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The role of lysosomal enzymes in killing of mammalian cells by the lysosomotropic detergent N-dodecylimidazole

Rockefeller University Press
Journal of Cell Biology (JCB)
Authors:
Patricia D Wilson at University College London
Patricia D Wilson
Raymond A Firestone at Independent Researcher
Raymond A Firestone
  • Independent Researcher
J Lenard
J Lenard
J Lenard
  • This person is not on ResearchGate, or hasn't claimed this research yet.
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Abstract and Figures

The sensitivity of cultured human and hamster fibroblast cells to killing by the lysosomotropic detergent N-dodecylimidazole (C12-Im) was investigated as a function of cellular levels of general lysosomal hydrolase activity, and specifically of cysteine cathepsin activity. Fibroblasts from patients with mucolipidosis II (I-cell disease) lack mannose-6-phosphate-containing proteins, and therefore possess only 10-15% of the normal level of most lysosomal hydrolases. I-cell fibroblasts are about one-half as sensitive to killing by C12-Im as are normal human fibroblasts. Overall lysosomal enzyme levels of CHO cells were experimentally manipulated in several ways without affecting cell viability: Growth in the presence of 10 mM ammonium chloride resulted in a gradual decrease in lysosomal enzyme content to 10-20% of control values within 3 d. Subsequent removal of ammonium chloride from the growth medium resulted in an increase in lysosomal enzymes, to approximately 125% of control values within 24 h. Treatment with 80 mM sucrose caused extensive vacuolization within 2 h; lysosomal enzyme levels remained at control levels for at least 6 h, but increased 15-fold after 24 h of treatment. Treatment with concanavalin A (50 micrograms/ml) also caused rapid (within 2 h) vacuolation with a sevenfold rise in lysosomal enzyme levels occurring only after 24 h. The sensitivity of these experimentally manipulated cells to killing by C12-Im always paralleled the measured intracellular lysosomal enzyme levels: lower levels were associated with decreased sensitivity while higher levels were associated with increased sensitivity, regardless of the degree of vacuolization of the cells. The cytotoxicity of the cysteine proteases (chiefly cathepsin L in our cells) was tested by inactivating them with the irreversible inhibitor E-64 (100 micrograms/ml). Cell viability, protein levels, and other lysosomal enzymes were unaffected, but cysteine cathepsin activity was reduced to less than 20% of control values. E-64-treated cells were almost completely resistant to C12-Im treatment, although lysosomal disruption appeared normal by fluorescent visualization of Lucifer Yellow CH-loaded cells. It is concluded that cysteine cathepsins are the major or sole cytotoxic agents released from lysosomes by C12-Im. These observations also confirm the previous conclusions that C12-Im kills cells as a consequence of lysosomal disruption.
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The role of lysosomal enzymes in killing of mammalian cells by the lysosomotropic detergent N-dodecylimidazo
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The role of lysosomal enzymes in killing of mammalian cells by the lysosomotropic detergent N-dodecylimidazo
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... For comparison, chloroquine also caused cell death in a concentration-and time-dependent manner ( Figure S2), indicating that lysosomotropic drugs may interfere with cell viability if used for a prolonged time at too high a concentration. In line with our observations, the cytotoxicity of N-dodecylimidazole has also been ascribed to the lysosomotropic features of this NH125-related compound [45][46][47]. To address this question, BHK-21 cells were first treated for 1 h with either NH125 or NH4Cl and subsequently incubated for 15 min with pHrodo green dextran. ...
... For comparison, chloroquine also caused cell death in a concentration-and time-dependent manner ( Figure S2), indicating that lysosomotropic drugs may interfere with cell viability if used for a prolonged time at too high a concentration. In line with our observations, the cytotoxicity of N-dodecylimidazole has also been ascribed to the lysosomotropic features of this NH125-related compound [45][46][47]. ...
