Niclosamide induces mitochondria fragmentation and promotes both apoptotic and autophagic cell death

Mitochondrial dynamics not only involves mitochondrial morphology but also mitochondrial biogenesis, mitochondrial distribution, and cell death. To identify specific regulators to mitochondria dynamics, we screened a chemical library and identified niclosamide as a potent inducer of mitochondria fission. Niclosamide promoted mitochondrial fragmentation but this was blocked by down-regulation of Drp1. Niclosamide treatment resulted in the disruption of mitochondria membrane potential and reduction of ATP levels. Moreover, niclosamide led to apoptotic cell death by caspase-3 activation. Interestingly, niclosamide also increased autophagic activity. Inhibition of autophagy suppressed niclosamide-induced cell death. Therefore, our findings suggest that niclosamide induces mitochondria fragmentation and may contribute to apoptotic and autophagic cell death.     

Oxyresveratrol activates parallel apoptotic and autophagic cell death pathways in neuroblastoma cells

BackgroundDrug resistance from apoptosis is a challenging issue with different cancer types, and there is an interest in identifying other means of inducing cytotoxicity. Here, treatment of neuroblastoma cells with oxyresveratrol (OXYRES), a natural antioxidant, led to dose-dependent cell death and increased autophagic flux along with activation of caspase-dependent apoptosis.MethodsFor cell viability, we performed the CCK-8 assay. Protein expression changes were with Western blot and immunocytochemistry. Silencing of proteins was with siRNA. The readouts for cell cycle, mitochondria membrane potential, caspase-3, autophagy and apoptosis were performed with flow cytometry.ResultsPhosphorylation of p38 MAPK increased with OXYRES treatment and inhibition of p38 reduced autophagy and cell death from OXYRES. In contrast, PI3K/AKT/mTOR signaling decreased in the target cells with OXYRES and inhibition of PI3K or mTOR enhanced OXYRES-mediated cytotoxicity with increased levels of autophagy. Modulation of either of the apoptosis and autophagy flux pathways affected the extent of cell death by OXYRES, but did not affect the indicators of these pathways with respect to each other. Both pathways were independent of ROS generation or p53 activation.ConclusionOXYRES led to cell death from autophagy, which was independent of apoptosis induction. The OXYRES effects were due to changes in the activity levels of p38 MAPK and PI3K/AKT/mTOR.General significanceWith two independent and parallel pathways for cytotoxicity induction in target cells, this study puts forward a potential utility for OXYRES or the pathways it represents as novel means of inducing cell death in neuroblastoma cells.     

Resveratrol protects astrocytes against traumatic brain injury through inhibiting apoptotic and autophagic cell death

Traumatic brain injury (TBI) is often caused by accidents that damage the brain. TBI can induce glutamate excitotoxicity and lead to neuronal and glial cell death. In this study, we investigated the mechanism of cell death during the secondary damage caused by TBI in vivo and in vitro, as well as the protective effect of resveratrol (RV). Here we report that glycogen synthase kinase-3β (GSK-3β) activation and microtubule-associated protein light chain 3 processing were induced in rat brains exposed to TBI. In the in vitro TBI model, apoptotic and autophagic cell death were induced through glutamate-mediated GSK-3β activation in normal CTX TNA2 astrocytes. The GSK-3β inhibitor SB216763 or transfection of GSK-3β small-interfering RNA increases cell survival. By contrast, overexpression of GSK-3β enhanced glutamate excitotoxicity. Administration of RV reduced cell death in CTX TNA2 astrocytes by suppressing reactive oxygen species (ROS)-mediated GSK-3β activation, the mechanism by which RV also exerted protective effects in vivo. Mitochondrial damages, including the opening of mitochondrial permeability transition pore (MPTP) and mitochondrial depolarization, were induced by glutamate through the ROS/GSK-3β pathway. Moreover, cyclosporine A, an MPTP inhibitor, suppressed mitochondrial damage and the percentages of cells undergoing autophagy and apoptosis and thereby increased cell survival. Taken together, our results demonstrated that cell death occurring after TBI is induced through the ROS/GSK-3β/mitochondria signaling pathway and that administration of RV can increase cell survival by suppressing GSK-3β-mediated autophagy and apoptosis. Therefore, the results indicated that resveratrol may serve as a potential therapeutic agent in the treatment of TBI.     

