Interaction of Iron Oxide Fe3O4 Nanoparticles and Alveolar Macrophages in Vivo

Aqueous suspension of magnetite nanoparticles with primary diameter of 10 nm were intratracheally administered into rat lungs. In 24 h, cells were isolated from bronchoalveolar lavage and examined under a transmission electron microscope. Alveolar macrophages demonstrated ability to actively uptake single nanoparticles and small aggregates composed of such particles, which then formed larger conglomerates inside fused phagosomes. Some of these mature phagosomes shed the membrane and free nanoparticles closely interacted with nuclear membrane and with cristae and mitochondrial membranes thereby inflicting pronounced damage to these intracellular structures. The loss of primary lysosomes can be viewed as indirect evidence attesting to the role played by diffusion of lysosomal hydrolytic enzymes in the final destruction of the alveolar macrophages provoked by nanoparticles.     

Lysosome Dysfunction Enhances Oxidative Stress‐Induced Apoptosis through Ubiquitinated Protein Accumulation in Hela Cells

The role of lysosomal system in oxidative stress‐induced apoptosis in cancer cells is not fully understood. Menadione is frequently used as oxidative stress model. It is indicated that menadione could induce autophagy in Hela cells. In the present study, we examined whether the lysosomal inhibitor, ammonium chloride (NH₄Cl) could prevent the autophagy flux by inhibiting the fusion of autophagosomes with lysosomes and enhance apoptosis induced by menadione via mitochondrial pathway. The results demonstrated generation and accumulation of reactive oxygen species and increased levels of ubiquitinated proteins and GRP78 in cells treated with both menadione and NH₄Cl. Our data indicates that lysosomal system through autophagy plays an important role in preventing menadione‐induced apoptosis in Hela cells by clearing misfolded proteins, which alleviates endoplasmic reticulum stress. Anat Rec, 2013. © 2012 Wiley Periodicals, Inc.     

Attenuation of the lysosomal death pathway by lysosomal cholesterol accumulation

In the past decade, lysosomal membrane permeabilization (LMP) has emerged as a significant component of cell death signaling. The mechanisms by which lysosomal stability is regulated are not yet fully understood, but changes in the lysosomal membrane lipid composition have been suggested to be involved. Our aim was to investigate the importance of cholesterol in the regulation of lysosomal membrane permeability and its potential impact on apoptosis. Treatment of normal human fibroblasts with U18666A, an amphiphilic drug that inhibits cholesterol transport and causes accumulation of cholesterol in lysosomes, rescued cells from lysosome-dependent cell death induced by the lysosomotropic detergent O-methyl-serine dodecylamide hydrochloride (MSDH), staurosporine (STS), or cisplatin. LMP was decreased by pretreating cells with U18666A, and there was a linear relationship between the cholesterol content of lysosomes and their resistance to permeabilization induced by MSDH. U18666A did not induce changes in expression or localization of 70-kDa heat shock proteins (Hsp70) or antiapoptotic Bcl-2 proteins known to protect the lysosomal membrane. Induction of autophagy also was excluded as a contributor to the protective mechanism. By using Chinese hamster ovary (CHO) cells with lysosomal cholesterol overload due to a mutation in the cholesterol transporting protein Niemann-Pick type C1 (NPC1), the relationship between lysosomal cholesterol accumulation and protection from lysosome-dependent cell death was confirmed. Cholesterol accumulation in lysosomes attenuates apoptosis by increasing lysosomal membrane stability.     

