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Google Scholar Articles by Das, G. Articles by Baehrecke, E. Search for related content. Subject Collections Cell Survival and Cell Death. Share CiteULike Delicious Digg Facebook Reddit Twitter What's this?

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Interview Click to see an interview with subject collection editor Lucy Shapiro. Interview Click to see an interview with subject collection editor Paolo Sassone-Corsi. Interview Click to see an interview with subject collection editor Richard Morimoto. In C cells there was an early but transient increase in the number of autophagosomes at 6 h following TR, which declined by 24 h Figure 2 B, bottom and C, right.

In PC3, however, autophagosome formation significantly increased only after 24 h TR Figure 2 B, bottom and C, left. Also, TR plus chloroquine CQ , which inhibits autophagic degradation, compared to CQ alone, resulted in a greater accumulation of autophagosomes in C compared to PC3 cells, suggesting that autophagic flux was higher in C cells Figure 2 B and C.

These results demonstrate that C, but not PC3 cells, exhibit a rapid induction and effective completion of autophagy in response to TR.

Next, we confirmed autophagic flux by determining LC3B localization using GFP-mCherry-LC3, which allows distinction between autophagosomal and autolysosomal LC3B as yellow indicating co-localization of GFP and mCherry and red signals, respectively.

By time-lapse confocal imaging, PC3 cells showed no change in green and red fluorescence for LC3B until 42 min, suggesting that autophagosomes remained intact following TR Figure 3 A and B; Additional file 1 : Movie S1 and Additional file 2 : Movie S2.

In contrast, C cells showed decreased green fluorescence intensity for LC3B at 21 min, with a small decrease, if any, in red fluorescence, indicating that autophagosomes were converting into autolysosomes Figure 3 A and C.

The autophagic flux is higher in C compared to PC3 cells. A-C Representative confocal time lapse images every 7 min and quantitation of green and red fluorescence for LC3B expression using GFP-mCherry-LC3 fusion protein stably expressed in PC3 and C cells following TR.

G Representative confocal images of fixed cells for p62 green immunostaining following 8 h TR in PC3 and C cells. Nuclei were stained with DAPI. Next, confocal imaging was performed to determine the total number of autophagosomes and autolysosomes, by scoring LC3B puncta following TR ± CQ.

PC3 cells displayed only autophagosomal LC3B-II yellow puncta on co-localization , whereas C showed more autolysosomal LC3B-II red within 2 h of TR Figure 3 D.

Co-treatment with CQ led to accumulation of TR-induced autophagosomal LC3B-II to a larger extent in C compared to PC3 cells Figure 3 D. Quantitation of autophagosomal and autolysosomal LC3B puncta also revealed much higher autophagic flux in response to TR in C compared to PC3 cells Figure 3 E and F.

Thus, autophagic flux observed in C cells was associated with cell survival whereas the low flux in PC3 cells was associated with TR-induced cell death. Immunofluorescence staining for endogenous LC3B showed an increase in the number of LC3B puncta in C but not PC3 cells following 8 h TR Additional file 3 : Figure S1A.

PC3 cells had, however, more LC3B puncta constitutively compared to C cells, indicating a greater autophagosomal content at baseline, but with no further induction of autophagy following TR.

Addition of CQ led to accumulation of LC3B puncta in both PC3 and C Additional file 3 : Figure S1A. In PC3 cells the number of LC3 puncta did not significantly increase following TR plus CQ compared to CQ alone. However, in C cells the number significantly increased following TR and was even higher upon addition of CQ Additional file 3 : Figure S1A.

Similar results were obtained with analysis of p62 levels. An autophagy-specific substrate that acts as a scaffold protein and forms protein aggregates, p62 is directed with its targets to autophagic degradation [ 22 , 23 ]. As shown by confocal immunostaining Figure 3 G and western blotting Additional file 3 : Figure S1B , after TR expression of cytoplasmic p62 aggregates are increased in PC3, whereas p62 expression decreased in C cells.

Pretreatment with CQ, however, prevented TR-induced degradation of p62 in C cells Additional file 3 : Figure S1B. These findings were extended to all additional PCa cell lines examined. Similar to C, LNCaP, DU, and CWRv Similar to C cells, p62 levels as analyzed by western blotting and immunostaining, were decreased in the other TR-resistant cell lines following TR treatment Additional file 3 : Figure S1D and S1E.

Nevertheless, CQ treatment inhibited TR—induced degradation of p62 Additional file 3 : Figure S1D and S1E. These results suggest that in response to TR, autophagy is induced and high autophagic flux is associated with cell survival whereas low flux is associated with cell death.

Next, pharmacological and genetic approaches were used to study the effect of autophagy induction and completion on TR-induced cell death in PCa cell lines.

Pharmacological inhibition of autophagy by 3-MA or CQ sensitized C cells to TR-induced cell death, with no significant effect on PC3 cells Figure 4 A and B. For genetic inhibition of autophagy, lentiviral-mediated shRNA-expressing stable clones of ATG7 and LAMP2 were generated in PC3 and C cells.

LC3B-II levels were reduced in shATG7, while they increased in shLAMP2-expressing cells Additional file 4 : Figure S2A and S2B. p62 levels were increased in both shATG7- and shLAMP2-expressing C and PC3 cells, suggesting that autophagy inhibition resulted in accumulation of p62 Additional file 4 : Figure S2A and S2B.

