Increase In Autophagy Markers Research Articles

Human filaggrin monomer does not seem to be a proteasome target.

Absence of a functional proteasome in the suprabasal layers of the epidermis is responsible for keratosis linearis with ichthyosis congenital and sclerosing keratoderma syndrome. Patient epidermis shows hypergranulosis associated with abnormally shaped keratohyalin granules and abnormal distribution of filaggrin in the Stratum granulosum and Stratum corneum. This suggests that the proteasome is involved in the degradation of filaggrin. To test this hypothesis, the proteasome proteolytic activity was inhibited in 3D reconstructed human epidermis (RHE) with the specific clasto-lactacystin β-lactone inhibitor. Confirming the efficacy of inhibition, ubiquitinated proteins accumulated in treated RHEs as compared to controls. Levels of urocanic acid (UCA) and pyrrolidone carboxylic acid (PCA), the end products of filaggrin degradation, were reduced. However, neither filaggrin accumulation nor appearance of filaggrin-derived peptides were observed. On the contrary, the amount of filaggrin was shown to decrease, and a similar tendency was observed for profilaggrin, its precursor. Accumulation of small cytoplasmic vesicles associated with a significant increase in autophagy markers indicated activation of the autophagy process upon proteasome inhibition. Taken together, these results suggest that the perturbation of UCA and PCA production after proteasome inhibition was probably due to down-regulation of filaggrin expression rather than to blocking of filaggrin proteolysis.

Role of apoptosis and autophagy in masseter muscle atrophy evoked by Botulinum Toxin injection

IntroductionMusculoskeletal disorders of the masticatory system impact the quality of life and have a high cost of diagnosis and treatment. Masseter muscle hyperactivity is a cause of pain in the chewing apparatus. Botulinum toxin type A (BoNTA) injection is widely used to induce paralysis of the masseter muscle, thereby decreasing impaired muscle activity. However, our laboratory has described in a preclinical model that the injection of BoNTA not only induces paralysis, but also muscle atrophy, which subsequently decreases bone quality. However, it is unknown whether apoptosis or autophagy mechanisms could contribute to muscle atrophy.AimTo evaluate the induction of apoptosis and autophagy in BoNTA‐injected masseter muscle of adult mice.MethodologyUnilateral injection of BoNTA (0.2U/10µl) in the masseter muscle was performed in adult BalbC mice (approved by IACUC‐Universidad de Chile, #21446‐ODO‐UCH). Apoptosis and autophagy markers were evaluated in masseter muscles by immunoblot at 2‐7d post‐injection and immunofluorescence at 7d post‐injection. Autophagy activity was blocked by i.p injection of chloroquine. The data were evaluated with t‐test, one‐way ANOVA test, Mann‐Whitney test, or Kruskal‐Wallis test, as appropriate. The results were expressed as mean ±SEM (n=4‐8; p <0.05).ResultsUnilateral injection of BoNTA did not change the relative levels of apoptosis‐related proteins like cleaved Caspase 3, PARP, and AIF. There was a significant increase in protein levels related to autophagy, such as lipidated LC3 (1.86‐fold), non‐lipidated LC3 (1.66‐fold), and P62 (1.24‐fold) at 7 d post‐injection. Also, there was a strong punctuated stain for LC3 in histological sections of masseter muscle 7d post injection, suggesting autophagic vesicles. However, the injection of chloroquine for blocking the autophagy flux did not improve the accumulation of autophagy proteins in masseter muscle evoked by BoNTA in the induction.ConclusionsThe atrophy of masseter muscle evoked by BoNTA injection is not related to autophagy‐ or apoptosis‐induction. The increase in autophagy markers after BoNTA injection, with no further increase after autophagic flux blockade, suggests that BoNTA may be blocking autophagy in the masseter muscle, perhaps favoring a proteasomal pathway of muscle atrophy.

Role of Sphingosine 1-Phosphate Signalling Axis in Muscle Atrophy Induced by TNFα in C2C12 Myotubes.

