Dissecting the dynamic turnover of GFP-LC3 in the autolysosome

Determination of autophagic flux is essential to assess and differentiate between the induction or suppression of autophagy. Western blot analysis for free GFP fragments resulting from the degradation of GFP-LC3 within the autolysosome has been proposed as one of the autophagic flux assays. However, the exact dynamics of GFP-LC3 during the autophagy process are not clear. Moreover, the characterization of this assay in mammalian cells is limited. Here we found that lysosomal acidity is an important regulating factor for the step-wise degradation of GFP-LC3, in which the free GFP fragments are first generated but accumulate only when the lysosomal acidity is moderate, such as during rapamycin treatment. When the lysosomal acidity is high, such as during starvation in Earle's balanced salt solution (EBSS), the GFP fragments are further degraded and thus do not accumulate. Much to our surprise, we found that the level of free GFP fragments increased in the presence of several late stage autophagy inhibitors, such as chloroquine or E64D plus pepstatin A. Furthermore, the amount of free GFP fragments depends on the concentrations of these inhibitors. Unsaturating concentrations of chloroquine or bafilomycin A1 increased the level of free GFP fragments while saturating concentrations did not. Data from the present study demonstrate that GFP-LC3 is degraded in a step-wise fashion in the autolysosome, in which the LC3 portion of the fusion protein appears to be more rapidly degraded than GFP. However, the amount of free GFP fragments does not necessarily correlate with autophagic flux if the lysosomal enzyme activity and pH are changed. Therefore, caution must be used when conducting the GFP-LC3 cleavage assay as a determinant of autophagic flux. In order to accurately assess autophagy, it is more appropriate to assess GFP-LC3 cleavage in the presence or absence of saturating or unsaturating concentrations of chloroquine or bafilomycin A1 together with other autophagy markers, such as levels of p62 and endogenous LC3-II.     

A new probe to measure autophagic flux in vitro and in vivo

Macroautophagy is a catabolic process that delivers cytoplasmic components via the autophagosome to lysosomes for degradation. Measuring autophagic activity is critical to dissect molecular mechanisms and functions of autophagy but remains challenging due to the lack of a definitive method. We have recently developed a new fluorescent probe, GFP-LC3-RFP-LC3ΔG, to assess autophagic flux. Upon intracellular expression, the probe is cleaved by ATG4 family proteases into equimolar amounts of GFP-LC3 and RFP-LC3ΔG. The former is degraded by autophagy while the latter persists as an internal control in the cytosol. Autophagic flux can thus be quantified by obtaining the ratio of GFP:RFP signals. Using this method, we have identified several autophagy-modulating drugs by screening an approved drug library. We have also demonstrated that induced and basal autophagic flux can be monitored in zebrafish and mice.     

Direct quantification of autophagic flux by a single molecule-based probe

Macroautophagy/autophagy remains a rapidly advancing research topic, and there continues to be a need for constantly evolving methodology to investigate this pathway at each individual step. Accordingly, new assays to measure autophagic flux in a robust and reliable manner are essential to understand the mechanism and physiological roles of autophagy. Kaizuka et al. recently reported a new fluorescence probe, GFP-LC3-RFP-LC3ΔG to directly demonstrate autophagic flux without being combined with lysosomal inhibitors (see the Kaizuka et al. punctum in this issue of the journal). When expressed in cells, the probe is cleaved into a degradable/quenchable part, GFP-LC3, and stable/cytosolic part, RFP-LC3ΔG. The latter serves as an internal control and a decrease of the GFP:RFP ratio indicates the occurrence of autophagy. Since the key index of this probe is the degradation of GFP-LC3, it can be used to measure the cumulative effect of basal autophagy. The assay is applicable to high-throughput drug discovery as well as in vivo analysis.     

Induction of genuine autophagy by cationic lipids in mammalian cells

Autophagy is a cellular stress response that results in the activation of a lysosomal degradation pathway. In this report, we showed that cationic lipids, a common-used class of transfection reagents, induced genuine autophagy in mammalian cells. Extensive LC3 dot formation was observed by treatment with cationic lipids (with or without DNA), but not neutral lipids, in a HeLa cell line stably expressing GFP-LC3 (HeLa-LC3). Further proofs for autophagy were obtained by the co-localization of the LC3 dots with lysosome-specific staining patterns, observation of LC3-I to LC3-II form conversion and appearance of autophagic vacuoles under TEM. The autophagic flux assay with bafilomycin A1 and degradation of p62/SQSTM1 suggested that the autophagy induced by cationic lipids was primarily due to increased formation of autophagosomes and not decreased turnover. Moreover, cationic lipids induced autophagy in an mTOR-independent manner.     