As with 3-MA and CQ, ATG7 and LAMP2-depleted C cells formed fewer colonies Figure 4 C and underwent cell death Additional file 5 : Figure S3A in response to TR, indicating that they were more sensitive to TR when autophagy was inhibited.

Interestingly, ATG7 depletion in PC3 cells inhibited TR-induced cell death significantly Figure 4 D and Additional file 5 : Figure S3A. Thus in TR-sensitive cells, with an intrinsic defect in TR-induced autophagic flux, inhibition of autophagosome formation at an early step was protective.

Autophagy inhibition prevents cell death in PC3 but enhances cell death in C cells. Overall, our data suggest that autophagic clearance of toxic cellular components is essential for the PCa cells to survive TR-induced cell death that is associated with autophagy induction.

In TR-sensitive cells TR induces autophagosome-formation; however, due to impaired autophagic flux, autophagosome-associated toxic cellular aggregates are formed, and this results in cell death.

Therefore, inhibiting autophagy induction could antagonize its effect. In TR-resistant cells that are proficient in autophagic flux, TR-induced accumulation of cellular aggregates is prevented and the cells survive.

Thus, inhibition of the autophagic pathway in TR-resistant cells leads to accumulation of protein aggregates and sensitizes these cells to TR. Thus, TR-induced autophagy causes cell death in TR-sensitive cells, whereas it has a prosurvival role in TR-resistant cells due to differential autophagic flux.

Caspase-8 can be proteolytically cleaved to a pkD fragment through its association with p62 aggregates, leading to its complete activation and ensuing apoptosis [ 13 ]. Since differential autophagic flux in PCa cells determined cell death in response to TR, we investigated whether the impaired or inhibited autophagic flux led to cell death in response to TR by accumulation of p62 and subsequent activation of caspase z-IETD-fmk inhibition of caspase-8 also prevented cell death in PC3 cells expressing shATG7 and shLAMP2 Figure 5 B.

Consistently, in C cells inhibition of autophagic flux using CQ pretreatment, as measured by inhibition of p62 degradation following TR treatment Figure 5 C , led to TR-induced accumulation of the fully activated pkD form of caspase-8 Figure 5 C.

Similarly, in PC3 cells both 3-MA pretreatment and siBECN1-expression led to a decrease in TR-induced cleaved caspase-8 levels Figure 5 D and E, respectively. These results confirmed that autophagy induction was required for TR-induced apoptosis in PC3 cells, which depended on caspase-8 activation.

Impaired autophagic flux causes apoptosis in PCa cells by caspase-8 activation. A Western blot analysis for caspase-8 activation following TR.

β-actin served as loading control. Western blot analysis of the indicated proteins in C C cells following TR ± CQ, and D PC3 cells following TR ± 3-MA or E PC3 cells expressing non-target siRNA or siBECN1 following TR.

Thus, a constitutive defect in autophagic flux in response to TR causes inhibition of autophagic clearance of p62 aggregates that, in turn, results in caspase-8 activation, leading to cell death in PC3 cells.

However, in TR-resistant C cells, complete autophagy signaling leads to clearance of p62 aggregates, and hence activation of caspase-8 is prevented, thereby facilitating cell survival. In this study we show that autophagy is critical for PCa pathogenesis, as p62 is overexpressed in the cytoplasm of high grade PCa.

In contrast, in benign tissue it is only expressed in the cell nuclei, suggesting that p62 has a more basic function apart from autophagy [ 17 ]. Interestingly, cytoplasmic p62 expression is positively associated with the aggressiveness of the disease. These findings suggest that p62 could be a potential molecular biomarker for PCa progression and that elevated autophagy might be an important factor for disease progression, maintenance of tumor homeostasis in higher grade PCa, or both.

In addition to its principal role of maintaining cellular homeostasis in health and disease, during chemotherapy, autophagy counterbalances the cellular stress generated by chemotherapeutic agents as well as provides energy to maintain cellular homeostasis [ 24 , 25 ].

Therefore, autophagy inhibition has recently emerged as a potential therapeutic approach to induce cell death in cancer cells. The dependence of PCa on this pathway is, therefore, exploitable for therapeutic benefit.

We have previously shown that the combination of CPT with TR increases apoptosis in C PCa cells, which are otherwise resistant to TR [ 19 , 20 ]. Autophagy mediates cell survival in tumor cells and serves as a mechanism of resistance against many chemotherapeutics, including TR [ 26 ].

Here, we have identified autophagy as a mediator of cell survival in four TR-resistant PCa cell lines. These TR-resistant PCa cells exhibited high autophagic flux, in contrast to TR-sensitive PC3 cells, in which autophagic flux was low, preventing completion of autophagy and leading to autophagosome accumulation.

TR led to degradation of p62 and prevented caspase-8 activation in TR-resistant cells. However, in TR-sensitive cells, accumulation of autophagosomes and p62 protein aggregates, which were associated with impaired autophagic degradation, led to caspase-8 activation and apoptosis. Consistently, inhibition of autophagy pharmacologically or genetically by shRNA-mediated knockdown of ATG7 and LAMP2 sensitized C cells to TR.