The role of lysosomal rupture in neuronal death

Apoptosis research in the past two decades has provided an enormous insight into its role in regulating cell death. However, apoptosis is only part of the story, and inhibition of neuronal necrosis may have greater impact than apoptosis, on the treatment of stroke, traumatic brain injury, and neurodegenerative diseases. Since the “calpain–cathepsin hypothesis” was first formulated, the calpain- and cathepsin-mediated regulation of necrotic cascades observed in monkeys, has been demonstrated to be a common neuronal death mechanism occurring from simpler organisms to humans. However, the detailed mechanism inducing lysosomal destabilization still remains poorly understood. Heat-shock protein-70 (Hsp70) is known to stabilize lysosomal membrane and protect cells from oxidative stress and apoptotic stimuli in many cell death pathways. Recent proteomics approach comparing pre- and post-ischemic hippocampal CA1 neurons as well as normal and glaucoma-suffered retina of primates, suggested that the substrate protein upon which activated calpain acts at the lysosomal membrane of neurons might be Hsp70. Understanding the interaction between activated calpains and Hsp70 will help to unravel the mechanism that destabilizes the lysosomal membrane, and will provide new insights into clarifying the whole cascade of neuronal necrosis. Although available evidence is circumferential, it is hypothesized that activated calpain cleaves oxidative stress-induced carbonylated Hsp70.1 (a major human Hsp70) at the lysosomal membrane, which result in lysosomal rupture/permeabilization. This review aims at highlighting the possible mechanism of lysosomal rupture in neuronal death by a modified “calpain–cathepsin hypothesis”. As the autophagy–lysosomal degradation pathway is a target of oxidative stress, the implication of autophagy is also discussed.     

The role of lysosomal rupture in neuronal death

Apoptosis research in the past two decades has provided an enormous insight into its role in regulating cell death. However, apoptosis is only part of the story, and inhibition of neuronal necrosis may have greater impact than apoptosis, on the treatment of stroke, traumatic brain injury, and neurodegenerative diseases. Since the “calpain–cathepsin hypothesis” was first formulated, the calpain- and cathepsin-mediated regulation of necrotic cascades observed in monkeys, has been demonstrated to be a common neuronal death mechanism occurring from simpler organisms to humans. However, the detailed mechanism inducing lysosomal destabilization still remains poorly understood. Heat-shock protein-70 (Hsp70) is known to stabilize lysosomal membrane and protect cells from oxidative stress and apoptotic stimuli in many cell death pathways. Recent proteomics approach comparing pre- and post-ischemic hippocampal CA1 neurons as well as normal and glaucoma-suffered retina of primates, suggested that the substrate protein upon which activated calpain acts at the lysosomal membrane of neurons might be Hsp70. Understanding the interaction between activated calpains and Hsp70 will help to unravel the mechanism that destabilizes the lysosomal membrane, and will provide new insights into clarifying the whole cascade of neuronal necrosis. Although available evidence is circumferential, it is hypothesized that activated calpain cleaves oxidative stress-induced carbonylated Hsp70.1 (a major human Hsp70) at the lysosomal membrane, which result in lysosomal rupture/permeabilization. This review aims at highlighting the possible mechanism of lysosomal rupture in neuronal death by a modified “calpain–cathepsin hypothesis”. As the autophagy–lysosomal degradation pathway is a target of oxidative stress, the implication of autophagy is also discussed.     

Lysosome is a primary organelle in B cell receptor-mediated apoptosis: an indispensable role of Syk in lysosomal function

To investigate the mechanism of B cell receptor (BCR)-mediated apoptosis, we utilized immature B cell lines, DT40 and WEHI-231. In both cell lines, BCR-crosslinking caused the increase in lysosomal pH with early apoptotic changes characterized by chromatin condensation and phosphatidylserine exposure. This increase was detected in c-Abl-deficient DT40 cells but not in Syk-deficient cells, which corresponded to the fact that the former cells but not the latter revealed BCR-induced apoptosis. In contrast, BCR-crosslinking caused no apparent change in mitochondrial transmembrane potential. Therefore, the lysosomal change might be a primary event in BCR-induced apoptosis in DT40 cells. The increased activity of cathepsin B and apoptosis-preventing effect of a cathepsin inhibitor suggested a significant role of lysosomal enzymes in this apoptosis. By microscopic studies, lysosomes of wild-type DT40 cells fused to BCR-carrying endosomes became enlarged and accumulated one another. In contrast, these changes of lysosomal dynamics did not occur in Syk-deficient cells but transfer of wild-type Syk restored the lysosomal changes and apoptosis. These results demonstrated that the lysosomal change accompanied with the activation of lysosomal enzymes is a primary step in BCR-crosslinking-mediated apoptosis and Syk is responsible for this step through the fusion of BCR-carrying endosomes to lysosomes.     

