Increased Autophagy Induction Research Articles

Trehalose Activates Hepatic and Myocardial Autophagy and Has Anti-Inflammatory Effects in db/db Diabetic Mice.

Db/db mice (carrying a mutation in the gene encoding leptin receptor) show autophagy suppression. Our aim was to evaluate the effect of autophagy inducer trehalose on liver and heart autophagy in db/db mice and to study inflammation dysregulation and the suitability of chitinases’ expression levels as diabetes markers. Thirty-eight male db/db mice and C57/BL mice (control) were used. The db/db model manifested inflammation symptoms: overexpression of TNF-α in the spleen and underexpression of IL-10 in the liver and spleen (cytokine imbalance). Simultaneously, we revealed decreased expression of chitotriosidase (CHIT1) and acid mammalian chitinase (CHIA) in the liver of db/db mice. CHIA expression in db/db mice is significantly lower only in the spleen. Trehalose treatment significantly reduced blood glucose concentration and glycated hemoglobin. Treatment of db/db mice by trehalose was followed by increased autophagy induction in the heart and liver (increased autolysosomes volume density studied by morphometric electron-microscopic method). Trehalose exerted beneficial cardiac effects possibly via increased lipophagy (uptake of lipid droplets). The autophagy activation by trehalose had several positive effects on the heart and liver of db/db mice; therefore, lipophagy activation seems to be a promising therapy for diabetes.

Contribution of TFEB-mediated autophagy to tubulointerstitial fibrosis in mice with adenine-induced chronic kidney disease

Autophagy has been implicated in the pathogenesis of chronic kidney disease (CKD). Transcription factor EB (TFEB) is a master controller of autophagy. However, the pathophysiological roles of TFEB in modulating autophagy and tubulointerstitial injury in CKD are unknown. This study aimed to determine whether TFEB-mediated autophagy contributed to the tubulointerstitial injury in mice with CKD. After the mice were treated with an adenine diet (0.2 % adenine) for 8 weeks, the development of CKD was observed to be characterised by increased levels of plasma blood urea nitrogen (BUN), creatinine (Cre), tubulointerstitial inflammation and fibrosis. Immunohistochemical and Western blot analysis further revealed that TFEB and autophagy genes were significantly up-regulated in the kidney of the mice with adenine-induced CKD, and this increase was mostly found in the tubular epithelial cells. Interestingly, a similar expression pattern of TFEB-autophagy genes was observed in tubular epithelial cells in the kidney tissue of patients with immunoglobulin A (IgA) nephropathy. Moreover, a pathogenic role of TFEB in adenine-induced CKD was speculated because the pharmacological activation of TFEB by trehalose failed to protect mice from tubulointerstitial injuries. In the epithelioid clone of normal rat kidney cells (NRK-52E), the activation of TFEB by trehalose increased autophagy induction, cell death and inflammatory cytokine (Interleukin-6, IL-6) release. Collectively, these results suggested that the activation of TFEB-mediated autophagy might cause autophagic cell death and inflammation in tubular epithelial cells, contributing to renal fibrosis in adenine-induced CKD. This study provided novel insights into the pathogenic role of TFEB in CKD associated with a high purine diet.

Lysosomal dysfunction-induced autophagic stress in diabetic kidney disease.

The catabolic process that delivers cytoplasmic constituents to the lysosome for degradation, known as autophagy, is thought to act as a cytoprotective mechanism in response to stress or as a pathogenic process contributing towards cell death. Animal and human studies have shown that autophagy is substantially dysregulated in renal cells in diabetes, suggesting that activating autophagy could be a therapeutic intervention. However, under prolonged hyperglycaemia with impaired lysosome function, increased autophagy induction that exceeds the degradative capacity in cells could contribute toward autophagic stress or even the stagnation of autophagy, leading to renal cytotoxicity. Since lysosomal function is likely key to linking the dual cytoprotective and cytotoxic actions of autophagy, it is important to develop novel pharmacological agents that improve lysosomal function and restore autophagic flux. In this review, we first provide an overview of the autophagic‐lysosomal pathway, particularly focusing on stages of lysosomal degradation during autophagy. Then, we discuss the role of adaptive autophagy and autophagic stress based on lysosomal function. More importantly, we focus on the role of autophagic stress induced by lysosomal dysfunction according to the pathogenic factors (including high glucose, advanced glycation end products (AGEs), urinary protein, excessive reactive oxygen species (ROS) and lipid overload) in diabetic kidney disease (DKD), respectively. Finally, therapeutic possibilities aimed at lysosomal restoration in DKD are introduced.