Importantly, inhibition of autophagy at the different steps in the autophagy pathway led to different outcomes for TR-induced cell death in PC3 cells. Thus, when autophagy was inhibited by 3-MA, siBECN1, or shATG7 before the association of LC3 with the autophagosomal membranes, p62 aggregate formation and subsequent caspase-8 activation was prevented, and cell death was inhibited.

In contrast, inhibiting autophagic degradation using CQ or shLAMP2 had no effect on TR-induced cell death in PC3 cells. These findings suggest that accumulation of autophagosomes and p62 protein aggregates in the absence of autophagic degradation is sufficient for TR-induced cell death.

Levels of LAMP2 were decreased in TR-sensitive compared to TR-resistant cells data not shown , supporting the notion that autophagic degradation was inhibited in TR-sensitive cells. This was also true in a small-lung carcinoma model, where LAMP2 was down-regulated in TR-sensitive as compared to TR-resistant groups [ 14 ].

These findings further support our conclusion that autophagic degradation is impaired in TR-sensitive tumor cell lines. Establishing the extent of autophagy in different grades of PCa will be useful for designing better therapeutic modalities by combining autophagy inhibitors currently in clinical trials [ 27 ].

These include autophagy inhibitors e. hydroxychloroquine and therapeutics that impact autophagy, either directly, such as mTOR inhibitors e. everolimus , or indirectly, such as inhibitors of PI3K and AKT. Moreover, in patients with caloric or metabolic deregulation such as those who are obese autophagy may have a greater impact, as might agents that modulate it and, therefore, based on our findings, such patients could be stratified for individualized treatments.

The following reagents were purchased: chloroquine CQ , 3-methyladenine 3-MA , acridine orange, and propidium iodide Sigma-Aldrich.

The GFP-mCherry-LC3B and pCL10 plasmids were a kind gift from Dr. Jayanta Debnath University of California San Francisco. The lentiviral packaging plasmids pVSVG and dr 8. siBECN1 sc and control siRNA Fluorescein Conjugate -A sc were from Santa Cruz Biotechnology. Fugene was from Roche Applied Science.

The following antibodies were used; p62 Fitzgerald Industries International ; LAMP2, p62, BECN1 Santa Cruz Biotechnologies ; ATG7 Cell Signaling Technologies , β -actin Sigma Aldrich ; Caspase-8 C; Alexis ; secondary anti-mouse HRP Millipore ; and secondary anti-rabbit HRP Fisher Scientific.

PCa cell lines PC3, LNCaP, DU, CWRv Benign adjacent prostate tissue was present in 40 of the 51 cases. Tissue was collected in the Department of Pathology at the Cleveland Clinic as part of an IRB-approved protocol. Immunohistochemistry with a monoclonal p62 antibody was performed on formalin-fixed paraffin-embedded TMA sections mounted on poly L-lysine-coated slides.

The sections were deparaffinized in xylene and rehydrated through graded alcohols into distilled water. Antigen retrieval was in 0. Non-immune pooled mouse immunoglobulin was used as a negative control. Slides were then counterstained with haematoxylin, rinsed, and dehydrated through graded alcohols into non-aqueous solution and cover-slipped with mounting media.

During autophagy, LC3B-II is recruited to the autophagosomal membranes and continues to be present on the membranes of completed autophagosomes, which can be visualized as a yellow signal because GFP and mCherry co-localize. In autolysosomes, because of the acidic pH, the GFP fluorescence is diminished while mCherry still remains stable.

Thus, the conversion of yellow LC3B-II puncta to red LC3-II puncta provides a readout for autophagic flux. oil immersion objective lens on a Leica TCS-SP2 confocal microscope Leica Microsystems AG.

LC3B puncta were quantified using the Red and Green Puncta Co-localization Macro with the Image J program, as described [ 29 , 30 ].

Immunostaining was performed as previously described [ 30 ]. Briefly, following the respective treatments, cells were fixed with 2.

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Cell death triggered by insulin withdrawal is switched from ACD to apoptosis by calpain 2 and VCP. High levels of corticosterone CORT induced by CRS cause ACD via SGK3, which has a PX domain for binding to PI3P and the initiation of autophagy. The dashed lines indicate that the process is not yet experimentally confirmed.

Recently, it was reported that autophagy activation is required for cell death and, thereby, the elimination of precancerous cells during replicative crisis caused by telomere dysfunction and that loss of ACD initiates tumorigenesis in fibroblasts and epithelial cells During replicative crises, apoptosis markers were not detected, whereas extensive cytoplasmic double-membrane autophagosomes and single-membrane autolysosomes were observed with a reduced level of p62, an autophagy cargo receptor.

Moreover, the accumulation of microtubule-associated protein 1 light chain 3-beta-II MAP1LC3B -II and p62 after treatment with the autophagy blocker bafilomycin A1 suggested increased autophagy flux during replicative crisis.

On the other hand, shRNA against ATG 3, ATG 5, or ATG7 promoted a bypass of the crisis, continued cell proliferation, and increased genome instability. It was also found that telomeric DNA damage activated ACD via the cGAS-STING pathway 57 Fig.