Essential roles of Atg5 and FADD in autophagic cell death: dissection of autophagic cell death into vacuole formation and cell death

Autophagic cell death is characterized by the accumulation of vacuoles in physiological and pathological conditions. However, its molecular event is unknown. Here, we show that Atg5, which is known to function in autophagy, contributes to autophagic cell death by interacting with Fas-associated protein with death domain (FADD). Down-regulation of Atg5 expression in HeLa cells suppresses cell death and vacuole formation induced by IFN-gamma. Inversely, ectopic expression of Atg5 using adenoviral delivery induces autophagic cell death. Deletion mapping analysis indicates that procell death activity resides in the middle and C-terminal region of Atg5. Cells harboring the accumulated vacuoles triggered by IFN-gamma or Atg5 expression become dead, and vacuole formation precedes cell death. 3-Methyladenine or expression of Atg5(K130R) mutant blocks both cell death and vacuole formation triggered by IFN-gamma, whereas benzyloxycarbonyl-VAD-fluoromethyl ketone (Z-VAD-fmk) inhibits only cell death but not vacuole formation. Atg5 interacts with FADD via death domain in vitro and in vivo, and the Atg5-mediated cell death, but not vacuole formation, is blocked in FADD-deficient cells. These results suggest that Atg5 plays a crucial role in IFN-gamma-induced autophagic cell death by interacting with FADD.     

Protection by Bcl-2 against apoptotic but not autophagic cell death after photodynamic therapy

Photodynamic therapy (PDT) induces apoptosis in many cell types. Recent reports identified autophagy as an alternative cell-death process following PDT. Here we investigated the occurrence of autophagy after PDT with the photosensitizer Pc 4 in human cancer cells that are deficient in the pro-apoptotic factor Bax (human prostate cancer DU145) or the apoptosis mediator caspase-3 (human breast cancer MCF-7v) and in apoptosis-competent cells (MCF-7c3 stably overexpressing human pro-caspase-3 and Chinese hamster ovary CHO 5A100). Further, each cell line was also studied with and without stably overexpressed Bcl-2. By electron microscopy and immunoblot analysis, autophagy was observed in all cells studied, whether or not they were capable of typical apoptosis or overexpressed Bcl-2. Bcl-2 overexpression protected against PDT-induced apoptosis and loss of clonogenicity in apoptosis-competent cells (MCF-7c3 and CHO); however, it did not protect against the development of autophagy or against loss of clonogenicity in apoptosis-deficient cells (MCF-7v and DU145). The results show that autophagy may be the dominant cell death pathway following PDT in cells that are incapable of undergoing normal apoptosis. In such cells, Bcl-2 does not protect against autophagic death.     

Mammalian target of rapamycin regulates isoliquiritigenin-induced autophagic and apoptotic cell death in adenoid cystic carcinoma cells

Previous studies, including those from our laboratory, have demonstrated that isoliquiritigenin (ISL), a flavonoid isolated from licorice, is a promising cancer chemotherapeutic agent. However the mechanisms underlying its anticancer effects are still far from clear. We now show, for the first time, that ISL triggers the mammalian target of rapamycin (mTOR)-dependent autophagic and apoptotic cell death in adenoid cystic carcinoma (ACC). Exposure of both ACC-2 and ACC-M cells to ISL resulted in several specific features for autophagy, including the appearance of membranous vacuoles, formation of acidic vesicular organelles, punctate pattern of LC3 immunostaining, and an increase in autophagic flux. Moreover, ISL treatment also resulted in significantly increased apoptosis in ACC cells. The ISL-mediated autophagic and apoptotic cell death were obviously attenuated by transfection with dominant negative Atg5 (DN-Atg5^K130R) plasmids or treatment with 3-methyladenine(3-MA). In additon, the data also revealed that the autophagic and apoptotic cell death induced by ISL occurred through a mTOR-dependent pathway. More importantly, the xenograft model using ACC-M cells provided further evidence of the occurrence of ISL-induced autophagy and apoptosis in vivo, correlating with the suppresson of mTOR activation as well as up-regulation of Atg5 expression. Taken together, these findings in our study suggest that induction of mTOR-dependent autophagic and apoptotic cell death may be an important mechanism in cancer chemotherapy by ISL.     