Prevention of hydrogen peroxide-induced apoptosis of human peripheral T cells by a lysosomotropic iron chelator, ammonium chloride

Human peripheral T cells are considered to be easily susceptible to oxidative stress because these cells lack peroxidase activity. Therefore, in a previous study, we investigated the site of ROS formation by utilizing Mito-Capture, H(2)DCFDA (succinimidyl ester of dichlorodihydrofluorescein diacetate), DAPI (4',6-diamidino-2-phenylindole), and LysoSensor. Our results showed that ROS formation was apparently diffusely distributed in T cells oxidatively stressed with 0.1 mM hydrogen peroxide. Moreover, lysosomal swelling and deformity, possibly revealing lysosomal membrane destabilization, were observed in these cells. Based on the above-mentioned results, we concluded that an apoptotic cascade involving early lysosomal membrane destabilization exists in the hydrogen peroxide-induced apoptosis of human peripheral T cells. Therefore, the possible involvement of lysosomal protease leakage caused by hydroxyl radical formation in lysosomes (possibly resulting in mitochondrial membrane dysfunction) is considered to play an important role in hydrogen peroxide-induced T cell apoptosis. Hydrogen peroxide-mediated destabilization of lysosomal membranes with release of hydrolytic enzymes such as many kinds of cathepsins into the cell cytoplasm can lead to a cascade eventuating in cell death. To assess the importance of the intralysosomal pool of redox-active iron, we examined the effect of blockade of lysosomal digestion by exposing T cells to the lysosomotropic alkalinizing agent ammonium chloride (NH(4)Cl). Preincubation of human peripheral T cells with 10 mM NH(4)Cl for 4 h dramatically decreased apoptotic death caused by subsequent exposure to hydrogen peroxide (H(2)O(2)), and lysosomes and mitochondria showed almost normally preserved appearance. Therefore, we concluded here that lysosomal protease leakage caused by hydrogen peroxide in T cells was prevented by preincubation with ammonium chloride (NH(4)Cl).     

The effects in the excess administration of prostaglandin E1 on the indicator enzymes of organella membrane in gastric mucosa

Administration of excess vitamin A to rats causes gastric ulceration. In this study the effects on the gastric mucosa of excess vitamin A and excess prostaglandin E1, alone and in combination, were studied. Prostaglandin E1 protected against ulceration by vitamin A. Vitamin A labilized marker enzymes from four different membrane systems, namely those of the lysosomes, mitochondria, endoplasmic reticulum and plasma membrane, whereas only the effect on lysosomes was prevented by prostaglandin E1. Indeed, the prostaglandin alone labilized the enzymes from plasma membrane and endoplasmic reticulum and also damaged mitochondrial membranes. Both vitamin A and prostaglandin E1 caused a reduction in the total number and an increase in irregularly-shaped mitochondria in the parietal cells and produced dilation of the endoplasmic reticulum in both parietal and chief cells. It is noteworthy that prostaglandin E1 effectively prevents ulceration by vitamin A despite the extent to which it damages these membrane systems. These findings lend support to the hypothesis that vitamin A ulceration of the gastric mucosa is mediated via release of lysosomal enzymes, following damage to the lysosomal membranes.     

The effects in the excess administration of prostaglandin E1 on the indicator enzymes of organella membrane in gastric mucosa.

Administration of excess vitamin A to rats causes gastric ulceration. In this study the effects on the gastric mucosa of excess vitamin A and excess prostaglandin E1, alone and in combination, were studied. Prostaglandin E1 protected against ulceration by vitamin A. Vitamin A labilized marker enzymes from four different membrane systems, namely those of the lysosomes, mitochondria, endoplasmic reticulum and plasma membrane, whereas only the effect on lysosomes was prevented by prostaglandin E1. Indeed, the prostaglandin alone labilized the enzymes from plasma membrane and endoplasmic reticulum and also damaged mitochondrial membranes. Both vitamin A and prostaglandin E1 caused a reduction in the total number and an increase in irregularly-shaped mitochondria in the parietal cells and produced dilation of the endoplasmic reticulum in both parietal and chief cells. It is noteworthy that prostaglandin E1 effectively prevents ulceration by vitamin A despite the extent to which it damages these membrane systems. These findings lend support to the hypothesis that vitamin A ulceration of the gastric mucosa is mediated via release of lysosomal enzymes, following damage to the lysosomal membranes.     