These findings highlight autophagy as an essential component in tumor-suppressive mechanisms. Melanoma cells, which are resistant to apoptosis-inducing drugs, can undergo ACD upon treatment with compounds targeting orphan nuclear receptor TR3 Upon treatment, TR3 is translocated to mitochondria via its interaction with the mitochondrial outer membrane protein Nix and dissipates mitochondrial membrane potential to induce massive mitochondrial clearance and ACD.

TR3 translocation-triggered autophagy requires TR3 to cross into the mitochondrial inner membrane; therefore, this nuclear receptor becomes integrated into a mitochondrial signaling pathway to induce ACD. However, further details are not yet clear, particularly those that might indicate whether selective removal of mitochondria through mitophagy is required for ACD or whether mitochondrial clearance is simply part of bulk autophagic degradation.

Despite the lack of detailed knowledge of the mode of action of TR3-targeting compounds, engagement of TR3 by compounds targeted to it demonstrated antimelanoma activity in the liver and lung in several mouse models The role of autophagy in tumorigenesis is complex, with autophagy having different consequences for cancer development and treatment depending on the types of tumors and their stages Autophagy may serve as a tumor-suppressor pathway through several mechanisms: by maintaining genomic stability; by eliminating defective subcellular organelles, including depolarized mitochondria, and thus by removing the cellular sources of oxidative stress; and by regulating inflammation.

All of these mechanisms may contribute to the prevention of cancer development. However, the survival- and death-promoting functions of autophagy make its association with cancer treatment very complicated.

In contrast to the antitumor roles of autophagy, whereby cancer cells are eradicated by ACD, autophagy can maintain cancer cells viability by providing metabolic substrates under nutrient-limited conditions, delaying the onset of apoptosis of cells challenged by chemotherapeutic drugs or irradiation, and enhancing cancer cell survival under stressful microenvironments, including hypoxia Kainate-induced excitotoxicity combined with hypoxia was used to mimic hypoxia—ischemia in vitro and induced cell death in primary rat cortical neurons.

Cell death was blocked by pharmacological autophagy inhibitors and genetic inhibition of autophagy by knocking down Beclin-1 or Atg7 , whereas overexpression of Beclin-1 or ATG7 enhanced hypoxic excitotoxicity In vivo knockdown via intrastriatal injection of lentivirus-expressing sh Beclin-1 reduced striatal damage in a rat model of neonatal hypoxia—ischemia No apoptosis activation was observed, and Bcl-2 overexpression or caspase inhibition prevented neuronal cell death.

FeTMPyP, a peroxynitrite decomposition catalyst, and Mdivi-1, a blocker of mitophagy activation, prevented mitophagy-induced cell death in the ischemia—reperfusion injured brain. In hippocampal neuronal cell death caused by neonatal hypoxia—ischemia, both caspasedependent and caspaseindependent cell death pathways are activated with the concomitant induction of autophagy 63 , Nestin-Cre-driven conditional knockout cKO of Atg7 in the nervous system prevented both caspase-dependent and caspase-independent neuronal death and reduced hippocampal damage.

Interestingly, neuronal death was both caspase-dependent and caspase-independent at the neonatal stage but caspase-independent with more-pronounced autophagy levels at the adult stage.

However, because mice deficient in Atg7 undergo neurodegeneration during development, whether neuronal cell death elicited by hypoxia—ischemia is truly attributable to ACD needs further study using an inducible KO adult mouse model.

Chronic stress or prolonged glucocorticoid administration leads to loss of hippocampal neurons and a reduction in hippocampus size 65 , Glucocorticoid receptors are enriched in the hippocampus 67 , and adult hippocampal neurogenesis continuous generation of new neurons in the adult hippocampus over a lifetime is highly susceptible to psychological stress and is greatly reduced in various models of stress 68 , However, most studies have failed to detect signs of apoptosis; therefore, PCD of hippocampal neurons or adult hippocampal NSCs has not been considered as a mechanism of stress-induced decline in adult hippocampal neurogenesis or hippocampal damage 70 , However, our recent genetic study using adult NSC-specific Atg7 -cKO mice demonstrated that chronic restraint stress CRS induced ACD in adult hippocampal NSCs in vivo and in vitro Fig.

As autophagy is essential for development and tissue homeostasis, deletion of key autophagy genes in the brain from an early developmental stage causes neurodegenerative symptoms and it is difficult to explore ACD in the adult mouse brain 73 , 74 , To overcome this obstacle and study the role of autophagy in the effects of psychological stress on adult hippocampal NSCs, a Nestin-Cre-ERT2 mouse line was crossed with Atg7 flox mice, and Atg7 deletion was induced in the offspring at 7 weeks of age; these NSC-specific cKO mice Atg7-NSC cKO mice were subjected to CRS.

Histological and electron microscopic examination revealed an increase in autophagy flux but not in apoptosis, in hippocampal NSCs. Loss of NSCs and decreases in adult neurogenesis were blocked by Atg7 deletion Furthermore, stress-triggered anxiety and depression, as well as cognitive deficits, were effectively prevented in the Atg7-NSC cKO mice.

These findings indicated that ACD is undoubtedly physiologically important in mammals and that autophagy in the adult hippocampus may provide a new therapeutic avenue for the treatment of stress-induced psychological disorders. The SGK family consists of three members: SGK1, SGK2, and SGK3 SGK3 contains a complete Phox homology PX domain 78 , 80 , which contains a phosphoinositide-binding site.

