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.

Imidacloprid Induces Lysosomal Dysfunction and Cell Death in Human Astrocytes and Fibroblasts-Environmental Implication of a Clinical Case Report.

Imidacloprid (IMI), a neonicotinoid insecticide, has potential cytotoxic and genotoxic effects on human and experimental models, respectively. While being an emerging environmental contaminant, occupational exposure and related cellular mechanisms are unknown. Herein, we were motivated by a specific patient case where occupational exposure to an IMI-containing plant protection product was associated with the diagnosis of Bell's palsy. The aim was to investigate the toxic effects and cellular mechanisms of IMI exposure on glial cells (D384 human astrocytes) and on human fibroblasts (AG01518). IMI-treated astrocytes showed a reduction in cell number and dose-dependent cytotoxicity at 24 h. Lower doses of IMI induced reactive oxygen species (ROS) and lysosomal membrane permeabilisation (LMP), causing apoptosis and autophagic dysfunction, while high doses caused significant necrotic cell death. Using normal fibroblasts, we found that IMI-induced autophagic dysfunction and lysosomal damage, activated lysophagy, and resulted in a compensatory increase in lysosomes. In conclusion, the observed IMI-induced effects on human glial cells and fibroblasts provide a possible link between IMI cytotoxicity and neurological complications observed clinically in the patient exposed to this neonicotinoid insecticide.

Screening for Inflammatory Markers Identifies IL-18Rα as a Potential Link between Exenatide and Its Anti-Inflammatory Effect: New Results from the Combat-JUDO Randomized Controlled Trial.

INTRODUCTION: Obesity is associated with chronic inflammation. Chronic inflammation has also been linked to insulin resistance and type 2 diabetes, metabolic associated fatty liver disease, and cardiovascular disease. Glucagon-like peptide-1 (GLP-1) receptor analogs (GLP-1RA) are clinically used to treat obesity, with known anti-inflammatory properties. How the GLP-1RA exenatide effects inflammation in adolescents with obesity is not fully investigated. METHODS: Forty-four patients were randomized to receive weekly subcutaneous injections with either 2 mg exenatide or placebo for 6 months. Plasma samples were collected at baseline and at the end of the study, and 92 inflammatory proteins were measured. RESULTS: Following treatment with exenatide, 15 out of the 92 proteins were decreased, and one was increased. However, after adjustment for multiple testing, only IL-18Rα was significantly lowered following treatment. CONCLUSIONS: Weekly injections with 2 mg of exenatide lowers circulating IL-18Rα in adolescents with obesity, which may be a potential link between exenatide and its anti-inflammatory effect in vivo. This contributes to exenatide's pharmaceutical potential as a treatment for obesity beyond weight control and glucose tolerance, and should be further studied mechanistically.

Real-Time Monitoring of Lysosomal Membrane Permeabilization Using Acridine Orange.

Loss of lysosomal membrane integrity results in leakage of lysosomal hydrolases to the cytosol which might harm cell function and induce cell death. Destabilization of lysosomes often precede apoptotic or necrotic cell death and occur during both physiological and pathological conditions. The weak base acridine orange readily enters cells and accumulates in the acidic environment of lysosomes. Vital staining with acridine orange is a well-proven technique to observe lysosomal destabilization using fluorescence microscopy and flow cytometry. These analyses are, however, time consuming and only adapted for discrete time points, which make them unsuitable for large-scale approaches. Therefore, we have developed a time-saving, high-throughput microplate reader-based method to follow destabilization of the lysosomal membrane in real-time using acridine orange. This protocol can easily be adopted for patient samples since the number of cells per sample is low and the time for analysis is short.

Lysosomal Function and Intracellular Position Determine the Malignant Phenotype in Malignant Melanoma.

Lysosomes are central in cell homeostasis and participate in macromolecular degradation, plasma membrane repair, exosome release, cell adhesion/migration, and apoptosis. In cancer, alterations in lysosomal function and spatial distribution may facilitate disease progression. In this study, we show enhanced lysosomal activity in malignant melanoma cells compared with that in normal human melanocytes. Most lysosomes show perinuclear location in melanocytes, while they are more dispersed in melanoma, with retained proteolytic activity and low pH also in the peripheral population. Rab7a expression is lower in melanoma cells than in melanocytes, and by increasing Rab7a, lysosomes are relocated to the perinuclear region in melanoma. Exposure to the lysosome-destabilizing drug L-leucyl-L-leucine methyl ester causes higher damage in the perinuclear subset of lysosomes in melanomas, whereas differences in subpopulation susceptibility cannot be found in melanocytes. Interestingly, melanoma cells recruit the endosomal sorting complex required for transport-III core protein CHMP4B, involved in lysosomal membrane repair, rather than initiate lysophagy. However, when the perinuclear lysosomal position is promoted by Rab7a overexpression or kinesore treatment, lysophagy is increased. In addition, Rab7a overexpression is accompanied by reduced migration capacity. Taken together, the study emphasizes that alterations in lysosomal properties facilitate the malignant phenotype and declares the targeting of lysosomal function as a future therapeutic approach.