Reactive oxygen species-producing site in radiation-induced apoptosis of human peripheral T cells: involvement of lysosomal membrane destabilization

In our previous studies, we have partly elucidated the mechanism of radiation-induced apoptosis of human peripheral T cells. The exact site of the ROS (reactive oxygen species) formation induced by irradiation has been so far unknown. Therefore, in this study, we investigated the site of ROS formation by utilizing MitoCapture, H2DCFDA (succinimidyl ester of dichlorodihydrofluorescein diacetate), DAPI, and Lysosensor. Our results showed that ROS formation apparently originated in the mitochondria and/or lysosomes instead of in the nuclei of irradiated T cells. Moreover, lysosomal swelling and deformity, possibly revealing lysosomal membrane instability, were observed at 1 h after 5 Gy irradiation of T cells. At 4 h after irradiation of 5 Gy, increase of fluorescence around the lysosomes, possibly revealing lysosomal rupture, was seen. Based on the above results, we concluded the possible existence of a new apoptotic cascade involving early lysosomal membrane destabilization in radiation-induced apoptosis of human peripheral T cells. Therefore, possible involvement of lysosomal protease leakage caused by hydroxyl radical formation in lysosomes (possibly resulting in mitochondrial membrane dysfunction) is considered to play an important role in radiation-induced T cell apoptosis.     

Organellar dysfunction in the pathogenesis of pancreatitis

SIGNIFICANCE: Acute pancreatitis is an inflammatory disease of exocrine pancreas that carries considerable morbidity and mortality; its pathophysiology remains poorly understood. During the past decade, new insights have been gained into signaling pathways and molecules that mediate the inflammatory response of pancreatitis and death of acinar cells (the main exocrine pancreas cell type). By contrast, much less is known about the acinar cell organellar damage in pancreatitis and how it contributes to the disease pathogenesis. RECENT ADVANCES: This review summarizes recent findings from our group, obtained on experimental in vivo and ex vivo models, which reveal disordering of key cellular organelles, namely, mitochondria, autophagosomes, and lysosomes, in pancreatitis. Our results indicate a critical role for mitochondrial permeabilization in determining the balance between apoptosis and necrosis in pancreatitis, and thus the disease severity. We further investigate how the mitochondrial dysfunction (and hence acinar cell death) is regulated by Ca(2+), reactive oxygen species, and Bcl-xL, in relation to specific properties of pancreatic mitochondria. Our results also reveal that autophagy, the principal cellular degradative, lysosome-driven pathway, is impaired in pancreatitis due to inefficient lysosomal function, and that impaired autophagy mediates two key pathological responses of pancreatitis-accumulation of vacuoles in acinar cells and the abnormal, intra-acinar activation of digestive enzymes such as trypsinogen. CRITICAL ISSUES AND FUTURE DIRECTIONS: The findings discussed in this review indicate critical roles for mitochondrial and autophagic/lysosomal dysfunctions in the pathogenesis of pancreatitis and delineate directions for detailed investigations into the molecular events that underlie acinar cell organellar damage.     

Autophagy, organelles and ageing

As a result of insufficient digestion of oxidatively damaged macromolecules and organelles by autophagy and other degradative systems, long-lived postmitotic cells, such as cardiac myocytes, neurons and retinal pigment epithelial cells, progressively accumulate biological 'garbage' ('waste' materials). The latter include lipofuscin (a non-degradable intralysosomal polymeric substance), defective mitochondria and other organelles, and aberrant proteins, often forming aggregates (aggresomes). An interaction between senescent lipofuscin-loaded lysosomes and mitochondria seems to play a pivotal role in the progress of cellular ageing. Lipofuscin deposition hampers autophagic mitochondrial turnover, promoting the accumulation of senescent mitochondria, which are deficient in ATP production but produce increased amounts of reactive oxygen species. Increased oxidative stress, in turn, further enhances damage to both mitochondria and lysosomes, thus diminishing adaptability, triggering mitochondrial and lysosomal pro-apoptotic pathways, and culminating in cell death.     

Lysosomal membrane permeabilization during apoptosis ‐ involvement of Bax?