Phosphatidylinositol 3-phosphate PtdIns3P is the most common lipid that binds to the PX domain, and it is enriched in endosomes and vacuoles; SGK3 binds PtdIns3P and is located mostly in endosomes PtdIns3P is a product of PI3K and regulates the initiation of autophagy A point mutation in which Arg is changed to Ala in SGK3 prevented ACD Therefore, SGK3 is a critical regulator of stress-induced ACD and has this role by interacting with PtdIns3P in adult hippocampal NSCs.

However, additional studies are required to elucidate the details of how SGK3 regulates ACD and to explore SGK3 as a potential therapeutic target for stress-induced psychological disorders. Why is ACD activated in normal cells equipped with intact apoptosis capability? This question can be answered by examining the molecular pathways that link ACD and apoptosis.

Detailed studies on insulin-deficient adult hippocampal NSCs have offered a few glimpses into the complicated intersection of ACD and apoptosis Fig. Calpain 2 is a major calpain in adult hippocampal NSCs and was identified as a key rheostat of apoptosis with respect to ACD, as suppression of calpain activity promoted ACD, whereas higher calpain activity switched the cell death program from ACD to apoptosis in insulin-deprived adult hippocampal NSCs Another interesting effector in the interplay between apoptosis and ACD in adult hippocampal NSCs is valosin-containing protein VCP , which positively regulates autophagosome maturation at the basal state.

However, under conditions of high autophagy flux following insulin withdrawal, VCP regulates the autophagy initiation step Of interest, pharmacological, and genetic inactivation of VCP led to apoptosis with a concomitant increase in calpain 2 levels in insulin-deprived adult hippocampal NSCs However, the switch from ACD to apoptosis and upregulation of calpain activity by inhibition or knockdown of VCP under insulin-deprived conditions were prevented by Atg7 knockdown, indicating that ACD is a prerequisite for the switch to apoptosis.

ATG5 and Beclin-1 were reported as substrates of calpain. Calpain cleaves ATG5 in HeLa, Jurkat, and MDA-MA cells in response to several apoptotic stimuli, including etoposide, doxorubicin, and staurosporine Cleaved ATG5 then translocates from the cytosol to mitochondria, where it associates with Bcl-X1, and triggers cytochrome c release and caspase activation.

Calpain-mediated cleavage of Beclin-1 following renal ischemia results in autophagy inhibition and extensive neuronal death However, we could not detect cleavage of ATG5 or Beclin-1 in insulin-deprived adult hippocampal NSCs. Therefore, understanding the molecular mechanism by which calpain regulates the switch from ACD to apoptosis in adult NSCs awaits further study.

The complex relationship between autophagy and apoptosis depends on the biological context and is not yet fully understood. Intriguingly, the two pathways share common components, such as Bcl-2 family proteins. Bcl-2 can directly bind to Beclin As Beclin-1 is a core component of the VPS34 complex, which is required for phagophore formation and initiation of autophagy through the generation of PtdIns3P, the binding ability of Bcl-2 to Beclin-1 confers, in addition to its well-known antiapoptotic function, another critical cellular function to Bcl an antiautophagic role.

Interestingly, the interaction of Bcl-2 with Beclin-1 does not interfere with the antiapoptotic potential of Bcl-2 However, this interaction can be disrupted by posttranslational modification of Bcl-2 or Beclin-1, including phosphorylation, ubiquitination, or caspase-mediated cleavage 87 , Nevertheless, whether the interaction of ACD with apoptosis is controlled by Bcl-2 family proteins is not yet clear.

This indication will be worth more attention in the near future. Our recent finding that caspase-9 is activated in an APAFindependent manner in insulin-deprived adult hippocampal NSCs provides another intriguing illustration of the interaction of autophagy and apoptosis Caspase-9 promotes ACD but not apoptosis following insulin withdrawal in adult NSCs Elucidation of the molecular mechanism by which autophagy directs caspase-9 into ACD rather than apoptosis will greatly advance our understanding of the interconnection between apoptosis and ACD.

Studies on the cell death mechanism in adult hippocampal NSCs following insulin withdrawal or psychological stress have greatly contributed to the elucidation of ACD at the molecular level.

Adult hippocampal NSCs have intact machinery for apoptosis and necroptosis subroutines, as indicated by staurosporine or H 2 O 2 treatment inducing apoptosis or necroptosis in these cells, and this machinery can be inhibited by appropriate pharmacological inhibitors Another conundrum is the nature of the signaling mechanisms that dictate the contradictory roles of autophagy in cell death and cell survival.

As a compromise, it has been assumed that basal, low-level autophagy is cytoprotective, whereas the sustained excessive level of autophagy flux causes cell death. However, this assumption has not yet been tested experimentally. As most techniques to measure autophagy flux are qualitative, quantitative comparisons of autophagy flux between different conditions, even in the same cell type, as well as between different cell types, is technically very challenging.

Therefore, the molecular mechanisms of ACD are far from being understood. Nevertheless, we have recently witnessed an increasing recognition of the critical roles of ACD in mammalian pathophysiology, including tumor suppression and mental disorders associated with psychological stress.

Elucidation of this uniquely programmed mechanism of cell death holds great potential for applications of autophagy in human health and the treatment of diseases. Yang, Z.