A proteolytic cleavage assay to monitor autophagy in Dictyostelium discoideum

Dictyostelium discoideum is a good model of autophagy. However, the lack of autophagic flux techniques hinders the assessment of new mutants or drugs. One of these techniques, which has been used successfully in yeast and mammalian cells, but has not yet been described in Dictyostelium, is based on the presence of proteolytic fragments derived from autophagic degradation of expressed fusion proteins. Lysosomotropic agents such as NH 4Cl penetrate acidic compartments and raise their pH, thus allowing the accumulation and measurement of these cleaved fragments, which otherwise would be rapidly degraded. We have used this property to detect the presence of free GFP fragments derived from the fusion protein GFP-Tkt-1, a cytosolic marker. We demonstrate that this proteolytic event is dependent on autophagy and can be used to detect differences in the level of autophagic flux among different mutant strains. Moreover, treatment with NH4Cl also facilitates the assessment of autophagic flux by confocal microscopy using the marker RFP-GFP-Atg8.     

Autophagy Benefits the Replication of Newcastle Disease Virus in Chicken Cells and Tissues

Newcastle disease virus (NDV) is an important avian pathogen. We previously reported that NDV triggers autophagy in U251 glioma cells, resulting in enhanced virus replication. In this study, we investigated whether NDV triggers autophagy in chicken cells and tissues to enhance virus replication. We demonstrated that NDV infection induced steady-state autophagy in chicken-derived DF-1 cells and in primary chicken embryo fibroblast (CEF) cells, evident through increased double- or single-membrane vesicles, the accumulation of green fluorescent protein (GFP)-LC3 dots, and the conversion of LC3-I to LC3-II. In addition, we measured autophagic flux by monitoring p62/SQSTM1 degradation, LC3-II turnover, and GFP-LC3 lysosomal delivery and proteolysis, to confirm that NDV infection induced the complete autophagic process. Inhibition of autophagy by pharmacological inhibitors and RNA interference reduced virus replication, indicating an important role for autophagy in NDV infection. Furthermore, we conducted in vivo experiments and observed the conversion of LC3-I to LC3-II in heart, liver, spleen, lung, and kidney of NDV-infected chickens. Regulation of the induction of autophagy with wortmannin, chloroquine, or starvation treatment affects NDV production and pathogenesis in tissues of both lung and intestine; however, treatment with rapamycin, an autophagy inducer of mammalian cells, showed no detectable changes in chicken cells and tissues. Moreover, administration of the autophagy inhibitor wortmannin increased the survival rate of NDV-infected chickens. Our studies provide strong evidence that NDV infection induces autophagy which benefits NDV replication in chicken cells and tissues.     

A proteolytic cleavage assay to monitor autophagy in Dictyostelium discoideum

Dictyostelium discoideum is a good model of autophagy. However, the lack of autophagic flux techniques hinders the assessment of new mutants or drugs. One of these techniques, which has been used successfully in yeast and mammalian cells, but has not yet been described in Dictyostelium, is based on the presence of proteolytic fragments derived from autophagic degradation of expressed fusion proteins. Lysosomotropic agents such as NH₄Cl penetrate acidic compartments and raise their pH, thus allowing the accumulation and measurement of these cleaved fragments, which otherwise would be rapidly degraded. We have used this property to detect the presence of free GFP fragments derived from the fusion protein GFP-Tkt-1, a cytosolic marker. We demonstrate that this proteolytic event is dependent on autophagy and can be used to detect differences in the level of autophagic flux among different mutant strains. Moreover, treatment with NH4Cl also facilitates the assessment of autophagic flux by confocal microscopy using the marker RFP-GFP-Atg8.     