Bcl-2 family members have long been known to control permeabilization of the mitochondrial membrane during apoptosis, but involvement of these proteins in lysosomal membrane permeabilization (LMP) was not considered until recently. The aim of this study was to investigate the mechanism underlying the release of lysosomal proteases to the cytosol seen during apoptosis, with special emphasis on the role of Bax. In human fibroblasts, exposed to the apoptosis-inducing drug staurosporine (STS), the release of the lysosomal protease cathepsin D to the cytosol was observed by immunocytochemistry. In response to STS treatment, there was a shift in Bax immunostaining from a diffuse to a punctate pattern. Confocal microscopy showed co-localization of Bax with both lysosomes and mitochondria in dying cells. Presence of Bax at the lysosomal membrane was confirmed by immuno-electron microscopy. Furthermore, when recombinant Bax was incubated with pure lysosomal fractions, Bax inserted into the lysosomal membrane and induced the release of lysosomal enzymes. Thus, we suggest that Bax is a mediator of LMP, possibly promoting the release of lysosomal enzymes to the cytosol during apoptosis.     

Reactive oxygen species-producing site in hydrogen peroxide-induced apoptosis of human peripheral T cells: involvement of lysosomal membrane destabilization

In our previous study, we examined the effect of exogenous hydrogen peroxide, which causes a potent oxidative stress and has been demonstrated to be a potent apoptosis-inducer in many kinds of cells. We found that the addition of 1 or 10 mM hydrogen peroxide induced reactive oxygen species (ROS) formation, oxidative DNA damage, dysfunction of the mitochondrial membrane potential, and early apoptotic changes in the human osteosarcoma cell line HS-Os-1. We therefore concluded that intracellular ROS formation was involved in the hydrogen peroxide-induced apoptosis of HS-Os-1 cells. In contrast to the osteosarcoma cell line HS-Os-1, human peripheral T cells are considered to be easily susceptible to oxidative stress, because these cells lack peroxidase activity. Therefore, in this study, we investigated the site of ROS formation by utilizing MitoCapture, H2DCFDA (succinimidyl ester of dichloro-dihydrofluorescein diacetate), DAPI (4',6-diamidino-2-phenylindole), and LysoSensor. Our results showed that ROS formation was apparently diffusely distributed in T cells oxidatively stressed with 0.1 mM hydrogen peroxide. Moreover, lysosomal swelling and deformity, possibly revealing lysosomal membrane destabilization, were observed in these cells. Based on the above results, there exists an apoptotic cascade involving early lysosomal membrane destabilization in the hydrogen peroxide-induced apoptosis of human peripheral T cells. Therefore, the possible involvement of lysosomal protease leakage caused by hydroxyl radical formation in lysosomes (possibly resulting in mitochondrial membrane dysfunction) is considered to play an important role in hydrogen peroxide-induced T cell apoptosis.     

芹菜素诱导人胃癌细胞凋亡作用及机制研究

目的：研究芹菜素（apigenin, API）致人胃癌细胞凋亡作用及其机制。方法：培养人胃癌BGC823细胞株，加入不同浓度的API，孵育48 h。PI染色流式细胞术（FCM）分析测定凋亡率；罗丹明染色FCM分析测定细胞线粒体跨膜电位(Δψm)；Caspase-9分光光度法检测试剂盒测定caspase-9活性；Western印迹检测线粒体凋亡信号转导通路相关蛋白的表达，包括bax，bcl-2，caspase-9和caspase-3。结果： API（20，40和80 μg/mL）作用48 h能呈浓度依赖性地诱导BGC823细胞凋亡。而且，API也能降低BGC823细胞的Δψm，增加caspase-9活性，促进细胞色素c（Cyt c）释放，上调bax，caspase-9和caspase-3蛋白的表达，同时下调bcl-2蛋白表达，且呈剂量依赖性。结论：API通过活化线粒体信号转导途径诱导人胃癌细胞凋亡。     