Glucosamine induces autophagic cell death through the stimulation of ER stress in human glioma cancer cells

Autophagy can promote cell survival or death, but the molecular basis of its dual role in cancer is not well understood. Here, we report that glucosamine induces autophagic cell death through the stimulation of endoplasmic reticulum (ER) stress in U87MG human glioma cancer cells. Treatment with glucosamine reduced cell viability and increased the expression of LC3 II and GFP-LC3 fluorescence puncta, which are indicative of autophagic cell death. The glucosamine-mediated suppression of cell viability was reversed by treatment with an autophagy inhibitor, 3-MA, and interfering RNA against Atg5. Glucosamine-induced ER stress was manifested by the induction of BiP, IRE1α, and phospho-eIF2α expression. Chemical chaperon 4-PBA reduced ER stress and thereby inhibited glucosamine-induced autophagic cell death. Taken together, our data suggest that glucosamine induces autophagic cell death by inducing ER stress in U87MG glioma cancer cells and provide new insight into the potential anticancer properties of glucosamine.     

Diclofenac induced in vivo nephrotoxicity may involve oxidative stress-mediated massive genomic DNA fragmentation and apoptotic cell death.

Diclofenac (DCLF) is a nonsteroidal anti-inflammatory drug that is widely used for the treatment of osteoarthritis, rheumatoid arthritis, ankylosing spondylitis, and acute muscle pain conditions. Toxic doses of DCLF can cause nephrotoxicity in humans and experimental animals. However, whether this DCLF-induced nephrotoxicity involves apoptotic cell death in addition to necrosis is unknown. The goals of this investigation were to determine whether DCLF-induced nephrotoxicity involves oxidative stress and apoptotic type genomic DNA fragmentation, and if so, whether DCLF-induced oxidative stress and DNA fragmentation cause apoptotic cell death in mouse kidneys. Male ICR mice (CD-1; 25-45 g), fed ad libitum, were administered nephrotoxic doses of DCLF (100, 200, 300 mg/Kg, po) and sacrificed 24 h later. Blood was collected to evaluate renal injury (BUN), lipid peroxidation (MDA: malondialdehyde levels), and superoxide dismutase (SOD) activity (a marker of oxidative stress). Kidney tissues were analyzed both quantitatively and qualitatively to determine the degree and type of DNA damage, and evaluated histopathologically for the presence of apoptotic characteristics in the nucleus of diverse types of kidney cells. Results show that diclofenac is a powerful nephrotoxicant (at 100, 200, and 300 mg/kg: 4.7-, 4.9-, and 5.0-fold increases in BUN compared to the control, respectively) and a strong inducer of oxidative stress (significant increase in MDA levels). Oxidative stress induced by DCLF was also coupled with massive kidney DNA fragmentation (100, 200, and 300 mg/kg: 3-, 8-, and 10-fold increases compared to control, respectively). A dose-dependent increase in MDA levels and SOD activity was also observed, which indicated a link between oxidative stress and nephrotoxicity. Qualitative analysis of DNA fragmentation by gel electrophoresis showed a DNA ladder indicative of Ca2+-Mg2+-endonuclease activation. Histopathological examination of kidney sections revealed numerous apoptotic nuclei across proximal and distal tubular cell linings. Collectively, these data for the first time suggest that DCLF-induced nephrotoxicity may involve production of reactive oxygen species leading to oxidative stress and massive genomic DNA fragmentation, and these two free radical mediated events may ultimately translate into apoptotic cell death of kidney cells in vivo, and reveal a DNA-active role for DCLF.     

Necrotic,apoptotic and autophagic cell fates triggered by nanoparticles

Nanomaterials have gained a rapid increase in use in a variety of applications that pertain to many aspects of human life. The majority of these innovations are centered on medical applications and a range of industrial and environmental uses ranging from electronics to environmental remediation. Despite the advantages of NPs, the knowledge of their toxicological behavior and their interactions with the cellular machinery that determines cell fate is extremely limited. This review is an attempt to summarize and increase our understanding of the mechanistic basis of nanomaterial interactions with the cellular machinery that governs cell fate and activity. We review the mechanisms of NP-induced necrosis, apoptosis and autophagy and potential implications of these pathways in nanomaterial-induced outcomes.