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In PC3, however, autophagosome formation significantly increased only after 24 h TR Figure 2 B, bottom and C, left. Also, TR plus chloroquine CQ , which inhibits autophagic degradation, compared to CQ alone, resulted in a greater accumulation of autophagosomes in C compared to PC3 cells, suggesting that autophagic flux was higher in C cells Figure 2 B and C.

These results demonstrate that C, but not PC3 cells, exhibit a rapid induction and effective completion of autophagy in response to TR. Next, we confirmed autophagic flux by determining LC3B localization using GFP-mCherry-LC3, which allows distinction between autophagosomal and autolysosomal LC3B as yellow indicating co-localization of GFP and mCherry and red signals, respectively.

By time-lapse confocal imaging, PC3 cells showed no change in green and red fluorescence for LC3B until 42 min, suggesting that autophagosomes remained intact following TR Figure 3 A and B; Additional file 1 : Movie S1 and Additional file 2 : Movie S2.

In contrast, C cells showed decreased green fluorescence intensity for LC3B at 21 min, with a small decrease, if any, in red fluorescence, indicating that autophagosomes were converting into autolysosomes Figure 3 A and C. The autophagic flux is higher in C compared to PC3 cells.

A-C Representative confocal time lapse images every 7 min and quantitation of green and red fluorescence for LC3B expression using GFP-mCherry-LC3 fusion protein stably expressed in PC3 and C cells following TR.

G Representative confocal images of fixed cells for p62 green immunostaining following 8 h TR in PC3 and C cells. Nuclei were stained with DAPI. Next, confocal imaging was performed to determine the total number of autophagosomes and autolysosomes, by scoring LC3B puncta following TR ± CQ.

PC3 cells displayed only autophagosomal LC3B-II yellow puncta on co-localization , whereas C showed more autolysosomal LC3B-II red within 2 h of TR Figure 3 D. Co-treatment with CQ led to accumulation of TR-induced autophagosomal LC3B-II to a larger extent in C compared to PC3 cells Figure 3 D.

Quantitation of autophagosomal and autolysosomal LC3B puncta also revealed much higher autophagic flux in response to TR in C compared to PC3 cells Figure 3 E and F. Thus, autophagic flux observed in C cells was associated with cell survival whereas the low flux in PC3 cells was associated with TR-induced cell death.

Immunofluorescence staining for endogenous LC3B showed an increase in the number of LC3B puncta in C but not PC3 cells following 8 h TR Additional file 3 : Figure S1A. PC3 cells had, however, more LC3B puncta constitutively compared to C cells, indicating a greater autophagosomal content at baseline, but with no further induction of autophagy following TR.

Addition of CQ led to accumulation of LC3B puncta in both PC3 and C Additional file 3 : Figure S1A. In PC3 cells the number of LC3 puncta did not significantly increase following TR plus CQ compared to CQ alone. However, in C cells the number significantly increased following TR and was even higher upon addition of CQ Additional file 3 : Figure S1A.

Similar results were obtained with analysis of p62 levels. An autophagy-specific substrate that acts as a scaffold protein and forms protein aggregates, p62 is directed with its targets to autophagic degradation [ 22 , 23 ].

As shown by confocal immunostaining Figure 3 G and western blotting Additional file 3 : Figure S1B , after TR expression of cytoplasmic p62 aggregates are increased in PC3, whereas p62 expression decreased in C cells.

Pretreatment with CQ, however, prevented TR-induced degradation of p62 in C cells Additional file 3 : Figure S1B. These findings were extended to all additional PCa cell lines examined.

Similar to C, LNCaP, DU, and CWRv Similar to C cells, p62 levels as analyzed by western blotting and immunostaining, were decreased in the other TR-resistant cell lines following TR treatment Additional file 3 : Figure S1D and S1E.

Nevertheless, CQ treatment inhibited TR—induced degradation of p62 Additional file 3 : Figure S1D and S1E.

These results suggest that in response to TR, autophagy is induced and high autophagic flux is associated with cell survival whereas low flux is associated with cell death. Next, pharmacological and genetic approaches were used to study the effect of autophagy induction and completion on TR-induced cell death in PCa cell lines.

Pharmacological inhibition of autophagy by 3-MA or CQ sensitized C cells to TR-induced cell death, with no significant effect on PC3 cells Figure 4 A and B. For genetic inhibition of autophagy, lentiviral-mediated shRNA-expressing stable clones of ATG7 and LAMP2 were generated in PC3 and C cells.

LC3B-II levels were reduced in shATG7, while they increased in shLAMP2-expressing cells Additional file 4 : Figure S2A and S2B. p62 levels were increased in both shATG7- and shLAMP2-expressing C and PC3 cells, suggesting that autophagy inhibition resulted in accumulation of p62 Additional file 4 : Figure S2A and S2B.

As with 3-MA and CQ, ATG7 and LAMP2-depleted C cells formed fewer colonies Figure 4 C and underwent cell death Additional file 5 : Figure S3A in response to TR, indicating that they were more sensitive to TR when autophagy was inhibited. Interestingly, ATG7 depletion in PC3 cells inhibited TR-induced cell death significantly Figure 4 D and Additional file 5 : Figure S3A.