Understanding the implications of engineered nanoparticle induced autophagy in human epidermal keratinocytes in vitro

Engineered nanoparticles (NPs) such as TiO2 and ZnO are key UV-blocking ingredients in sunscreens. While toxicological risks of applying these materials are generally regarded as low due to minute levels of penetration across the skin, our understanding of the physiological influence of potential cell-nanoparticle interactions in the skin is limited. This study plugs the current knowledge gap by profiling TiO2 and ZnO NP interaction with primary human epidermal keratinocytes, based on potential levels of NP penetration across the skin. Specific attention was given to profiling real-life relevant levels of exposure, and accurate dosimetry measurements in vitro. ZnO was expectedly more cytotoxic than TiO2. Although both NPs generated Reactive Oxygen Species (ROS) and Mitochondrial Superoxide (MSO) within 4 h exposure to sub-lethal concentrations, ZnO induction of these oxidative stress markers increased much more significantly after 24 h exposure. Exposure to increasing NP concentrations increased autophagy induction along with activation of inflammatory responses in the keratinocytes, primarily through the TRAF6-mediated pathway. Sustained induction of autophagy led to degradation of TRAF6 and, only in the case of TiO2, reduced NF-κB activation. This pro-survival mode of autophagy induction provides further insights into the on-going debate on the use of these NPs in consumer products.

Vanadium(IV)-chlorodipicolinate alleviates hepatic lipid accumulation by inducing autophagy via the LKB1/AMPK signaling pathway in vitro and in vivo

Numerous studies have demonstrated that vanadium compounds are able to improve lipemia and triglyceridemia in both humans and animals. However, the molecular mechanism remains elusive. The present study was conducted to investigate the anti-hyperlipidemic effect of vanadium(IV) complex with 4-chlorodipicolinic acid (VOdipic-Cl)-induced autophagy on hepatic lipid accumulation. To explore the possible underlying mechanisms, primary rat hepatocytes, human hepatoma cell line HepG2, and liver tissue from C57BL/6 mice fed a high-fat diet (HFD) were used. In vitro, cultured primary rat hepatocytes were treated with palmitate (0.25, 0.5 and 0.75 mM) prior to VOdipic-Cl (50, 100, and 200 μM) for 24 h, respectively. In vivo, C57BL/6 mice were fed with high-fat diet for 16 weeks. VOdipic-Cl (10 mg V/kg body weight) was given by daily gavage for 4 weeks. In vitro results showed that VOdipic-Cl significantly inhibited lipid droplet formation by increasing the level of conversion and punctuation of microtubule-associated proteins light chain 3 (LC3) in a dose-dependent manner, and activated liver kinase B-1 (LKB1) and adenosine monophosphate-activated protein kinase (AMPK) phosphorylation. Confocal microscopy images also showed that VOdipic-Cl induced sequestration of lipid droplets (LDs) by autophagy. In vivo, VOdipic-Cl attenuated the increase in serum and liver triglyceride levels in the mice fed with high-fat diet, while significantly increased autophagy induction and activated LKB1 and AMPK phosphorylation in the liver. Taken together, these results suggest that VOdipic-Cl reduces hepatic lipid accumulation by inducing autophagy via the activation of LKB1/AMPK-dependent signaling pathway.

Effect of erythropoietin production induced by hypoxia on autophagy in HepG2 cells

The induction of hypoxia inducible factor (HIF) and autophagy are important cellular responses to hypoxia. The production of erythropoietin (EPO) regulated by HIF is increased by hypoxia and participates in cell protection in various organs and tissues. Signal Transducers and Activator of Transcription 3 (STAT3) is a regulatory factor that is common to autophagy induction and EPO-EPO receptor signaling. In this study, we analyzed the promotion of EPO production and autophagy, and the participation of STAT3, in HepG2 cells under hypoxia. Treatment with EPO siRNA (si-EPO) significantly increased autophagy induction by hypoxia, while treatment with recombinant EPO inhibited the effect of si-EPO. NSC74859, an inhibitor of the phosphorylation of STAT3, increased autophagy induction to the same extent as si-EPO treatment. Even when 3-Methyladenine, an inhibitor of autophagy, was added, the increase of EPO mRNA expression due to hypoxia was not affected. Hypoxia-induced EPO restrained autophagy induction through the EPO receptor and phosphorylation of STAT3. Because cell viability with treatment of si-EPO under hypoxia did not increase over the control, our results suggested that EPO produced by hypoxia prevented excess autophagy induction.