1-Benzyl-3-cetyl-2-methylimidazolium Iodide (NH125) Is a Broad-Spectrum Inhibitor of Virus Entry with Lysosomotropic Features
Article
Full-text available
  • Jun 2018
Cellular kinases are crucial for the transcription/replication of many negative-strand RNA viruses and might serve as targets for antiviral therapy. In this study, a library comprising 80 kinase inhibitors was screened for antiviral activity against vesicular stomatitis virus (VSV), a prototype member of the family Rhabdoviridae. 1-Benzyl-3-cetyl-2-methylimidazolium iodide (NH125), an inhibitor of eukaryotic elongation factor 2 (eEF2) kinase, significantly inhibited entry of single-cycle VSV encoding a luciferase reporter. Treatment of virus particles had only minimal effect on virus entry, indicating that the compound primarily acts on the host cell rather than on the virus. Accordingly, resistant mutant viruses were not detected when the virus was passaged in the presence of the drug. Unexpectedly, NH125 led to enhanced, rather than reduced, phosphorylation of eEF2, however, it did not significantly affect cellular protein synthesis. In contrast, NH125 revealed lysosomotropic features and showed structural similarity with N-dodecylimidazole, a known lysosomotropic agent. Related alkylated imidazolium compounds also exhibited antiviral activity, which was critically dependent on the length of the alkyl group. Apart from VSV, NH125 inhibited infection by VSV pseudotypes containing the envelope glycoproteins of viruses that are known to enter cells in a pH-dependent manner, i.e. avian influenza virus (H5N1), Ebola virus, and Lassa virus. In conclusion, we identified an alkylated imidazolium compound which inhibited entry of several viruses not because of the previously postulated inhibition of eEF2 kinase but most likely because of its lysosomotropic properties.
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... 9 Siramesine destabilizes the lysosomal membrane, 11 but is structurally different from alkyl LDs, and while it partitions into bilayers, it also binds to acidic phospholipid headgroups with high affinity, and sequestering of acidic lipids has been suggested as an alternative mechanism by which it contributes to cell toxicity. 15 The sigmoidal concentration dependence of toxicity on LD concentration, 5,7,12,16 indicates cooperative behavior 5,12 and has been linked to aggregation, or the critical micelle concentration (CMC), of the protonated LD. 3 In the prevailing picture, LDs are assumed not to harm plasma membranes of cells, but diffuse freely across these, because as free bases they are lipophilic and not surface active, but become powerful detergents upon protonation, 13 or phrased differently, "the largely un-ionized amine will be simply an oily substance without surface-active properties, but upon ionization within the lysosome it will become a detergent". 3 This is questionable on physicochemical grounds, since LDs will still have more or less polar headgroups under neutral conditions, and charging of headgroups usually counteracts aggregation due to headgroup Coulomb repulsion. ...
... Lysosomotropic agents are also used for fluorescence-guided surgery, 21 as lysosomal markers in fluorescence microscopy (acridine orange, LysoTracker), or used as lysosomal transporters via covalent conjugation. 22 The synthetic LD O-methyl-serine dodecylamide hydrochloride (MSDH) (see Figure 1) is one in a series of LDs initially designed as anticancer drugs, 3,16 and has also been tested for its ability to inhibit DNA synthesis. 4 It selectively ruptures lysosomes, but is otherwise biochemically inactive, facilitating study of the specific mechanisms of lysosomedependent cell death. ...
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Article
Full-text available
  • Nov 2016
Lysosomotropic detergents (LDs) selectively rupture lysosomal membranes through mechanisms that have yet to be characterized. A consensus view, currently, holds that LDs, which are weakly basic, diffuse across cellular membranes as monomers in an uncharged state, and via protonation in the acidic lysosomal compartment, they become trapped and accumulate, and subsequently solubilize the membrane and induce lysosomal membrane permeabilization. Here, we demonstrate that the lysosomotropic detergent O-methyl-serine dodecylamide hydrochloride (MSDH) spontaneously assembles into vesicles at, and above, cytosolic pH, and that the vesicles disassemble as pH reaches 6.4 or lower. The aggregation commences at concentrations below the range of those used in cell studies. Assembly and disassembly of the vesicles was studied via dynamic light scattering, zeta potential measurements, cryo-TEM and fluorescence correlation spectroscopy, and was found to be reversible via control of the pH. Aggregation of MSDH into closed vesicles under cytosolic conditions is at variance with the commonly held view of LD behaviour, and we propose that endocytotic pathways should be considered as possible routes of LD entry into lysosomes. We further demonstrate that MSDH vesicles can be loaded with fluorophores via a solution transition from low to high pH, for subsequent release when the pH is lowered again. The ability to encapsulate molecular cargo into MSDH vesicles together with its ability to disaggregate at low pH and to permeabilize the lysosomal membrane presents an intriguing possibility to use MSDH as a delivery system.
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... By placing imidazole, an amine of intermediate pK, onto a long hydrocarbon chain, the compound was endowed with the ability to diffuse across lysosomal membrane, where in the low pH environment it becomes protonated and trapped. NDI then develops surfactant properties and disrupts the lysosomal membrane, causing death of both normal and cancer cells by releasing the lysosomal contents into the cytoplasm (5)(6)(7). Depending on the concentration of lysosomal detergent and the extent of lysosomal damage, cells eventually die by apoptosis or necrosis (8)(9)(10)(11). Since NDI acquires detergent properties under acidic conditions, it was suggested that it might be useful in inducing lysosomal membrane permeabilization (LMP) and selective killing of cells in regions of solid tumors which have reduced extracellular pH due to anaerobic metabolism (12). ...
