracellular transport, digestion and metabolism, and maintenance of homeostasis. The endolysosomal ion channels regulate the function of the endolysosomal system through four basic mechanisms: calcium release, control of membrane potential, pH change, and osmolarity regulation. In this review, we put particular emphasis on the endolysosomal cation channels, including TPC2 and TRPML2, which are particularly important in monocyte function. We discuss existing endogenous and synthetic ligands of these channels and summarize current knowledge of their impact on channel activity and function in different cell types. Moreover, we summarize recent findings on the importance of TPC2 and TRPML2 channels as potential drug targets for the prevention and treatment of the emerging infectious diseases and cancer.

Keywords:  Cancer; Endolysosomal trafficking; Infection; MCOLN; TPC2; TRPML2

DOI:  https://doi.org/10.1016/j.biopha.2022.112751