Thus in TR-sensitive cells, with an intrinsic defect in TR-induced autophagic flux, inhibition of autophagosome formation at an early step was protective. Autophagy inhibition prevents cell death in PC3 but enhances cell death in C cells. Overall, our data suggest that autophagic clearance of toxic cellular components is essential for the PCa cells to survive TR-induced cell death that is associated with autophagy induction.

In TR-sensitive cells TR induces autophagosome-formation; however, due to impaired autophagic flux, autophagosome-associated toxic cellular aggregates are formed, and this results in cell death.

Therefore, inhibiting autophagy induction could antagonize its effect. In TR-resistant cells that are proficient in autophagic flux, TR-induced accumulation of cellular aggregates is prevented and the cells survive.

Thus, inhibition of the autophagic pathway in TR-resistant cells leads to accumulation of protein aggregates and sensitizes these cells to TR.

Thus, TR-induced autophagy causes cell death in TR-sensitive cells, whereas it has a prosurvival role in TR-resistant cells due to differential autophagic flux. Caspase-8 can be proteolytically cleaved to a pkD fragment through its association with p62 aggregates, leading to its complete activation and ensuing apoptosis [ 13 ].

Since differential autophagic flux in PCa cells determined cell death in response to TR, we investigated whether the impaired or inhibited autophagic flux led to cell death in response to TR by accumulation of p62 and subsequent activation of caspase z-IETD-fmk inhibition of caspase-8 also prevented cell death in PC3 cells expressing shATG7 and shLAMP2 Figure 5 B.

Consistently, in C cells inhibition of autophagic flux using CQ pretreatment, as measured by inhibition of p62 degradation following TR treatment Figure 5 C , led to TR-induced accumulation of the fully activated pkD form of caspase-8 Figure 5 C. Similarly, in PC3 cells both 3-MA pretreatment and siBECN1-expression led to a decrease in TR-induced cleaved caspase-8 levels Figure 5 D and E, respectively.

These results confirmed that autophagy induction was required for TR-induced apoptosis in PC3 cells, which depended on caspase-8 activation. Impaired autophagic flux causes apoptosis in PCa cells by caspase-8 activation.

A Western blot analysis for caspase-8 activation following TR. β-actin served as loading control. Western blot analysis of the indicated proteins in C C cells following TR ± CQ, and D PC3 cells following TR ± 3-MA or E PC3 cells expressing non-target siRNA or siBECN1 following TR.

Thus, a constitutive defect in autophagic flux in response to TR causes inhibition of autophagic clearance of p62 aggregates that, in turn, results in caspase-8 activation, leading to cell death in PC3 cells.

However, in TR-resistant C cells, complete autophagy signaling leads to clearance of p62 aggregates, and hence activation of caspase-8 is prevented, thereby facilitating cell survival. In this study we show that autophagy is critical for PCa pathogenesis, as p62 is overexpressed in the cytoplasm of high grade PCa.

In contrast, in benign tissue it is only expressed in the cell nuclei, suggesting that p62 has a more basic function apart from autophagy [ 17 ]. Interestingly, cytoplasmic p62 expression is positively associated with the aggressiveness of the disease.

These findings suggest that p62 could be a potential molecular biomarker for PCa progression and that elevated autophagy might be an important factor for disease progression, maintenance of tumor homeostasis in higher grade PCa, or both. In addition to its principal role of maintaining cellular homeostasis in health and disease, during chemotherapy, autophagy counterbalances the cellular stress generated by chemotherapeutic agents as well as provides energy to maintain cellular homeostasis [ 24 , 25 ].

Therefore, autophagy inhibition has recently emerged as a potential therapeutic approach to induce cell death in cancer cells. The dependence of PCa on this pathway is, therefore, exploitable for therapeutic benefit. We have previously shown that the combination of CPT with TR increases apoptosis in C PCa cells, which are otherwise resistant to TR [ 19 , 20 ].

Autophagy mediates cell survival in tumor cells and serves as a mechanism of resistance against many chemotherapeutics, including TR [ 26 ]. Here, we have identified autophagy as a mediator of cell survival in four TR-resistant PCa cell lines.

These TR-resistant PCa cells exhibited high autophagic flux, in contrast to TR-sensitive PC3 cells, in which autophagic flux was low, preventing completion of autophagy and leading to autophagosome accumulation.

TR led to degradation of p62 and prevented caspase-8 activation in TR-resistant cells. However, in TR-sensitive cells, accumulation of autophagosomes and p62 protein aggregates, which were associated with impaired autophagic degradation, led to caspase-8 activation and apoptosis.

Consistently, inhibition of autophagy pharmacologically or genetically by shRNA-mediated knockdown of ATG7 and LAMP2 sensitized C cells to TR. Importantly, inhibition of autophagy at the different steps in the autophagy pathway led to different outcomes for TR-induced cell death in PC3 cells.

Thus, when autophagy was inhibited by 3-MA, siBECN1, or shATG7 before the association of LC3 with the autophagosomal membranes, p62 aggregate formation and subsequent caspase-8 activation was prevented, and cell death was inhibited.

In contrast, inhibiting autophagic degradation using CQ or shLAMP2 had no effect on TR-induced cell death in PC3 cells. These findings suggest that accumulation of autophagosomes and p62 protein aggregates in the absence of autophagic degradation is sufficient for TR-induced cell death.