General Anesthetics Regulate Autophagy via Modulating the Inositol 1,4,5-Trisphosphate Receptor: Implications for Dual Effects of Cytoprotection and Cytotoxicity

General anesthetics are both neuroprotective and neurotoxic with unclear mechanisms. General anesthetics may control cell survival via their effects on autophagy by activation of type 1 inositol triphosphate receptor (InsP3R-1). DT40 or SH-SY5Y cells with only or over 99% expression of InsP3R-1 were treated with isoflurane or propofol. Cell viability was determined by MTT reduction or LDH release assays. Apoptosis was determined by measuring Caspase-3 or by TUNEL assay. Autophagy activity was determined by measuring LC3 II and P62. We evaluated mitochondrial integrity using MitoTracker Green and cytosolic ATP levels. Fura2-AM was used to measure the concentrations of cytosolic calcium ([Ca2+]c). Propofol significantly increased peak and integrated calcium response (P < 0.001) in cells with InsP3R-1 but not in cells with triple knockout of InsP3R. Both propofol and isoflurane increased autophagy induction (P < 0.05) in an mTOR- and InsP3R- activity dependent manner. Short exposure to propofol adequately activated InsP3-1 to provide sufficient autophagy for cytoprotection, while prolonged exposure to propofol induced cell apoptosis via impairment of autophagy flux through over activation of InsP3-1. Propofol damaged mitochondria and decreased cytosolic ATP. The effects of general anesthetics on apoptosis and autophagy are closely integrated; both are caused by differential activation of the type 1 InsP3R.

Role of mechanistic target of rapamycin (mTOR) in renal function and ischaemia-reperfusion induced kidney injury.

Despite the presence of many studies on the role of mechanistic target of rapamycin (mTOR) in cardiorenal tissues, the definitive role of mTOR in the pathogenesis of renal injury subsequent to ischaemia-reperfusion (IR) remains unclear. The aims of the current study were to characterize the role of mTOR in normal kidney function and to investigate the role of mTOR activation in IR-induced kidney injury. In euvolemic anaesthetized rats, treatment with the mTOR inhibitor rapamycin increased blood pressure (121±2 to 144±3mmHg; P<.05), decreased glomerular filtration rate (GFR; 1.6±0.3 to 0.5±0.2mL/min; P<.05) and increased urinary sodium excretion (UNaV; 14±1 to 109±25mmol/L per hour; P<.05). In rats subjected to IR, autophagy induction, p-mTOR expression and serum creatinine increased (1.9±0.2 to 3±0.3mg/dL; P<.05); treatment with rapamycin blunted p-mTOR expression but further increased autophagy induction and serum creatinine (3±0.3 to 5±0.6mg/dL; P<.05). In contrast, clenbuterol, an mTOR activator, blunted the effect of rapamycin on serum creatinine (4±0.6 vs 2.3±0.3mg/dL; P<.05), autophagy induction and p-mTOR expression. IR also increased 24hour protein excretion (9±3 to 17±2mg/day; P<.05) and kidney injury molecule-1 (KIM-1) expression, and rapamycin treatment further increased KIM-1 expression. Clenbuterol exacerbated protein excretion (13±2 to 26±4mg/day; P<.05) and antagonized the effect of rapamycin on KIM-1 expression. Histopathological data demonstrated kidney injury in IR rats that was worsened by rapamycin treatment but attenuated by clenbuterol treatment. Thus, mTOR signalling is crucial for normal kidney function and protecting the kidney against IR injury through autophagy suppression.

Synergistic antitumor effects of radiation and proteasome inhibitor treatment in pancreatic cancer through the induction of autophagy and the downregulation of TRAF6

Ninety percent of human pancreatic cancer is characterized by activating K-RAS mutations. TRAF6 is an oncogene that plays a vital role in K-RAS-mediated oncogenesis. We investigated the synergistic effect of combining ionizing radiation (IR) and proteasome inhibitor (MG132). Furthermore, following combined treatment with IR and MG132, we analyzed the expression of TRAF6 and the mechanism of human pancreatic cancer cell death in vitro and in an orthotopic pancreatic cancer mouse model. The combined treatment groups displayed synergistic cell killing effects and induced endoplasmic reticulum stress in human pancreatic cancer cells. The combined treatment groups were characterized by enhanced cytotoxicity, which resulted from increased autophagy induction through the inhibition of TRAF6. Significantly reduced cytotoxicity was observed following MG132 and IR treatment of MIA PaCa-2 cells pre-treated with 3-MA (an autophagy inhibitor). Down-regulation of TRAF6 led to a significant increase in apoptosis and autophagy. In an orthotopic xenograft model of SCID mice, combination MG132 and IR therapy resulted in a significant increase in the tumor growth delay time and a decreased tumor tissue expression of TRAF6. IR combined with a proteasome inhibitor or TRAF6 inhibition could represent a new therapeutic strategy for human pancreatic cancer.