Skeletal muscle atrophy is characterized by a decrease in muscle mass causing reduced agility, increased fatigability and higher risk of bone fractures. Inflammatory cytokines, such as tumor necrosis factor-alpha (TNFα), are strong inducers of skeletal muscle atrophy. The bioactive sphingolipid sphingosine 1-phoshate (S1P) plays an important role in skeletal muscle biology. S1P, generated by the phosphorylation of sphingosine catalyzed by sphingosine kinase (SK1/2), exerts most of its actions through its specific receptors, S1P1–5. Here, we provide experimental evidence that TNFα induces atrophy and autophagy in skeletal muscle C2C12 myotubes, modulating the expression of specific markers and both active and passive membrane electrophysiological properties. NMR-metabolomics provided a clear picture of the deep remodelling of skeletal muscle fibre metabolism induced by TNFα challenge. The cytokine is responsible for the modulation of S1P signalling axis, upregulating mRNA levels of S1P2 and S1P3 and downregulating those of SK2. TNFα increases the phosphorylated form of SK1, readout of its activation. Interestingly, pharmacological inhibition of SK1 and specific antagonism of S1P3 prevented the increase in autophagy markers and the changes in the electrophysiological properties of C2C12 myotubes without affecting metabolic remodelling induced by the cytokine, highlighting the involvement of S1P signalling axis on TNFα-induced atrophy in skeletal muscle.

Androgen depletion alters the diurnal patterns to signals that regulate autophagy in the limb skeletal muscle.

Hypogonadism contributes to limb skeletal muscle atrophy by increasing rates of muscle protein breakdown. Androgen depletion increases markers of the autophagy protein breakdown pathway in the limb muscle that persist throughout the diurnal cycle. However, the regulatory signals underpinning the increase in autophagy markers remain ill-defined. The purpose of this study was to characterize changes to autophagy regulatory signals in the limb skeletal muscle following androgen depletion. Male mice were subjected to a castration surgery or a sham surgery as a control. Seven weeks post-surgery, a subset of mice from each group was sacrificed every 4hr over a 24hr period. Protein and mRNA from the Tibialis Anterior (TA) were subjected to Western blot and RT-PCR. Consistent with an overall increase in autophagy, the phosphorylation pattern of Uncoordinated Like Kinase 1 (ULK1) (Ser555) was elevated throughout the diurnal cycle in the TA of castrated mice. Factors that induce the progression of autophagy were also increased in the TA following androgen depletion including an increase in the phosphorylation of c-Jun N-terminal Kinase (JNK) (Thr183/Tyr185) and an increase in the ratio of BCL-2 Associated X (BAX) to B-cell lymphoma 2 (BCL-2). Moreover, we observed an increase in the protein expression pattern of p53 and the mRNA of the p53 target genes Cyclin-Dependent Kinase Inhibitor 1A (p21) and Growth Arrest and DNA Damage Alpha (Gadd45a), which are known to increase autophagy and induce muscle atrophy. These data characterize novel changes toautophagy regulatory signals in the limb skeletal muscle following androgen deprivation.

Protective Effect of Angiotensin 1-7 on Sarcopenia Induced by Chronic Liver Disease in Mice.

Sarcopenia associated with chronic liver disease (CLD) is one of the more common extrahepatic features in patients with these pathologies. Among the cellular alterations observed in the muscle tissue under CLD is the decline in the muscle strength and function, as well as the increased fatigue. Morphological changes, such as a decrease in the fiber diameter and transition in the fiber type, are also reported. At the molecular level, sarcopenia for CLD is characterized by: (i) a decrease in the sarcomeric protein, such as myosin heavy chain (MHC); (ii) an increase in the ubiquitin–proteasome system markers, such as atrogin-1/MAFbx1 and MuRF-1/TRIM63; (iii) an increase in autophagy markers, such as LC3II/LC3I ratio. Among the regulators of muscle mass is the renin-angiotensin system (RAS). The non-classical axis of RAS includes the Angiotensin 1–7 [Ang-(1-7)] peptide and its receptor Mas, which in skeletal muscle has anti-atrophic effect in models of muscle wasting induced by immobilization, lipopolysaccharide, myostatin or angiotensin II. In this paper, we evaluated the effect of Ang-(1-7) on the sarcopenia by CLD in a murine model induced by the 5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) hepatotoxin administered through diet. Our results show that Ang-(1-7) administration prevented the decline of the function and strength of muscle and increased the fatigue detected in the DDC-fed mice. Besides, we observed that the decreased fiber diameter and MHC levels, as well as the transition of fiber types, were all abolished by Ang-(1-7) in mice fed with DDC. Finally, Ang-(1-7) can decrease the atrogin-1 and MuRF-1 expression as well as the autophagy marker in mice treated with DDC. Together, our data support the protective role of Ang-(1-7) on the sarcopenia by CLD in mice.

Aggregatibacter Actinomycetemcomitans Induces Autophagy in Human Junctional Epithelium Keratinocytes.