Synergistic Anticancer Action of Lysosomal Membrane Permeabilization and Glycolysis Inhibition
Article
Full-text available
  • Sep 2016
  • J BIOL CHEM
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... the pro-apoptotic member of the Bcl-protein family, BH3 interacting-domain death agonist [BID], to form truncated-BID [t-BID], which are eventuated in apoptosis [40][41][42]26] . Similarly, photo-oxidized the sarcoplasmic reticulum provides a burst of calcium that has been shown to play a substantial role in inducing programmed necrosis and necroptosis signaling pathway [43][44][45] . ...
An Alternative Treatment Modality of Diseases Using Photodynamic Therapy with a Wide Range Biological Targeting Possibility
Article
Full-text available
  • Nov 2015
An Alternative Treatment Modality of Diseases Using Photodynamic Therapy with a Wide Range Biological Targeting Possibility Mehmet VAROL 1,2,* Department of Biology, Faculty of Science, Yunusemre Campus, Anadolu University, Eskisehir TR26470, Turkey Department of Molecular Biology and Genetics, Kotekli Campus, Mugla Sitki Kocman University, Mugla TR 48000, Turkey Corresponding Author: Mehmet VAROL, Department of Molecular Biology and Genetics, Faculty of Science, Kotekli Campus, Mugla Sitki Kocman University, Mugla TR48000, Tel: 0090-252-211-31-32 / 4839; Fax: +90-252-211-92-80 Turkey. E-mail: mehmetvarol@mu.edu.tr Received: 16/11/2015 Accepted: 18/11/2015 Published: 25/11/2015 Related article at Pubmed, Scholar Google Visit for more related articles at Research & Reviews: Research Journal of Biology
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... Oxidative modification of lipid constituents is associated with phospholipid packing defects and membrane permeabilization [73,74], ultimately leading to necrosis. Lysosome-targeted PSs may induce ROS-triggered cell death in two ways: (1) via the discharge of cathepsins from lysosomes due to lipid (per) oxidation-mediated lysosome rupture and/or (2) PDT-induced relocalization of the PS to other organelles and subsequent induction of oxidative damage [75][76][77]. With respect to the first pathway, cathepsins exhibit proteolytic activity and are able to cleave BH3 interacting-domain death agonist (BID) to form truncated-BID (t-BID), which ultimately culminates in apoptosis. ...
Enhancing photodynamic therapy of refractory solid cancers: Combining second-generation photosensitizers with multi-targeted liposomal delivery
Article
  • Jun 2015
  • J PHOTOCH PHOTOBIO C
Contemporary photodynamic therapy (PDT) for the last-line treatment of refractory cancers such as nasopharyngeal carcinomas, superficial recurrent urothelial carcinomas, and non-resectable extrahepatic cholangiocarcinomas yields poor clinical outcomes and may be associated with adverse events. This is mainly attributable to three factors: (1) the currently employed photosensitizers exhibit suboptimal spectral properties, (2) the route of administration is associated with unfavorable photosensitizer pharmacokinetics, and (3) the upregulation of survival pathways in tumor cells may impede cell death after PDT. Consequently, there is a strong medical need to improve PDT of these recalcitrant cancers. An increase in PDT efficacy and reduction in clinical side-effects may be achieved by encapsulating second-generation photosensitizers into liposomes that selectively target to pharmacologically important tumor locations, namely tumor cells, tumor endothelium, and tumor interstitial spaces. In addition to addressing the drawbacks of clinically approved photosensitizers, this review addresses the most relevant pharmacological aspects that dictate clinical outcome, including photosensitizer biodistribution and intracellular localization in relation to PDT efficacy, the mechanisms of PDT-induced cell death, and PDT-induced antitumor immune responses. Also, a rationale is provided for the use of second-generation photosensitizers such as diamagnetic phthalocyanines (e.g., zinc or aluminum phthalocyanine), which exhibit superior photophysical and photochemical properties, in combination with a multi-targeted liposomal photosensitizer delivery system. The rationale for this PDT platform is corroborated by preliminary experimental data and proof-of-concept studies. Finally, a summary of the different nanoparticulate photosensitizer delivery systems is provided followed by a section on phototriggered release mechanisms in the context of liposomal photosensitizer delivery systems.