Abbreviations: Ag, silver; CdTe, cadmium telluride; CNTs, carbon nanotubes; EC, endothelial cell; GFP, green fluorescent protein; GO, graphene oxide; GSH, glutathione; HUVECs, human umbilical vein endothelial cells; NP, nanoparticle; PEI, polyethylenimine; PVP, polyvinylpyrrolidone; QD, quantum dot; ROS, reactive oxygen species; SiO₂, silicon dioxide; SPIONs, superparamagnetic iron oxide nanoparticles; SWCNT, single-walled carbon nanotubes; TiO₂, titanium dioxide; USPION, ultra-small super paramagnetic iron oxide; ZnO, zinc oxide.     

Another way to die: autophagic programmed cell death

Programmed cell death (PCD) is one of the important terminal paths for the cells of metazoans, and is involved in a variety of biological events that include morphogenesis, maintenance of tissue homeostasis, and elimination of harmful cells. Dysfunction of PCD leads to various diseases in humans, including cancer and several degenerative diseases. Apoptosis is not the only form of PCD. Recent studies have provided evidence that there is another mechanism of PCD, which is associated with the appearance of autophagosomes and depends on autophagy proteins. This form of cell death most likely corresponds to a process that has been morphologically defined as autophagic PCD. The present review summarizes recent experimental evidence about autophagic PCD and discusses some aspects of this form of cell death, including the mechanisms that may distinguish autophagic death from the process of autophagy involved in cell survival.     

ER stress-induced cell death mechanisms

The endoplasmic-reticulum (ER) stress response constitutes a cellular process that is triggered by a variety of conditions that disturb folding of proteins in the ER. Eukaryotic cells have developed an evolutionarily conserved adaptive mechanism, the unfolded protein response (UPR), which aims to clear unfolded proteins and restore ER homeostasis. In cases where ER stress cannot be reversed, cellular functions deteriorate, often leading to cell death. Accumulating evidence implicates ER stress-induced cellular dysfunction and cell death as major contributors to many diseases, making modulators of ER stress pathways potentially attractive targets for therapeutics discovery. Here, we summarize recent advances in understanding the diversity of molecular mechanisms that govern ER stress signaling in health and disease. This article is part of a Special Section entitled: Cell Death Pathways. Guest Editors: Frank Madeo and Slaven Stekovic.     

Rational incorporation of selenium into temozolomide elicits superior antitumor activity associated with both apoptotic and autophagic cell death

Background

The DNA alkylating agent temozolomide (TMZ) is widely used in the treatment of human malignancies such as glioma and melanoma. On the basis of previous structure-activity studies, we recently synthesized a new TMZ selenium analog by rationally introducing an N-ethylselenocyanate extension to the amide functionality in TMZ structure.

Principal Findings

This TMZ-Se analog showed a superior cytotoxicity to TMZ in human glioma and melanoma cells and a more potent tumor-inhibiting activity than TMZ in mouse glioma and melanoma xenograft model. TMZ-Se was also effective against a TMZ-resistant glioma cell line. To explore the mechanism underlying the superior antitumor activity of TMZ-Se, we compared the effects of TMZ and TMZ-Se on apoptosis and autophagy. Apoptosis was significantly increased in tumor cells treated with TMZ-Se in comparison to those treated with TMZ. TMZ-Se also triggered greater autophagic response, as compared with TMZ, and suppressing autophagy partly rescued cell death induced by TMZ-Se, indicating that TMZ-Se-triggered autophagy contributed to cell death. Although mRNA level of the key autophagy gene, Beclin 1, was increased, Beclin 1 protein was down-regulated in the cells treated with TMZ-Se. The decrease in Beclin 1 following TMZ-Se treatment were rescued by the calpain inhibitors and the calpain-mediated degradation of Beclin1 had no effect on autophagy but promoted apoptosis in cells treated with TMZ-Se.