Integrin β1对胃癌细胞SGC7901多药耐药性的影响

目的:探究整合素β1(integrinβ1)对胃癌多药耐药性的影响及可能的作用机制。方法:Western blot法及q PCR实验检测胃癌细胞株SGC-7901及胃癌耐药细胞株SGC-7901/DDP中integrinβ1的表达情况。采用integrinβ1反义寡核苷酸转染,敲减胃癌耐药细胞株SGC-7901/DDP中integrinβ1的表达,CCK-8法检测细胞活力,流式细胞术检测细胞凋亡,Western blot法检测integrinβ1、Bcl-2/Bax、cleaved caspase-3/caspase-3、细胞色素C(CytC)和p-AKT/AKT的蛋白水平。结果:耐药细胞株SGC7901/DDP中integrinβ1的mRNA及蛋白表达水平均明显高于亲本细胞株;并且在亲本细胞株SGC7901中加入顺铂、长春新碱及5-氟尿嘧啶等化疗药物刺激后,integrinβ1的蛋白表达水平明显升高。敲减integrinβ1的表达可诱导胃癌耐药细胞SGC7901/DDP的凋亡,增加细胞对化疗药物的敏感性;此外下调Bcl-2/Bax、p-AKT~(Ser473)和p-AKT~(Thr308)的蛋白水平,同时促进线粒体Cyt-C的释放,上调cleaved caspase-3的蛋白水平。结论:敲减胃癌顺铂耐药细胞SGC7901/DDP的integrinβ1表达可恢复细胞对化疗药物的敏感性,促进细胞经线粒体路径的凋亡,其机制可能与抑制AKT的磷酸化,阻断该信号通路有关。     

A review of the role of enhanced apoptosis in the pathophysiology of cystinosis

The role of lysosomal cystine in development of the phenotype in cystinosis is problematic, in that the cystine is effectively isolated from the rest of cellular metabolism. Several models have been proposed, but most do not provide a mechanism for such an interaction. During early apoptosis the lysosomes are permeablized, providing such access. We have shown that lysosomal cystine enhances apoptosis in cultured normal and cystinotic fibroblasts and cultured renal proximal tubule epithelial cells, that the process occurs via mixed disulfide (cysteinylation) formation, and that PKC delta is involved. Further, the "swan neck" deformity of proximal renal tubules, long a hallmark of cystinosis, is explicable via this model, as is the renal failure that results from progression of tubule cell loss to atubular glomeruli. Modification of this process by other genes may explain the milder forms of the disease.     

Aminoglutethimide-induced lysosomal changes in adrenal gland in mice

Aminoglutethimide is a steroidogenesis inhibitor and inhibits a cholesterol side-chain cleavage enzyme (CYP11A1) that converts cholesterol to pregnenolone in mitochondria. We investigated histopathological changes induced by 5-day administration of AG in mice. Cytoplasmic vacuoles of various sizes and single cell necrosis were found in zona fasciculata cells in AG-treated mice. Some vacuoles were positive for adipophilin, whereas others were positive for lysosome-associated membrane protein-2 on immunohistochemical staining, indicating they were enlarged lipid droplets and lysosomes, respectively. Electron microscopy revealed enlarged lysosomes containing damaged mitochondria and lamellar bodies in zona fasciculata cells, and they were considered to reflect the intracellular protein degradation processes, mitophagy and lipophagy. From these results, we showed that AG induces excessive lipid accumulation and mitochondrial damage in zona fasciculata cells, which leads to an accelerated lysosomal degradation in mice.     

Adjustment of the lysosomal-mitochondrial axis for control of cellular senescence

Mitochondria and lysosomes undergo the most marked senescence-related alterations among all cellular organelles. Whereas mitochondria undergo gradual structural changes associated with reduced function, lysosomes exhibit progressively deteriorated function along with the accumulation of lipofuscins. Lysosomal dysfunction induces the deterioration of mitochondrial turnover, resulting in the generation of more reactive oxygen species (ROS), with the increased ROS levels in turn targeting lysosomes. This vicious feedback loop between lysosomes and mitochondria thus aggravates senescence phenotypes. Based on findings that lysosomal activity is diminished in senescent cells and that the resultant oxidative stress correlates with mitochondrial damage, the existence of a lysosomal–mitochondrial axis with a functional role in senescence has been proposed. In this review, we interrogate the interplay between lysosomes and mitochondria during senescence and propose the lysosomal-mitochondrial axis to serve a potential function as an inducer of senescence alleviation. Thus, learning how to control the lysosomal-mitochondrial axis should represent an important research directive for developing therapeutics toward ageing-related disease as well as the aging process itself. Further research focusing on the lysosomal-mitochondrial axis will add to our knowledge regarding aging and age-related pathologies, as well as provide new strategies for anti-aging intervention.