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Inhibition of PLA2G4E/cPLA2 promotes survival of random skin flaps by alleviating Lysosomal membrane permeabilization-Induced necroptosis
Article
  • Dec 2021
Necrosis that appears at the ischemic distal end of random-pattern skin flaps increases the pain and economic burden of patients. Necroptosis is thought to contribute to flap necrosis. Lysosomal membrane permeabilization (LMP) plays an indispensable role in the regulation of necroptosis. Nonetheless, the mechanisms by which lysosomal membranes become leaky and the relationship between necroptosis and lysosomes are still unclear in ischemic flaps. Based on Western blotting, immunofluorescence, enzyme-linked immunosorbent assay, and liquid chromatography-mass spectrometry (LC-MS) analysis results, we found that LMP was presented in the ischemic distal portion of random-pattern skin flaps, which leads to disruption of lysosomal function and macroautophagic/autophagic flux, increased necroptosis, and aggravated necrosis of the ischemic flaps. Moreover, bioinformatics analysis of the LC-MS results enabled us to focus on the role of PLA2G4E/cPLA2 (phospholipase A2, group IVE) in LMP of the ischemic flaps. In vivo inhibition of PLA2G4E with an adeno-associated virus vector attenuated LMP and necroptosis, and promoted flap survival. In addition, microRNA-seq helped us determine that Mir504-5p was differentially expressed in ischemic flaps. A string of in vitro and in vivo tests was employed to verify the inhibitory effect of Mir504-5p on PLA2G4E, LMP and necroptosis. Finally, we concluded that the inhibition of PLA2G4E by Mir504-5p reduced LMP-induced necroptosis, thereby promoting the survival of random-pattern skin flaps.
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Intracellular cathepsin C levels determine sensitivity of cells to leucyl‐leucine methyl ester‐triggered apoptosis
Article
Full-text available
L‐leucyl‐leucine methyl ester (LLOMe) is a lysosomotropic detergent, which was evaluated in clinical trials in graft‐vs‐host disease because it very efficiently killed monocytic cell lines. It was also shown to efficiently trigger apoptosis in cancer cells, suggesting that the drug might have potential in anticancer therapy. Using U‐937 and THP‐1 promonocytes as models for monocytic cells, U‐87‐MG and HeLa cells as models for cancer cells, and noncancerous HEK293 cells, we show that the drug triggers rapid cathepsin C‐dependent lysosomal membrane permeabilization, followed by the release of other cysteine cathepsins into the cytosol and subsequent apoptosis. However, monocytes were found to be far more sensitive to the drug than the cancer and noncancer cells, which is most likely a consequence of the much higher intracellular levels of cathepsin C—the most upstream molecule in the pathway—in monocytic cell lines as compared to cancer cells. Overexpression of cathepsin C in HEK293 cells substantially enhances their sensitivity to the drug, consistent with the crucial role of cathepsin C. Major involvement of cysteine cathepsins B, S, and L in the downstream signaling pathway to mitochondrial cell death was confirmed in two gene ablation models, including the ablation of the major cytosolic inhibitor of cysteine cathepsins, stefin B, in primary mouse cancer cells, and simultaneous ablation of two major cathepsins, B and L, in mouse embryonic fibroblasts (MEFs). Deletion of stefin B resulted in sensitizing primary murine breast cancer cells to cell death without affecting the release of cathepsins, whereas simultaneous ablation of cathepsins B and L largely protected MEFs against cell death. However, due to the extreme sensitivity of monocytes to LLOMe, it appears that the drug may not be suitable for anticancer therapy due to risk of systemic toxicity.
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Lysosomal Membrane Permeabilization in Cell Death: Biology, Diseases, and Therapeutics
Chapter
  • Aug 2016
This chapter discusses lysosomal membrane permeabilization (LMP) in the context of cell death together with the agents that cause it. In multicellular organisms, cell death process has evolved into an array of regulated signaling pathways that can respond to external and internal signals and limit their potentially harmful consequences for the sake of preserving the homeostasis of the organism. Apoptotic cells show chromatin and cytoplasm condensation, membrane blebbing, and apoptotic body formation. In the context of cell death, LMP commonly refers to the permeabilization of the limiting membrane of lysosomes, late endosomes, and hybrid organelles between these two, as they all harbor hydrolases, which can promote cell death. The chapter describes exogenous compounds that directly destabilize lysosomal membrane, the endogenous signaling events that lead to LMP. Finally, it summarizes cell death signaling downstream of LMP.