BIX-01294 induces autophagy-associated cell death via EHMT2/G9a dysfunction and intracellular reactive oxygen species production

We screened a chemical library in MCF-7 cells stably expressing green fluorescent protein (GFP)-conjugated microtubule-associated protein 1 light chain 3 (LC3) (GFP-LC3-MCF-7) using cell-based assay, and identified BIX-01294 (BIX), a selective inhibitor of euchromatic histone-lysine N-methyltransferase 2 (EHMT2), as a strong autophagy inducer. BIX enhanced formation of GFP-LC3 puncta, LC3-II, and free GFP, signifying autophagic activation. Inhibition of these phenomena with chloroquine and increasement in punctate dKeima ratio (550/438) signal indicated that BIX activated autophagic flux. BIX-induced cell death was suppressed by the autophagy inhibitor, 3-methyladenine, or siRNA against BECN1 (VPS30/ATG6), ATG5, and ATG7, but not by caspase inhibitors. Moreover, EHMT2 siRNA augmented GFP-LC3 puncta, LC3-II, free GFP, and cell death, implying that inhibition of EHMT2 caused autophagy-mediated cell death. Treatment with EHMT2 siRNA and BIX accumulated intracellular reactive oxygen species (ROS). BIX augmented mitochondrial superoxide via NADPH oxidase activation. In addition, BIX increased hydrogen peroxide and glutathione redox potential in both cytosol and mitochondria. Treatment with N-acetyl-L-cysteine (NAC) or diphenyleneiodonium chloride (DPI) decreased BIX-induced LC3-II, GFP-LC3 puncta, and cell death, indicating that ROS instigated autophagy-dependent cell death triggered by BIX. We observed that BIX potentiated autophagy-dependent and caspase-independent cell death in estrogen receptor (ESR)-negative SKBr3 and ESR-positive MCF-7 breast cancer cells, HCT116 colon cancer cells, and importantly, in primary human breast and colon cancer cells. Together, the results suggest that BIX induces autophagy-dependent cell death via EHMT2 dysfunction and intracellular ROS accumulation in breast and colon cancer cells, therefore EHMT2 inhibition can be an effective therapeutic strategy for cancer treatment.     

A method to measure cardiac autophagic flux in vivo

Autophagy, a highly conserved cellular mechanism wherein various cellular components are broken down and recycled through lysosomes, has been implicated in the development of heart failure. However, tools to measure autophagic flux in vivo have been limited. Here, we tested whether monodansylcadaverine (MDC) and the lysosomotropic drug chloroquine could be used to measure autophagic flux in both in vitro and in vivo model systems. Using HL-1 cardiac-derived myocytes transfected with GFP-tagged LC3 to track changes in autophagosome formation, autophagy was stimulated by mTOR inhibitor rapamycin. Administration of chloroquine to inhibit lysosomal activity enhanced the rapamycin-induced increase in the number of cells with numerous GFP-LC3-positive autophagosomes. The chloroquine-induced increase of autophagosomes occurred in a dose-dependent manner between 1 microM and 8 microM, and reached a maximum 2 hour after treatment. Chloroquine also enhanced the accumulation of autophagosomes in cells stimulated with hydrogen peroxide, while it attenuated that induced by Bafilomycin A1, an inhibitor of V-ATPase that interferes with fusion of autophagosomes with lysosomes. The accumulation of autophagosomes was inhibited by 3-methyladenine, which is known to inhibit the early phase of the autophagic process. Using transgenic mice expressing 3 mCherry-LC3 exposed to rapamycin for 4 hr, we observed an increase in mCherry-LC3-labeled autophagosomes in myocardium, which was further increased by concurrent administration of chloroquine, thus allowing determination of flux as a more precise measure of autophagic activity in vivo. MDC injected 1 hr before sacrifice colocalized with mCherry-LC3 puncta, validating its use as a marker of autophagosomes. This study describes a method to measure autophagic flux in vivo even in non-transgenic animals, using MDC and chloroquine.     