Abstract B18: Targeting rapamycin-induced autophagy in cancer

Abstract Mechanistic target of rapamycin complex 1 (mTORC1) is an important regulator of cell cycle progression and proliferation, and is a target of the inhibitor rapamycin. The 40S ribosomal S6 Kinase 1 (S6K1) is one of the best characterized downstream targets of mTORC1. The mTORC1/S6K1 pathway is hyperactivated in many types of cancer, contributing to cancer pathogenesis and progression. Autophagy is a cellular mechanism conserved in all eukaryotes, whose role is to maintain homeostasis of the cell under stress-induced conditions by recycling intracellular components. In cancer, autophagy may serve to protect cancer cells from cell death and may contribute to acquired drug resistance. The mTORC1/S6K1 signaling pathway is an important regulator of autophagy. One of the concerns that arise with the use of mTORC1 inhibitors, such as rapamycin analogs (or rapalogs), is that rapalogs promote autophagy and cause cell senescence, which protects cancer cells from undergoing apoptosis. Recent evidence suggests that a possible mechanism by which mTORC1 regulates autophagy may involve the tumor suppressor programmed cell death protein 4 (PDCD4), which is a downstream target of S6K1. Inhibition of mTORC1/S6K1 signaling causes upregulation of PDCD4 expression, which correlates with increased autophagy induction. We observed that suppressing PDCD4 expression in combination with rapamycin reduces autophagy and causes cancer cells to undergo cell death via apoptosis. Our results indicate that such strategy prevents rapamycin-induced upregulation of autophagy, while maintaining inhibition of mTORC1/S6K1 signaling, and promotes progression to apoptosis. Citation Format: Anya Alayev, Rose B. Snyder, Marina K. Holz. Targeting rapamycin-induced autophagy in cancer. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr B18.

The pan erbB inhibitor PD168393 enhances lysosomal dysfunction-induced apoptotic death in malignant peripheral nerve sheath tumor cells

Malignant peripheral nerve sheath tumors (MPNSTs) are rapidly progressive Schwann cell neoplasms. The erbB family of membrane tyrosine kinases has been implicated in MPNST mitogenesis and invasion and, thus, is a potential therapeutic target. However, tyrosine kinase inhibitors (TKIs) used alone have limited tumoricidal activity. Manipulating the autophagy lysosomal pathway in cells treated with cytostatic agents can promote apoptotic cell death in some cases. The goal of this study was to establish a mechanistic basis for formulating drug combinations to effectively trigger death in MPNST cells. We assessed the effects of the pan erbB inhibitor PD168393 on MPNST cell survival, caspase activation, and autophagy. PD168393 induced a cytostatic but not a cytotoxic response in MPNST cells that was accompanied by suppression of Akt and mTOR activation and increased autophagic activity. The effects of autophagy modulation on MPNST survival were then assessed following the induction of chloroquine (CQ)-induced lysosomal stress. In CQ-treated cells, suppression of autophagy was accompanied by increased caspase activation. In contrast, increased autophagy induction by inhibition of mTOR did not trigger cytotoxicity, possibly because of Akt activation. We thus hypothesized that dual targeting of mTOR and Akt by PD168393 would significantly increase cytotoxicity in cells exposed to lysosomal stress. We found that PD168393 and CQ in combination significantly increased cytotoxicity. We conclude that combinatorial therapies with erbB inhibitors and agents inducing lysosomal dysfunction may be an effective means of treating MPNSTs.

Abstract A33: Pan erbB inhibition enhances lysosomal dysfunction-induced death in malignant peripheral nerve sheath tumor cells.

Abstract Malignant peripheral nerve sheath tumors (MPNSTs) are highly aggressive Schwann cell-derived sarcomas. The erbB family of membrane tyrosine kinases has been implicated in stimulating MPNST cell proliferation and invasion rendering it an attractive therapeutic target. However, tyrosine kinase inhibitors (TKIs) used as single agents are ineffective tumoricidal agents. Manipulating the autophagy lysosomal pathway in cells treated with cytostatic agents can promote apoptotic cell death in some settings. The goal of this study was to establish a mechanistic basis for formulating drug combinations to effectively trigger death in MPNST cells. We assessed the effects of the pan erbB inhibitor PD168393 on MPNST cell survival. PD168393 induced a cytostatic but not a cytotoxic response in MPNST cells which was accompanied by suppression of Akt and mTOR activation and increased autophagic activity. The effects of autophagy modulation on MPNST survival were then assessed by pharmacologic and genetic manipulations following the induction of chloroquine (CQ)-induced lysosomal stress. We observed that CQ-induced death is atleast in part apoptotic. In contrast, increased autophagy induction by inhibition of mTOR did not trigger cytotoxicity, possibly due to Akt activation. We thus hypothesized that dual targeting of mTOR and Akt by PD168393 would significantly increase cytotoxicity in cells exposed to lysosomal stress. We found that PD168393 and CQ in combination significantly increased cytotoxicity. We conclude that combinatorial therapies with erbB inhibitors and agents inducing lysosomal dysfunction may be an effective means of treating MPNSTs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A33.