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Photonanomedicine: A convergence of photodynamic therapy and nanotechnology
Article
  • Jun 2016
  • Nanoscale
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Etude des interactions du photosensibilisant méta-tétra(hydroxyphényl)chlorine avec les protéines de plasma et les cellules
Article
  • Oct 2006
The investigation of the influence of mTHPC distribution in tumor, plasma and leukocytes on PDT response shows that photosensitizer accumulation in leukocytes exhibits a good correlation with PDT efficacy. This result suggests that leukocytes could play an important role in the mechanism of PDT-induced vascular damage.The study of mTHPC monomerisation in the course of interactions with plasma proteins demonstrates slow rate of disaggregation kinetics. The fraction of aggregated mTHPC at equilibrium and sensitizer disaggregation rates strongly depends on protein content and incubation temperature. The data obtained show that in aqueous media mTHPC forms free large-scale aggregates with strong interaction between sensitizer molecules.Kinetic analysis demonstrates that mTHPC is characterized by very slow redistribution rates from the complexes with plasma proteins. Low redistribution rates as compared to other sensitizers are consistent with the unique binding properties of mTHPC. The existence of both collisional and aqueous mediated transfer of mTHPC was supposed. Confocal microscopy study reveals diffuse mTHPC localization pattern at 3h and formation of highly fluorescent spots of sensitizer at 24h incubation in MCF-7 cells. The parameters of sensitizer absorption, fluorescence lifetime and photobleaching have shown that at 24h incubation mTHPC is much more aggregated compared to 3h. The yield of cellular photoinactivation is about 2 times higher for 3h time point. Such difference is attributed to different sensitizer aggregation states and interactions with cellular components. Theoretical and spectroscopic study of mTHPC, mTHPP and mTHPBC allowed us to define their aggregates structure in aqueous media. For this purpose new quantum mechanic semi-empirical method was developed on the basis of which the spectral shifts in different solvents were calculated. mTHPC and mTHPP form linear dimers in aqueous media, whereas mTHPBC form zigzag dimers.
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Rapid acidification of endocytic vesicles containing ??
Article
  • Mar 1982
  • CELL
We have used fluorescein-labeled alpha 2-macroglobulin (F-alpha 2M) to measure pH changes in the microenvironment of internalized ligands following receptor-mediated endocytosis. Fluorescence intensities of single BALB/c 3T3 mouse fibroblasts were measured by using a microscope spectrofluorometer with narrow bandpass excitation filters. The pH was determined from the ratio of fluorescein fluorescence intensities with 450 nm and 490 nm excitation. A standard pH curve was obtained by incubating cells with F-alpha 2M for 30 min at 37 degrees C followed by fixation and incubation in buffers of varying pH. To measure the pH of endocytic vesicles, cells were incubated with F-alpha 2M for 15 min at 37 degrees C. Fluorescence intensities were measured on living cells within 5 min of rinsing. Under these conditions, the pH of the F-alpha 2M microenvironment was 5.0 +/- 0.2. Using colloidal gold-alpha 2M for electron microscopic localizations we have verified that, under these conditions, alpha 2M is predominantly in uncoated vesicles that are negative for acid phosphatase activity. With further incubation for 1/2 hr, we obtained a pH of 5.0 +/- 0.2 for the F-alpha 2M. Using fluorescein dextran, we obtained a lysosomal pH of 4.6 +/- 0.2. These results indicate that endocytic vesicles become acidic prior to fusion with lysosomes.
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Fluorescence Probe Measurement of the Intralysosomal pH in Living Cells and the Perturbation of pH by Various Agents
Article
  • Aug 1978
A quantitative method is described for the measurement of intralysosomal pH in living cells. Fluorescein isothiocyanate-labeled dextran (FD) is endocytized and accumulates in lysosomes where it remains without apparent degradation. The fluorescence spectrum of this compound changes with pH in the range 4-7 and is not seriously affected by FD concentration, ionic strength, or protein concentration. Living cells on coverslips are mounted in a spectrofluorometer cell and can be perfused with various media. The normal pH inside macrophage lysosomes seems to be 4.7-4.8, although it can drop transiently as low as 4.5. Exposure of the cells to various weak bases and to acidic potassium ionophores causes the pH to increase. The changes in pH are much more rapid than is the intralysosomal accumulation of the weak bases. Inhibitors of glycolysis (2-deoxyglucose) and of oxidative phosphorylation (cyanide or azide) added together, but not separately, cause the intralysosomal pH to increase. These results provide evidence for the existence of an active proton accumulation mechanism in the lysosomal membrane and support the theory of lysosomal accumulation of weak bases by proton trapping.
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