The adverse environmental conditions found in the periodontium during periodontitis pathogenesis stimulate local autophagy responses, mainly due to a continuous inflammatory response against the dysbiotic subgingival microbiome. The junctional epithelium represents the main site of the initial interaction between the host and the dysbiotic biofilm. Here, we investigated the role of autophagy in junctional epithelium keratinocytes (JEKs) in response to Aggregatibacter actinomycetemcomitans or its purified lipopolysaccharides (LPS). Immunofluorescence confocal analysis revealed an extensive nuclear translocation of transcription factor EB (TFEB) and consequently, an increase in autophagy markers and LC3-turnover assessed by immunoblotting and qRT-PCR. Correspondingly, challenged JEKs showed a punctuate cytosolic profile of LC3 protein contrasting with the diffuse distribution observed in untreated controls. Three-dimensional reconstructions of confocal images displayed a close association between intracellular bacteria and LC3-positive vesicles. Similarly, a close association between autophagic vesicles and the protein p62 was observed in challenged JEKs, indicating that p62 is the main adapter protein recruited during A. actinomycetemcomitans infection. Finally, the pharmacological inhibition of autophagy significantly increased the number of bacteria-infected cells as well as their death, similar to treatment with LPS. Our results indicate that A. actinomycetemcomitans infection induces autophagy in JEKs, and this homeostatic process has a cytoprotective effect on the host cells during the early stages of infection.

Akirin Is Required for Muscle Function and Acts Through the TGF-β Sma/Mab Signaling Pathway in Caenorhabditis elegans Development.

Akirin, a conserved metazoan protein, functions in muscle development in flies and mice. However, this was only tested in the rodent and fly model systems. Akirin was shown to act with chromatin remodeling complexes in transcription and was established as a downstream target of the NFκB pathway. Here we show a role for Caenorhabditis elegans Akirin/AKIR-1 in the muscle and body length regulation through a different pathway. Akirin localizes to somatic tissues throughout the body of C. elegans, including muscle nuclei. In agreement with its role in other model systems, Akirin loss of function mutants exhibit defects in muscle development in the embryo, as well as defects in movement and maintenance of muscle integrity in the C. elegans adult. We also have determined that Akirin acts downstream of the TGF-β Sma/Mab signaling pathway in controlling body size. Moreover, we found that the loss of Akirin resulted in an increase in autophagy markers, similar to mutants in the TGF-β Sma/Mab signaling pathway. In contrast to what is known in rodent and fly models, C. elegans Akirin does not act with the SWI/SNF chromatin-remodeling complex, and is instead involved with the NuRD chromatin remodeling complex in both movement and regulation of body size. Our studies define a novel developmental role (body size) and a new pathway (TGF-β Sma/Mab) for Akirin function, and confirmed its evolutionarily conserved function in muscle development in a new organism.

Remote Ischemic Pre-Conditioning Attenuates Adverse Cardiac Remodeling and Mortality Following Doxorubicin Administration in Mice

ObjectivesBecause of its multifaceted cardioprotective effects, remote ischemic pre-conditioning (RIPC) was examined as a strategy to attenuate doxorubicin (DOX) cardiotoxicity. BackgroundThe use of DOX is limited by dose-dependent cardiotoxicity and heart failure. Oxidative stress, mitochondrial dysfunction, inflammation, and autophagy modulation have been proposed as mediators of DOX cardiotoxicity. MethodsAfter baseline echocardiography, adult male CD1 mice were randomized to either sham or RIPC protocol (3 cycles of 5 min femoral artery occlusion followed by 5 min reperfusion) 1 h before receiving DOX (20 mg/kg, intraperitoneal). The mice were observed primarily for survival over 85 days (86 mice). An additional cohort of 50 mice was randomized to either sham or RIPC 1 h before DOX treatment and was followed for 25 days, at which time cardiac fibrosis, apoptosis, and mitochondrial oxidative phosphorylation were assessed, as well as the expression profiles of apoptosis and autophagy markers. ResultsSurvival was significantly improved in the RIPC cohort compared with the sham cohort (p = 0.007). DOX-induced cardiac fibrosis and apoptosis were significantly attenuated with RIPC compared with sham (p < 0.05 and p < 0.001, respectively). Although no mitochondrial dysfunction was detected at 25 days, there was a significant increase in autophagy markers with DOX that was attenuated with RIPC. Moreover, DOX caused a 49% decline in cardiac BCL2/BAX expression, which was restored with RIPC (p < 0.05 vs. DOX). DOX also resulted in a 17% reduction in left ventricular mass at 25 days, which was prevented with RIPC (p < 0.01), despite the lack of significant changes in left ventricular ejection fraction. ConclusionsOur preclinical results suggested that RIPC before DOX administration might be a promising approach for attenuating DOX cardiotoxicity.