A reporter cell system to monitor autophagy based on p62/SQSTM1

Macroautophagy (hereafter referred to as autophagy) is a catabolic pathway to isolate and transport cytosolic components to the lysosome for degradation. Recently, autophagy receptors, like p62/SQSTM1 and NBR1, which physically link autophagic cargo to ATG8/MAP1-LC3/GABARAP family members located on the forming autophagic membranes, have been identified. To identify conditions or compounds that affect autophagy cell systems that efficiently report on autophagic flux are required. Here we describe reporter cell systems based on induced expression of GFP-p62, GFP-NBR1 or GFP-LC3B. The degradation of the fusion proteins was followed after promoter shut off by flow cytometry of live cells. All three fusion proteins were degraded at a basal rate by autophagy. Surprisingly, the basal degradation rate varied for the three reporter fusion proteins. GFP-LC3B was the most stable protein. GFP-NBR1 was most efficiently degraded under basal conditions while degradation of GFP-p62 displayed the strongest response to amino acid starvation. GFP-p62 was found to perform best of the tested reporters. Single cell analysis of autophagic flux by flow cytometry allows estimates of heterogeneous cell populations. The feasibility of this approach was demonstrated using transient overexpression of a dominant negative ULK1 kinase and siRNA-mediated knock-down of LC3B to inhibit autophagic degradation of GFP-p62. The inducible GFP-p62 cell system allows quantification by several approaches and will be useful in screening for compounds or conditions that affect the rate of autophagy. Inducers of autophagy can be identified using rich medium whereas inhibitors are identified under starvation conditions.     

A method to measure cardiac autophagic flux in vivo

Autophagy, a highly conserved cellular mechanism wherein various cellular components are broken down and recycled through lysosomes, has been implicated in the development of heart failure. However, tools to measure autophagic flux in vivo have been limited. Here, we tested whether monodansylcadaverine (MDC) and the lysosomotropic drug chloroquine could be used to measure autophagic flux in both in vitro and in vivo model systems. Using HL-1 cardiac-derived myocytes transfected with GFP-tagged LC3 to track changes in autophagosome formation, autophagy was stimulated by mTOR inhibitor rapamycin. Administration of chloroquine to inhibit lysosomal activity enhanced the rapamycin-induced increase in the number of cells with numerous GFP-LC3-positive autophagosomes. The chloroquine-induced increase of autophagosomes occurred in a dose-dependent manner between 1 µM and 8 µM, and reached a maximum 2 hour after treatment. Chloroquine also enhanced the accumulation of autophagosomes in cells stimulated with hydrogen peroxide, while it attenuated that induced by Bafilomycin A₁, an inhibitor of V-ATPase that interferes with fusion of autophagosomes with lysosomes. The accumulation of autophagosomes was inhibited by 3-methyladenine, which is known to inhibit the early phase of the autophagic process. Using transgenic mice expressing mCherry-LC3 exposed to rapamycin for 4 hr, we observed an increase in mCherry-LC3-labeled autophagosomes in myocardium, which was further increased by concurrent administration of chloroquine, thus allowing determination of flux as a more precise measure of autophagic activity in vivo. MDC injected 1 hr before sacrifice colocalized with mCherry-LC3 puncta, validating its use as a marker of autophagosomes. This study describes a method to measure autophagic flux in vivo even in non-transgenic animals, using MDC and chloroquine.     

Suppression of autophagy is protective in high glucose-induced cardiomyocyte injury

Hyperglycemia is linked to increased heart failure among diabetic patients. However, the mechanisms that mediate hyperglycemia-induced cardiac damage remain poorly understood. Autophagy is a cellular degradation pathway that plays important roles in cellular homeostasis. Autophagic activity is altered in the diabetic heart, but its functional role has been unclear. In this study, we determined if mimicking hyperglycemia in cultured cardiomyocytes from neonatal rats and adult mice could affect autophagic activity and myocyte viability. High glucose (17 or 30 mM) reduced autophagic flux compared with normal glucose (5.5 mM) as indicated by the difference in protein levels of LC3-II (microtubule-associated protein 1 light chain 3 form II) or the changes of punctate fluorescence patterns of GFP-LC3 and mRFP-LC3 in the absence and presence of the lysosomal inhibitor bafilomycin A(1). Unexpectedly, the inhibited autophagy turned out to be an adaptive response that functioned to limit high glucose cardiotoxicity. Indeed, suppression of autophagy by 3-methyladenine or short hairpin RNA-mediated silencing of the Becn1 or Atg7 gene attenuated high glucose-induced cardiomyocyte death. Conversely, upregulation of autophagy with rapamycin or overexpression of Becn1 or Atg7 predisposed cardiomyocytes to high glucose toxicity. Mechanistically, the high glucose-induced inhibition of autophagy was mediated at least partly by increased mTOR signaling that likely inactivated ULK1 through phosphorylation at serine 467. Together, these findings demonstrate that high glucose inhibits autophagy, which is a beneficial adaptive response that protects cardiomyocytes against high glucose toxicity. Future studies are warranted to determine if autophagy plays a similar role in diabetic heart in vivo.     