Interactions of the Lysosomotropic Detergent O-Methyl-Serine Dodecylamide Hydrochloride (MSDH) with Lipid Bilayer Membranes-Implications for Cell Toxicity.

O-methyl-serine dodecylamine hydrochloride (MSDH) is a detergent that accumulates selectively in lysosomes, a so-called lysosomotropic detergent, with unexpected chemical properties. At physiological pH, it spontaneously forms vesicles, which disassemble into small aggregates (probably micelles) below pH 6.4. In this study, we characterize the interaction between MSDH and liposomes at different pH and correlate the findings to toxicity in human fibroblasts. We find that the effect of MSDH on lipid membranes is highly pH-dependent. At neutral pH, the partitioning of MSDH into the liposome membrane is immediate and causes the leakage of small fluorophores, unless the ratio between MSDH and lipids is kept low. At pH 5, the partitioning of MSDH into the membrane is kinetically impeded since MSDH is charged and a high ratio between MSDH and the lipids is required to permeabilize the membrane. When transferred to cell culture conditions, the ratio between MSDH and plasma membrane lipids must therefore be low, at physiological pH, to maintain plasma membrane integrity. Transmission electron microscopy suggests that MSDH vesicles are taken up by endocytosis. As the pH of the endosomal compartment progressively drops, MSDH vesicles disassemble, leading to a high concentration of increasingly charged MSDH in small aggregates inside the lysosomes. At sufficiently high MSDH concentrations, the lysosome is permeabilized, the proteolytic content released to the cytosol and apoptotic cell death is induced.

Restoration of lysosomal function after damage is accompanied by recycling of lysosomal membrane proteins.

Lysosomes are central organelles for cellular degradation and energy homeostasis. In addition, lysosomal membrane permeabilization (LMP) and subsequent release of lysosomal content to the cytosol can initiate programmed cell death. The extent of LMP and available repair mechanisms determine the cell fate after lysosomal damage. In this study, we aimed to investigate the premises for lysosomal membrane repair after LMP and found that lysosomal membrane damage initiated by L-leucyl-L-leucine methyl ester (LLOMe) caused caspase-dependent apoptosis in almost 50% of the cells, while the rest recovered. Immediately after LLOMe addition, lysosomal proteases were detected in the cytosol and the ESCRT-components ALIX and CHMP4B were recruited to the lysosomal membrane. Next, lysophagic clearance of damaged lysosomes was evident and a concentration-dependent translocation of several lysosomal membrane proteins, including LAMP2, to the cytosol was found. LAMP2 was present in small vesicles with the N-terminal protein chain facing the lumen of the vesicle. We conclude that lysophagic clearance of damaged lysosomes results in generation of lysosomal membrane protein complexes, which constitute small membrane enclosed units, possibly for recycling of lysosomal membrane proteins. These lysosomal membrane complexes enable an efficient regeneration of lysosomes to regain cell functionality.

Extracellular vesicles released by melanocytes after UVA irradiation promote intercellular signaling via miR21.

Skin pigmentation is controlled by complex crosstalk between melanocytes and keratinocytes and is primarily induced by exposure to ultraviolet (UV) irradiation. Several aspects of UVA-induced signaling remain to be explored. In skin cells, UVA induces plasma membrane damage, which is repaired by lysosomal exocytosis followed by instant shedding of extracellular vesicles (EVs) from the plasma membrane. The released EVs are taken up by neighboring cells. To elucidate the intercellular crosstalk induced by UVA irradiation, EVs were purified from UVA-exposed melanocytes and added to keratinocytes. Transcriptome analysis of the keratinocytes revealed the activation of TGF-ß and IL-6/STAT3 signaling pathways and subsequent upregulation of microRNA (miR)21. EVs induced phosphorylation of ERK and JNK, reduced protein levels of PDCD4 and PTEN, and augment antiapoptotic signaling. Consequently, keratinocyte proliferation and migration were stimulated and UV-induced apoptosis was significantly reduced. Interestingly, melanoma cells and melanoma spheroids also generate increased amounts of EVs with capacity to stimulate proliferation and migration upon UVA. In conclusion, we present a novel intercellular crosstalk mediated by UVA-induced lysosome-derived EVs leading to the activation of proliferation and antiapoptotic signaling via miR21.