Conclusions

Our study indicates that incorporation of Se into TMZ can render greater potency to this chemotherapeutic drug.     

A calpain-like protease inhibits autophagic cell death

Programmed cell death (PCD) plays critical roles during development and in disease states. One form of programmed cell death utilizes autophagy--a cellular mechanism of degrading bulk cytosolic components--to destroy cells. Previously, the broad-spectrum caspase inhibitor z-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD) was shown to induce autophagic cell death. The mechanism of Zvad-induced cell death was proposed to require caspase-8 inhibition. In our report, we extend these findings to show that--as is the case for apoptosis--induction of autophagic cell death in response to zVAD results in phosphatidylserine exposure prior to loss of membrane integrity. Additionally, we show that caspase-8 inhibition is insufficient to cause autophagic cell death. Rather, the activity of a calpain-like protease must also be blocked. These results reveal the existence of an autophagic PCD-inhibiting calpain-like cysteine protease.     

Simultaneous induction of apoptotic, autophagic, and necrosis-like cell death by monoclonal antibodies recognizing chicken transferrin receptor

Programmed cell death (PCD) is categorized as apoptotic, autophagic, or necrosis-like. Although the possibility that plural (two or three) death signals could be induced by a given stimulus has been reported, the precise mechanisms regulating PCD are not well understood. Recently, we have obtained two anti-chicken transferrin receptor (TfR) monoclonal antibodies (mAbs; D18 and D19) inducing a unique cell death. Although the cell death had several features of apoptosis, autophagic and necrosis-like morphological alterations were simultaneously observed in electron microphotographs. In addition to cells with condensed chromatin and an intact plasma membrane (apoptotic cells), cells having many vacuoles in the cytoplasm (autophagic cells), and enlarged cells with ruptured plasma membranes (necrosis-like cells) were observed in DT40 cells treated with the mAbs, however, the latter two types of dead cells were not detected upon treatment with staurosporine, a typical apoptosis inducer. In autophagic cells, numerous membrane-bound vesicles occupying most of the cytoplasmic space, which frequently contained electron-dense materials from cytoplasmic fragments and organelles, were observed. The simultaneous induction of multiple death signals from a stimulus via the TfR is of great interest to those researching cell death. In addition, activation of caspases was observed in DT40 cells treated with D19, however, the cell death was not inhibited with z-VAD-fmk, a pan-caspase inhibitor, suggesting that at least in part, a caspase-independent pathway is involved in the TfR-mediated cell death.     

Autosis and autophagic cell death: the dark side of autophagy

It is controversial whether cells truly die via autophagy or whether — in dying cells — autophagy is merely an innocent bystander or a well-intentioned ‘Good Samaritan'' trying to prevent inevitable cellular demise. However, there is increasing evidence that the genetic machinery of autophagy may be essential for cell death in certain settings. We recently identified a novel form of autophagy gene-dependent cell death, termed autosis, which is mediated by the Na⁺,K⁺-ATPase pump and has unique morphological features. High levels of cellular autophagy, as occurs with treatment with autophagy-inducing peptides, starvation, or in vivo during certain types of ischemia, can trigger autosis. These findings provide insights into the mechanisms and strategies for prevention of cell death during extreme stress conditions.     

Paclitaxel resistance is associated with switch from apoptotic to autophagic cell death in MCF-7 breast cancer cells

Taxanes remain first line chemotherapy in management of metastatic breast cancer and have a key role in epithelial ovarian cancer, with increasingly common use of weekly paclitaxel dosing regimens. However, their clinical utility is limited by the development of chemoresistance. To address this, we modelled in vitro paclitaxel resistance in MCF-7 cells. We show that at clinically relevant drug doses, emerging paclitaxel resistance is associated with profound changes in cell death responses and a switch from apoptosis to autophagy as the principal mechanism of drug-induced cytotoxicity. This was characterised by a complete absence of caspase-mediated apoptotic cell death (using the pan-caspase-inhibitor Z-VAD) in paclitaxel-resistant MCF-7TaxR cells, compared with parent MCF-7 or MDA-MB-231 cell lines on paclitaxel challenge, downregulation of caspase-7, caspase-9 and BCl2-interacting mediator of cell death (BIM) expression. Silencing with small interfering RNA to BIM in MCF-7 parental cells was sufficient to confer paclitaxel resistance, inferring the significance in downregulation of this protein in contributing to the resistant phenotype of the MCF-7TaxR cell line. Conversely, there was an increased autophagic response in the MCF-7TaxR cell line with reduced phospho-mTOR and relative resistance to the mTOR inhibitors rapamycin and RAD001. In conclusion, we show for the first time that paclitaxel resistance is associated with profound changes in cell death response with deletion of multiple apoptotic factors balanced by upregulation of the autophagic pathway and collateral sensitivity to platinum.     