Autophagy maturation associated with CD38‐mediated regulation of lysosome function in mouse glomerular podocytes

Podocytes are highly differentiated glomerular epithelial cells that contribute to the glomerular barrier function of kidney. A role for autophagy has been proposed in maintenance of their cellular integrity, but the mechanisms controlling autophagy in podocytes are not clear. The present study tested whether CD38‐mediated regulation of lysosome function contributes to autophagic flux or autophagy maturation in podocytes. Podocytes were found to exhibit a high constitutive level of LC3‐II, a robust marker of autophagosomes (APs), suggesting a high basal level of autophagic activity. Treatment with the mTOR inhibitor, rapamycin, increased LC3‐II and the content of both APs detected by Cyto‐ID Green staining and autophagolysosomes (APLs) measured by acridine orange staining and colocalization of LC3 and Lamp1. Lysosome function inhibitor bafilomycin A1 increased APs, but decreased APLs content under both basal and rapamycin‐induced conditions. Inhibition of CD38 activity by nicotinamide or silencing of CD38 gene produced the similar effects to that bafilomycin A1 did in podocytes. To explore the possibility that CD38 may control podocyte autophagy through its regulation of lysosome function, the fusion of APs with lysosomes in living podocytes was observed by co‐transfection of GFP‐LC3B and RFP‐Lamp1 expression vectors. A colocalization of GFP‐LC3B and RFP‐Lamp1 upon stimulation of rapamycin became obvious in transfected podocytes, which could be substantially blocked by nicotinamide, CD38 shRNA, and bafilomycin. Moreover, blockade of the CD38‐mediated regulation by PPADS completely abolished rapamycin‐induced fusion of APs with lysosomes. These results indicate that CD38 importantly control lysosomal function and influence autophagy at the maturation step in podocytes.     

Autophagy activation reduces renal tubular injury induced by urinary proteins

Autophagy is shown to be beneficial for renal tubular injury caused by nephrotoxic drugs. To investigate whether autophagy could protect renal tubular epithelial cells (TECs) from injury induced by urinary proteins, we studied the activity and action of autophagy in TECs after urinary protein overload in vivo and in vitro. We found that autophagic vacuoles increased in TECs from patients with minimal change nephrotic syndrome (MCNS) and rat models with severe proteinuria induced by cationic BSA. In HK-2 cells, exposure to urinary proteins extracted from patients with MCNS led to a significant increase in autophagosome and autolysosome formation and decrease in SQSTM1/p62 protein level. Urinary protein addition also induced lysosomal turnover of LC3-II and perinuclear clustering of lysosomes. These changes were mediated by a reactive oxygen species (ROS)-dependent mechanism. Furthermore, pretreatment of HK-2 cells with rapamycin reduced the production of LCN2/NGAL and HAVCR1/KIM-1 and the level of apoptosis induced by urinary proteins. In contrast, blocking autophagy with chloroquine or BECN1 siRNAs exerted an opposite effect. Similar results were also observed in animal models with proteinuria after treatments with rapamycin and chloroquine. Taken together, our results indicated an increase in autophagic flux, which mounts an adaptive response in TECs after urinary protein overload.     

The natural compound oblongifolin C inhibits autophagic flux and enhances antitumor efficacy of nutrient deprivation

Metabolic stress induces autophagy as an alternative source of energy and metabolites. Insufficient autophagy in nutrient-deprived cancer cells would be beneficial for cancer therapy. Here, we performed a functional screen in search of novel autophagy regulators from natural products. We showed that oblongifolin C (OC), a natural small molecule compound extracted from Garcinia yunnanensis Hu, is a potent autophagic flux inhibitor. Exposure to OC results in an increased number of autophagosomes and impaired degradation of SQSTM1/p62. Costaining of GFP-LC3B with LysoTracker Red or LAMP1 antibody demonstrates that autophagosome-lysosome fusion is blocked by OC treatment. Furthermore, OC inhibits lysosomal proteolytic activity by altering lysosomal acidification and downregulating the expression of lysosomal cathepsins. Importantly, OC can eliminate the tolerance of cancer cells to nutrient starvation. Starvation dramatically increases the susceptibility of cancer cells to OC-induced CASP3-dependent apoptosis in vitro. Subsequent studies in xenograft mouse model showed that OC has anticancer potency as revealed by increased staining of cleaved CASP3, LC3 puncta, and SQSTM1, as well as reduced expression of lysosomal cathepsins. Combined treatment with OC and caloric restriction potentiates anticancer efficacy of OC in vivo. Collectively, these data demonstrated that OC is a novel autophagic flux inhibitor and might be useful in anticancer therapy.     