UV radiation promotes melanoma dissemination mediated by the sequential reaction axis of cathepsins-TGF-ß1-FAP-α.

BACKGROUND: Ultraviolet radiation (UVR) is the major risk factor for development of malignant melanoma. Fibroblast activation protein (FAP)-α is a serine protease expressed on the surface of activated fibroblasts, promoting tumour invasion through extracellular matrix (ECM) degradation. The signalling mechanism behind the upregulation of FAP-α is not yet completely revealed. METHODS: Expression of FAP-α was analysed after UVR exposure in in vitro co-culture systems, gene expression arrays and artificial skin constructs. Cell migration and invasion was studied in relation to cathepsin activity and secretion of transforming growth factor (TGF)-ß1. RESULTS: Fibroblast activation protein-α expression was induced by UVR in melanocytes of human skin. The FAP-α expression was regulated by UVR-induced release of TGF-ß1 and cathepsin inhibitors prevented such secretion. In melanoma cell culture models and in a xenograft tumour model of zebrafish embryos, FAP-α mediated ECM degradation and facilitated tumour cell dissemination. CONCLUSIONS: Our results provide evidence for a sequential reaction axis from UVR via cathepsins, TGF-ß1 and FAP-α expression, promoting cancer cell dissemination and melanoma metastatic spread.

Analysis of Lysosomal pH by Flow Cytometry Using FITC-Dextran Loaded Cells.

The acidic environment of the lysosomal lumen provides an optimal milieu for the acid hydrolases and is also essential for fusion/fission of endo-lysosomal compartments and sorting of cargo. Evidence suggests that maintaining lysosomal acidity is essential to avoid disease. In this chapter, we describe a protocol for analyzing the lysosomal pH in cultured cells using the fluorescent probe fluorescein isothiocyanate (FITC)-dextran together with a dual-emission ratiometric technique suitable for flow cytometry. Fluorescence-labeled dextran is endocytosed and accumulated in the lysosomal compartment. FITC shows a pH-dependent variation in fluorescence when analyzed at maximum emission wavelength and no variation when analyzing at the isosbestic point, thereby the ratio can be used to determine the lysosomal pH. A standard curve is obtained by equilibrating intralysosomal pH with extracellular pH using the ionophore nigericin. The protocol also includes information regarding procedures to induce lysosomal alkalinization and lysosomal membrane permeabilization.

True's beaked whale (Mesoplodon mirus) in Macaronesia.

The True's beaked whale (Mesoplodon mirus, True 1913) is a poorly known member of the Ziphiidae family. Its distribution in the northern hemisphere is thought to be restricted to the temperate or warm temperate waters of the North Atlantic, while a few stranding records from the southern hemisphere suggest a wider and antitropical distribution, extending to waters from the Atlantic coast of Brazil to South Africa, Mozambique, Australia and the Tasman Sea coast of New Zealand. This paper (i) reports the first molecular confirmation of the occurrence of the True's beaked whale at the southern limit of its distribution recorded in the northeast Atlantic: the Azores and Canary Islands (macaronesian ecoregion); (ii) describes a new colouration for this species using evidence from a whale with molecular species confirmation; and (iii) contributes to the sparse worldwide database of live sightings, including the first underwater video recording of this species and close images of a calf. Species identification was confirmed in two cases using mitochondrial DNA control region and cytochrome b gene markers: a subadult male True's beaked whale that stranded in El Hierro, Canary Islands, in November 2012, and a subadult male found floating dead near Faial, the Azores, in July 2004. The whale that stranded in the Canary Islands had a clearly delimited white area on its head, extending posteriorly from the tip of the beak to cover the blowhole dorsally and the gular grooves ventrally. This colouration contrasts with previous descriptions for the species and it may be rare, but it exemplifies the variability of the colouration of True's beaked whales in the North Atlantic, further confirmed here by live sightings data. The recording of several observations of this species in deep but relatively coastal waters off the Azores and the Canary Islands suggests that these archipelagos may be unique locations to study the behaviour of the enigmatic True's beaked whale.

Impact of high cholesterol in a Parkinson's disease model: Prevention of lysosomal leakage versus stimulation of α-synuclein aggregation.