Abstract 4844: Dual effect of Akt on autophagy in pancreatic cancer cells

Abstract Background &amp; Aims: Key steps in autophagic protein degradation are formation of autophagosomes (induction) and their fusion with lysosomes (progression). Akt is a major anti-apoptotic factor in cancer cells; however, its role in autophagy in general, and in pancreatic cancer (PaCa) in particular, is poorly understood. We recently showed that phosphatases PHLPP-I and -II are key negative regulators of Akt in PaCa cells. The present study aimed to elucidate the effects of Akt and PHLPPs on individual steps of autophagy and the role of autophagy in the prosurvival function of Akt in PaCa cells. Methods: We measured apoptosis by DNA fragmentation and caspase-3 activity; autophagy, by conversion of cytosolic LC3-I to autophagic LC3-II (with immunoblot) and LC3 immunofluorescence; autophagy induction, in cells pretreated with NH4Cl to block autophagy progression; autophagosomal fusion with lysosomes, by co-localization of the lysosomal marker LAMP-1 with LC3-II; and efficiency of protein degradation, by level of p62 protein, a specific autophagy target. Results: Akt inactivation by AktVIII inhibitor or siRNA, or by overexpression of PHLPP-I and -II all stimulated autophagic vacuole formation in MiaPaCa-2 and PANC-1 cells. Akt inactivation greatly facilitated LC3-I/LC3-II conversion both in the presence and absence of NH4Cl, indicating increased autophagy induction. At the same time, Akt inactivation inhibited autophagosomal fusion with lysosomes and efficiency of protein degradation, indicating decreased autophagy progression. Thus, Akt has dual effect on autophagy in PaCa cells: it inhibits autophagy induction but promotes autophagosomal fusion with lysosomes and hence autophagy progression. The overall effect of Akt is decreased accumulation of autophagic vacuoles, i.e., inhibition of autophagic death. To study the role of autophagy in apoptosis, we used 3-methyladenine or beclin siRNA to inhibit autophagy induction, and bafilomycin or chloroquine to block the fusion of autophagosomes with lysosomes. All these treatments stimulated apoptosis in PaCa cells, indicating the anti-apoptotic role of autophagy. In cells with inactivated Akt, blocking autophagy induction (e.g., with 3-methyladenine) further greatly stimulated apoptosis. This indicates that the ability of Akt to inhibit autophagy induction greatly diminishes its anti-apoptotic function. Differently, blocking autophagosomal fusion with lysosomes had little effect on apoptosis in cells in which Akt was inactivated, likely because in these cells autophagy progression was already inhibited. Conclusions: Akt inhibition hampers autophagy progression, thus promoting vacuolization and autophagic cell death. At the same time, Akt inhibition greatly stimulates autophagy induction, thus counteracting apoptosis. Therefore, to maximally stimulate apoptosis in PaCa cells we propose to apply Akt inhibitors in combination with inhibitors of autophagy. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4844.

Autophagy, amyloidogenesis and Alzheimer disease

Autophagy is the sole pathway for organelle turnover in cells and is a vital pathway for degrading normal and aggregated proteins, particularly under stress or injury conditions. Recent evidence has shown that the amyloid beta peptide is generated from amyloid beta precursor protein (APP) during autophagic turnover of APP-rich organelles supplied by both autophagy and endocytosis. Abeta generated during normal autophagy is subsequently degraded by lysosomes. Within neurons, autophagosomes and endosomes actively form in synapses and along neuritic processes but efficient clearance of these compartments requires their retrograde transport towards the neuronal cell body, where lysosomes are most concentrated. In Alzheimer disease, the maturation of autophagolysosomes and their retrograde transport are impeded, which leads to a massive accumulation of ;autophagy intermediates' (autophagic vacuoles) within large swellings along dystrophic and degenerating neurites. The combination of increased autophagy induction and defective clearance of Abeta-generating autophagic vacuoles creates conditions favorable for Abeta accumulation in Alzheimer disease.