TLR3 contributes to persistent autophagy and heart failure in mice after myocardial infarction

Toll‐like receptors (TLRs) are essential immunoreceptors involved in host defence against invading microbes. Recent studies indicate that certain TLRs activate immunological autophagy to eliminate microbes. It remains unknown whether TLRs regulate autophagy to play a role in the heart. This study examined this question. The activation of TLR3 in cultured cardiomyocytes was observed to increase protein levels of autophagic components, including LC3‐II, a specific marker for autophagy induction, and p62/SQSTM1, an autophagy receptor normally degraded in the final step of autophagy. The results of transfection with a tandem mRFP‐GFP‐LC3 adenovirus and use of an autophagic flux inhibitor chloroquine both suggested that TLR3 in cardiomyocytes promotes autophagy induction without affecting autophagic flux. Gene‐knockdown experiments showed that the TRIF‐dependent pathway mediated the autophagic effect of TLR3. In the mouse model of chronic myocardial infarction, persistent autophagy was observed, concomitant with up‐regulated TLR3 expression and increased TLR3‐Trif signalling. Germline knockout (KO) of TLR3 inhibited autophagy, reduced infarct size, attenuated heart failure and improved survival. These protective effects were abolished by in vivo administration of an autophagy inducer rapamycin. Similar to the results obtained in cultured cardiomyocytes, TLR3‐KO did not prevent autophagic flux in mouse heart. Additionally, this study failed to detect the involvement of inflammation in TLR3‐KO‐derived protection, as wild‐type and TLR3‐KO hearts were comparable in inflammatory activity. It is concluded that up‐regulated TLR3 expression and signalling contributes to persistent autophagy following MI, which promotes heart failure and lethality.     

Autophagy plays a protective role during zVAD-induced necrotic cell death

The aim of this study is to examine the role of autophagy in cell death by using a well-established system in which zVAD, a pan-caspase inhibitor, induces necrotic cell death in L929 murine fibrosarcoma cells. First, we observed the presence of autophagic hallmarks, including an increased number of autophagosomes and the accumulation of LC3-II in zVAD-treated L929 cells. Since the presence of such autophagic hallmarks could be the result of either increased flux of autophagy or blockage of autophagosome maturation (lysosomal fusion and degradation), we next tested the effect of rapamycin, a specific inhibitor for mTOR, and chloroquine, a lysosomal enzyme inhibitor, on zVAD-induced cell death. To our surprise, rapamycin, known to be an autophagy inducer, blocked zVAD-induced cell death, whereas chloroquine greatly sensitized zVAD-induced cell death in L929 cells. Moreover, similar results with rapamycin and chloroquine were also observed in U937 cells when challenged with zVAD. Consistently, induction of autophagy by serum starvation offered significant protection against zVAD-induced cell death, whereas knockdown of Atg5, Atg7 or Beclin 1 markedly sensitized zVAD-induced cell death in L929 cells. More importantly, Atg genes knockdown completely abolished the protective effect of serum starvation on zVAD-induced cell death. Finally, we demonstrated that zVAD was able to inhibit lysosomal enzyme cathepsin B activity, and subsequently blocked autophagosome maturation. Taken together, in contrast to the previous conception that zVAD induces autophagic cell death, here we provide compelling evidence suggesting that autophagy serves as a cell survival mechanism and suppression of autophagy via inhibition of lysosomal function contributes to zVAD-induced necrotic cell death.     