Parkinson's disease is characterized by accumulation of intraneuronal cytoplasmic inclusions, Lewy bodies, which mainly consist of aggregated α-synuclein. Controversies exist as to whether high blood cholesterol is a risk factor for the development of the disease and whether statin treatment could have a protective effect. Using a model system of BE(2)-M17 neuroblastoma cells treated with the neurotoxin 1-methyl-4-phenylpyridinium (MPP+), we found that MPP+-induced cell death was accompanied by cholesterol accumulation in a lysosomal-like pattern in pre-apoptotic cells. To study the effects of lysosomal cholesterol accumulation, we increased lysosomal cholesterol through pre-treatment with U18666A and found delayed leakage of lysosomal contents into the cytosol, which reduced cell death. This suggests that increased lysosomal cholesterol is a stress response mechanism to protect lysosomal membrane integrity in response to early apoptotic stress. However, high cholesterol also stimulated the accumulation of α-synuclein. Treatment with the cholesterol-lowering drug lovastatin reduced MPP+-induced cell death by inhibiting the production of reactive oxygen species, but did not prevent lysosomal cholesterol increase nor affect α-synuclein accumulation. Our study indicates a dual role of high cholesterol in Parkinson's disease, in which it acts both as a protector against lysosomal membrane permeabilization and as a stimulator of α-synuclein accumulation.

Extracellular vesicles are transferred from melanocytes to keratinocytes after UVA irradiation.

Ultraviolet (UV) irradiation induces skin pigmentation, which relies on the intercellular crosstalk of melanin between melanocytes to keratinocytes. However, studying the separate effects of UVA and UVB irradiation reveals differences in cellular response. Herein, we show an immediate shedding of extracellular vesicles (EVs) from the plasma membrane when exposing human melanocytes to UVA, but not UVB. The EV-shedding is preceded by UVA-induced plasma membrane damage, which is rapidly repaired by Ca(2+)-dependent lysosomal exocytosis. Using co-cultures of melanocytes and keratinocytes, we show that EVs are preferably endocytosed by keratinocytes. Importantly, EV-formation is prevented by the inhibition of exocytosis and increased lysosomal pH but is not affected by actin and microtubule inhibitors. Melanosome transfer from melanocytes to keratinocytes is equally stimulated by UVA and UVB and depends on a functional cytoskeleton. In conclusion, we show a novel cell response after UVA irradiation, resulting in transfer of lysosome-derived EVs from melanocytes to keratinocytes.

Sunbathing: What've lysosomes got to do with it?

Solar radiation is an important risk factor for skin cancer, the incidence of which is increasing, especially in the fair-skinned populations of the world. While the ultraviolet (UV)B component has direct DNA damaging ability, UVA-induced effects are currently mainly attributed to the production of reactive oxygen species. In our recent study, we compared the effects of UVA and UVB radiation on human keratinocytes and found that UVA-induced plasma membrane damage was rapidly repaired by lysosomal exocytosis, which was detected based on the expression of lysosomal membrane associated protein-1 (LAMP-1) on the plasma membrane of non-permeabilized cells. Later, the keratinocytes died through caspase-8 mediated apoptosis. In contrast, the plasma membranes of keratinocytes exposed to UVB showed no LAMP-1 expression, and, although the cells died by apoptosis, no initial caspase-8 activity was detected. We have also demonstrated the occurrence of UVA-induced lysosomal exocytosis in reconstructed skin and shown the relocation of lysosomes from the center of cells to the vicinity of the plasma membrane. Thus, we suggest that lysosomal exocytosis also occurs in keratinocytes covered by the stratum corneum following exposure to UVA. Our findings provide new insight into the mechanism of UVA-induced skin damage.

Comparison of gamma (Anger) camera systems in terms of detective quantum efficiency using Monte Carlo simulation.

AIM: The aim of the present study was to perform an extensive evaluation of available gamma camera systems in terms of their detective quantum efficiency (DQE) and determine their dependency on relevant parameters such as collimator type, imaging depth, and energy window using the Monte Carlo technique. MATERIALS AND METHODS: The modulation transfer function was determined from a simulated (99m)Tc point source and was combined with the system sensitivity and photon yield to obtain the DQE of the system. The simulations were performed for different imaging depths in a water phantom for 13 gamma camera systems from four manufacturers. RESULTS: Except at very low spatial frequencies, the highest DQE values were found with a lower energy window threshold of around 130 keV for all systems. The height and shape of the DQE curves were affected by the collimator design and the intrinsic properties of the gamma camera systems. High-sensitivity collimators gave the highest DQE at low spatial frequencies, whereas the high-resolution and ultrahigh-resolution collimators showed higher DQE values at higher frequencies. The intrinsic resolution of the system mainly affected the DQE curve at superficial depths. CONCLUSION: The results indicate that the manufacturers have succeeded differently in their attempts to design a system constituting an optimal compromise between sensitivity and spatial resolution.