Levels of LAMP2 were decreased in TR-sensitive compared to TR-resistant cells data not shown , supporting the notion that autophagic degradation was inhibited in TR-sensitive cells.

This was also true in a small-lung carcinoma model, where LAMP2 was down-regulated in TR-sensitive as compared to TR-resistant groups [ 14 ].

These findings further support our conclusion that autophagic degradation is impaired in TR-sensitive tumor cell lines. Establishing the extent of autophagy in different grades of PCa will be useful for designing better therapeutic modalities by combining autophagy inhibitors currently in clinical trials [ 27 ].

These include autophagy inhibitors e. hydroxychloroquine and therapeutics that impact autophagy, either directly, such as mTOR inhibitors e. everolimus , or indirectly, such as inhibitors of PI3K and AKT.

Moreover, in patients with caloric or metabolic deregulation such as those who are obese autophagy may have a greater impact, as might agents that modulate it and, therefore, based on our findings, such patients could be stratified for individualized treatments.

The following reagents were purchased: chloroquine CQ , 3-methyladenine 3-MA , acridine orange, and propidium iodide Sigma-Aldrich. The GFP-mCherry-LC3B and pCL10 plasmids were a kind gift from Dr.

Jayanta Debnath University of California San Francisco. The lentiviral packaging plasmids pVSVG and dr 8. siBECN1 sc and control siRNA Fluorescein Conjugate -A sc were from Santa Cruz Biotechnology.

Fugene was from Roche Applied Science. The following antibodies were used; p62 Fitzgerald Industries International ; LAMP2, p62, BECN1 Santa Cruz Biotechnologies ; ATG7 Cell Signaling Technologies , β -actin Sigma Aldrich ; Caspase-8 C; Alexis ; secondary anti-mouse HRP Millipore ; and secondary anti-rabbit HRP Fisher Scientific.

PCa cell lines PC3, LNCaP, DU, CWRv Benign adjacent prostate tissue was present in 40 of the 51 cases. Tissue was collected in the Department of Pathology at the Cleveland Clinic as part of an IRB-approved protocol.

Immunohistochemistry with a monoclonal p62 antibody was performed on formalin-fixed paraffin-embedded TMA sections mounted on poly L-lysine-coated slides. The sections were deparaffinized in xylene and rehydrated through graded alcohols into distilled water.

Antigen retrieval was in 0. Non-immune pooled mouse immunoglobulin was used as a negative control. Slides were then counterstained with haematoxylin, rinsed, and dehydrated through graded alcohols into non-aqueous solution and cover-slipped with mounting media. During autophagy, LC3B-II is recruited to the autophagosomal membranes and continues to be present on the membranes of completed autophagosomes, which can be visualized as a yellow signal because GFP and mCherry co-localize.

In autolysosomes, because of the acidic pH, the GFP fluorescence is diminished while mCherry still remains stable. Thus, the conversion of yellow LC3B-II puncta to red LC3-II puncta provides a readout for autophagic flux.

oil immersion objective lens on a Leica TCS-SP2 confocal microscope Leica Microsystems AG. LC3B puncta were quantified using the Red and Green Puncta Co-localization Macro with the Image J program, as described [ 29 , 30 ]. Immunostaining was performed as previously described [ 30 ]. Briefly, following the respective treatments, cells were fixed with 2.

The coverslips were then immunostained using the antibodies diluted in blocking buffer, followed by fluorescently-conjugated secondary antibody. DAPI was added to stain nuclei before the penultimate washing.

They were then mounted in Vectashield Vector Laboratories. Cells were immediately fixed in 2. After en bloc staining and dehydration with ethanol, the samples were embedded with eponate 12 medium Ted Pella Inc, Redding, CA.

Thin sections 85 nm were cut with a diamond knife, double-stained with uranyl acetate and lead citrate, and analyzed using a Philips CM12 electron microscope FEI Company operated at 60 kv.

Cells with more than 10 vacuoles were scored as autophagy positive. The autophagic area was quantified as described previously [ 30 ]. At least 10 cells per sample were used for quantitation. The size of autophagic structures was represented as relative area values calculated by selecting specific areas using Image J.

The autophagic area was calculated as the percentage fraction normalized to the total cytoplasmic area. Media containing viral particles was then collected 24 h after transfection, passed through a 0.

For control pLKO. Cell viability was determined by staining with annexinV-FITC and propidium iodide PI followed by flow cytometric analysis on a FACScan with a nm argon laser BD Biosciences. Analyses were performed with the Cell Quest program.

For clonogenic survival, cells were plated in 6-well plates in triplicate. The absolute number of colonies was plotted. Cells were grown in mm glass bottom dishes MatTek and imaged using an UltraVIEW VoX spinning disc confocal microscope Perkin Elmer equipped with a high-sensitivity cooled bit EMCCD C camera Hamamatsu.

During imaging, cells were kept in a heated incubation chamber at 37°C with CO 2 LiveCell, Pathology Devices, Inc.

Volocity image acquisition software was used to capture the images Perkin Elmer. The track analysis and intensity measurements were done with Image Pro 7. All the remaining data were obtained from at least three independent experiments carried out in triplicate with the error bars denoting SEM.

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