Andrographolide sensitizes cisplatin-induced apoptosis via suppression of autophagosome-lysosome fusion in human cancer cells

Suppression of autophagy has been increasingly recognized as a novel cancer therapeutic approach. Andrographolide (Andro), a diterpenoid lactone isolated from an herbal plant Andrographis paniculata, is known to possess anti-inflammatory and anticancer activity. In this study, we sought to examine the effect of Andro on autophagy, and to evaluate whether such effect is relevant to the sensitization effect of Andro on apoptosis induced by DNA damage agents in cancer cells. First, we found that Andro is able to significantly enhance autophagic markers in various cancer cell lines, including GFP-LC3 puncta and LC3-II level. Interestingly, Andro treatment also led to marked increase of p62 protein level and addition of chloroquine (CQ) failed to further enhance either LC3-II or p62 level, indicating that Andro is likely to suppress autophagic flux at the maturation and degradation stage. Next, we provided evidence that Andro inhibits autophagosome maturation not by affecting the lysosomal function, but by impairing autophagosome-lysosome fusion. Lastly, we demonstrated that treatment with cisplatin, a DNA damage agent, induces autophagy in cancer cells. Importantly, Andro is capable of sensitizing cisplatin-induced cell killing determined with both short-term apoptosis assays and long-term clonogenic test, via suppression of autophagy, a process independent of p53. In summary, these observations collectively suggest that Andro could be a promising anti-cancer agent in combination therapy via its potent inhibitory effect on autophagy by disrupting autophagosome-lysosome fusion.     

Inhibition of Protein Deubiquitination by PR-619 Activates the Autophagic Pathway in OLN-t40 Oligodendroglial Cells

Protein aggregate formation may be the result of an impairment of the protein quality control system, e.g., the ubiquitin proteasome system (UPS) and the lysosomal autophagic pathway. For proteasomal degradation, proteins need to be covalently modified by ubiquitin and deubiquitinated before the substrates are proteolytically degraded. Deubiquitination is performed by a large family of proteases, the deubiquitinating enzymes (DUBs). DUBs display a variety of functions and their inhibition may have pathological consequences. Using the broad specificity DUB inhibitor PR-619 we previously have shown that DUB inhibition leads to an overload of ubiquitinated proteins, to protein aggregate formation and subsequent inhibition of the UPS. This study was undertaken to investigate whether PR-619 modulates autophagic functions to possibly compensate the failure of the proteasomal system. Using the oligodendroglial cell line OLN-t40 and a new oligodendroglial cell line stably expressing GFP-LC3, we show that DUB inhibition leads to the activation of autophagy and to the recruitment of LC3 and of the ubiquitin binding protein p62 to the forming aggresomes without impairing the autophagic flux. Furthermore, PR-619 induced the transport of lysosomes to the forming aggregates in a process requiring an intact microtubule network. Further stimulation of autophagy by rapamycin did not prevent PR-619 aggregate formation but rather exerted cytotoxic effects. Hence, inhibition of DUBs by PR-619 activated the autophagic pathway supporting the hypothesis that the UPS and the autophagy–lysosomal pathway are closely linked together.     

Autophagy plays a protective role during zVAD-induced necrotic cell death

The aim of this study is to examine the role of autophagy in cell death by using a well-established system in which zVAD, a pan-caspase inhibitor, induces necrotic cell death in L929 murine fibrosarcoma cells. First, we observed the presence of autophagic hallmarks, including an increased number of autophagosomes and the accumulation of LC3-II in zVAD-treated L929 cells. Since the presence of such autophagic hallmarks could be the result of either increased flux of autophagy or blockage of autophagosome maturation (lysosomal fusion and degradation), we next tested the effect of rapamycin, a specific inhibitor for mTOR, and chloroquine, a lysosomal enzyme inhibitor, on zVAD-induced cell death. To our surprise, rapamycin, known to be an autophagy inducer, blocked zVAD-induced cell death, whereas chloroquine greatly sensitized zVAD-induced cell death in L929 cells. Moreover, similar results with rapamycin and chloroquine were also observed in U937 cells when challenged with zVAD. Consistently, induction of autophagy by serum starvation offered significant protection against zVAD-induced cell death, whereas knockdown of Atg5, Atg7 or Beclin 1 markedly sensitized zVAD-induced cell death in L929 cells. More importantly, Atg genes knockdown completely abolished the protective effect of serum starvation on zVAD-induced cell death. Finally, we demonstrated that zVAD was able to inhibit lysosomal enzyme cathepsin B activity, and subsequently blocked autophagosome maturation. Taken together, in contrast to the previous conception that zVAD induces autophagic cell death, here we provide compelling evidence suggesting that autophagy serves as a cell survival mechanism and suppression of autophagy via inhibition of lysosomal function contributes to zVAD-induced necrotic cell death.