Some autophagic and apoptotic features of programmed cell death in the anterior silk glands of the silkworm, Bombyx mori

Programmed cell death has been subdivided into two major groups: apoptosis and autophagic cell death. The anterior silk gland of Bombyx mori degenerates during larval-pupal metamorphosis. Our findings indicate that two types of programmed cell death features are observed during this physiological process. During the prepupal period, pyknosis of the nucleus, cell detachment,and membrane blebbing occur and they are the first signs of programmed cell death in the anterior silk glands. According to previous studies, all of these morphological appearances are common for both cell-death types. Autophagy features are also exhibited during the prepupal period. Levels of one of the lysosomal marker enzymes-acid phosphatase-are high during this period then decrease gradually. Vacuole formation begins to appear first at the basal surface of the cell, then expands to the apical surface just before the larval pupal ecdysis. After larval-pupal ecdysis, DNA fragmentation, which is the obvious biochemical marker of apoptosis, is detected in agarose gel electrophoresis, which also shows that caspase-like enzyme activities occur during the programmed cell death process of the anterior silk glands. Apoptosis and autophagic cell death interact with each other during the degeneration process of the anterior silk gland in Bombyx mori and this interaction occurs at a late phase of cell death. We suggest that apoptotic cell death only is not enough for whole gland degeneration and that more effective degeneration occurs with this cooperation.     

Mitochondrial regulation of apoptotic cell death

Mitochondria play a decisive role in the regulation of both apoptotic and necrotic cell death. Permeabilization of the outer mitochondrial membrane and subsequent release of intermembrane space proteins are important features of both models of cell death. The mechanisms by which these proteins are released depend presumably on cell type and the nature of stimuli. Of the mechanisms involved, mitochondrial permeability transition appears to be associated mainly with necrosis, whereas the release of caspase activating proteins during early apoptosis is regulated primarily by the Bcl-2 family of proteins. However, there is increasing evidence for interaction and co-operation between these two mechanisms. The multiple mechanisms of mitochondrial permeabilization may explain diversities in the response of mitochondria to numerous apoptotic stimuli in different types of cells.     

Deficiency in apoptotic effectors Bax and Bak reveals an autophagic cell death pathway initiated by photodamage to the endoplasmic reticulum

Efficient exploitation of cell death mechanisms for therapeutic purpose requires the identification of the molecular events committing cancer cells to death and the intracellular elements of the pro-death and pro-survival machinery activated in response to the anticancer therapy. Photodynamic therapy (PDT) is a paradigm of anticancer therapy utilizing the generation of reactive oxygen species to kill the cancer cells. In this study we have identified the photodamage to the sarco(endo)plasmic-reticulum Ca(2+)-ATPase (SERCA) pump and consequent loss in the ER-Ca(2+) homeostasis as the most apical molecular events leading to cell death in hypericin-photosensitized cells. Downstream of the ER-Ca(2+) emptying, both caspase-dependent and -independent pathways are activated to ensure cell demise. The induction of apoptosis as a cell death modality is dependent on the availability of proapopototic Bax and Bak proteins, which are essential effectors of the mitochondrial outer membrane permeabilization (MOMP) and subsequent caspase activation. In Bax(-/-)/Bak(-/-) cells a nonapoptotic pathway dependent on sustained autophagy commits the oxidatively damaged cells to death. These results argue that the decision to die in this paradigm of oxidative stress is taken upstream of Bax-dependent MOMP and that the irreversible photodamage to the ER induced by hypericin-PDT acts as a trigger for an autophagic cell death pathway in apoptosis-deficient cells.