Lysosomal exocytosis and caspase-8-mediated apoptosis in UVA-irradiated keratinocytes.

Ultraviolet (UV) irradiation is a major environmental carcinogen involved in the development of skin cancer. To elucidate the initial signaling during UV-induced damage in human keratinocytes, we investigated lysosomal exocytosis and apoptosis induction. UVA, but not UVB, induced plasma membrane damage, which was repaired by Ca(2+)-dependent lysosomal exocytosis. The lysosomal exocytosis resulted in extracellular release of cathepsin D and acid sphingomyelinase (aSMase). Two hours after UVA irradiation, we detected activation of caspase-8, which was reduced by addition of anti-aSMAse. Furthermore, caspase-8 activation and apoptosis was reduced by prevention of endocytosis and by the use of cathepsin inhibitors. We conclude that lysosomal exocytosis is part of the keratinocyte response to UVA and is followed by cathepsin-dependent activation of caspase-8. The findings have implications for the understanding of UV-induced skin damage and emphasize that UVA and UVB initiate apoptosis through different signaling pathways in keratinocytes.

TNF-α-stimulated macrophages protect A549 lung cells against iron and oxidation.

Previously, we have shown that TNF-α protects iron-exposed J774 macrophages against iron-catalyzed oxidative lysosomal disruption and cell death by increasing reduced glutathione and H-ferritin in cells. Because J774 cells are able to harbor large amounts of iron, which is potentially harmful in a redox-active state, we hypothesized that TNF-α-stimulated J774 macrophages will prevent iron-driven oxidative killing of alveolar epithelial A549 cells in co-culture. In the present study, iron trichloride (which is endocytosed by cells as hydrated iron-phosphate complexes) was mainly deposited inside the lysosomes of J774 macrophages, while A549 cells, equally iron exposed, accumulated much less iron. When challenged by oxidants, however, reactive lysosomal iron in A549 cells promoted lysosomal disruption and cell death, particularly in the presence of TNF-α. This effect resulted from an elevation in ROS generation by TNF-α, while a compensatory upregulation of protective molecules (H-ferritin and/or reduced glutathione) by TNF-α was absent. A549 cell death was particularly pronounced when iron and TNF-α were present in the conditioned medium during oxidant challenge; thus, iron-driven oxidative reactions in the culture medium were a much greater hazard to A549 cells than those taking place inside their lysosomes. Consequently, the iron chelator, deferoxamine, efficiently prevented A549 cell death when added to the culture medium during an oxidant challenge. In co-cultures of TNF-α-stimulated lung cells, J774 macrophages sequestered iron inside their lysosomes and protected A549 cells from oxidative reactions and cell death. Thus, the collective effect of TNF-α on co-cultured lung cells was mainly cytoprotective.

Determination of the detective quantum efficiency of gamma camera systems: a Monte Carlo study.

The purpose of the present work was to investigate the validity of using the Monte Carlo technique for determining the detective quantum efficiency (DQE) of a gamma camera system and to use this technique in investigating the DQE behaviour of a gamma camera system and its dependency on a number of relevant parameters. The Monte Carlo-based software SIMIND, simulating a complete gamma camera system, was used in the present study. The modulation transfer function (MTF) of the system was determined from simulated images of a point source of (99m)Tc, positioned at different depths in a water phantom. Simulations were performed using different collimators and energy windows. The MTF of the system was combined with the photon yield and the sensitivity, obtained from the simulations, to form the frequency-dependent DQE of the system. As figure-of-merit (FOM), the integral of the 2D DQE was used. The simulated DQE curves agreed well with published data. As expected, there was a strong dependency of the shape and magnitude of the DQE curve on the collimator, energy window and imaging position. The highest FOM was obtained for a lower energy threshold of 127 keV for objects close to the detector and 131 keV for objects deeper in the phantom, supporting an asymmetric window setting to reduce scatter. The Monte Carlo software SIMIND can be used to determine the DQE of a gamma camera system from a simulated point source alone. The optimal DQE results in the present study were obtained for parameter settings close to the clinically used settings.