Overexpression of a Novel E3 Ligase leads to a Skeletal Muscle Myopathy through Alterations in Cytoskeleton Proteins and Enhanced Protein Degradation

Multiple E3 ubiquitin ligases have been identified to be transcriptionally increased in skeletal muscle under atrophy‐inducing conditions. We have recently observed a novel E3 ligase, F‐box and Leucine‐rich protein 22 (FbxL22), to be elevated during denervation‐induced muscle atrophy. Little is known about the role of FbxL22 in skeletal muscle, with recent research highlighting its impact on cardiac muscle function and sarcomeric protein turnover (Spaich et al., 2012).Subsequently, male C57BL/6 mice (12‐week old) were subjected to in vivo electroporation with a mouse specific FbxL22 plasmid (20ug) in the tibialis anterior (TA) muscle with the contralateral leg serving as an empty vector control (pcDNA3.1). After 14 days, TA muscles were sectioned for H&amp;E stain and GFP visualisation to confirm plasmid incorporation. Muscle mass and cross‐sectional area (CSA) were determined. Western blotting was performed to determine intermediate filament, autophagy and poly‐ubiquitin protein levels in the transfected TA muscles.Interestingly, we observed no difference in muscle mass after 14 days, but we did observe increased numbers of large and small fibers in the FbxL22 transfected muscle. At the protein level, there was a 75% reduction in alpha‐actinin‐2, which was accompanied by 4‐fold increase in desmin and vimentin. We additionally observed a 2‐fold increase in polyubquitination, and 1.5‐fold increase in autophagy markers (p62 and LCB II).Our data highlights the powerful impact of FbxL22 on skeletal muscle structure. Future studies will seek to investigate the chronic effect of FbxL22 on skeletal muscle and characterize its role during the process of muscle atrophy.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Muscle PGC-1α in exercise and fasting-induced regulation of hepatic UPR in mice.

To provide a detailed time course of hepatic autophagy and all UPR branches in response to an acute bout of exercise and 24hours of fasting and test the hypothesis that muscle-specific PGC-1α overexpression dampens the UPR and autophagy responses to these metabolic challenges. Muscle-specific PGC-1α overexpression (TG) and wild-type (WT) mice (a) performed a single bout of exercise, where the liver was obtained immediately after exercise, 2, 6 or 10hours into recovery as well as from resting mice or (b) fasted for 24hours or remained fed and the liver was obtained. In both genotypes, hepatic PERK and eIF2α phosphorylation increased immediately after exercise, with no change in IRE1α phosphorylation and cleaved ATF6 protein. Fasting decreased PERK, eIF2α and IRE1α phosphorylation as well as increased cleaved ATF6 protein in both genotypes. Hepatic p62 was unchanged, while LC3II/LC3I ratio increased immediately after exercise and LC3II protein increased in response to fasting in both genotypes. TG mice had lower eIF2α phosphorylation after exercise, a blunted fasting-induced CHOP and HSP72 mRNA response and in fasted mice lower GADD34 and BiP mRNA as well as FAS protein in the liver than WT mice. This study provides for the first time evidence for transient pathway-specific activation of hepatic UPR and increase in markers of autophagy in the liver with acute exercise. On the other hand, fasting both increased and decreased UPR branches and seemed to increase autophagy. In addition, muscle PGC-1α seemed to dampen some of these responses.

Jaboticaba peel extract decrease autophagy in white adipose tissue and prevents metabolic disorders in mice fed with a high-fat diet

The increase in autophagy markers in the white adipose tissue is associated with adipocyte hypertrophy and obesity physiopathology. The jaboticaba peel is rich in bioactive compounds as polyphenols, which have been demonstrating anti-oxidant and inflammatory effects, and possibly interferes in adipose tissue metabolism and obesity. Thus, this study aimed to evaluate the effects of jaboticaba peel extract (FJE) treatment in autophagy markers of epididymal and inguinal white adipose tissue (eWAT and iWAT) of mice fed a normocaloric or high-fat diet for 6 weeks. The FJE treatment modulates autophagy markers in the eWAT of animals fed a high-fat diet by the reduction of Beclin-1 and LC3BII, and an increase in SQSTM-p62, accompanied by a decrease in tissue and total body weight, indicating the importance of autophagy in obesity development. These alterations were not followed by lower cytokines concentration, suggesting that autophagy may be fundamental to adipocyte remodeling at the beginning of adipocyte hypertrophy, before the onset of low-grade inflammation. Furthermore, the treatment reduced total serum triglycerides and liver fat accumulation. Thus, the results suggest that FJE could be considered a strategy against metabolic disorders caused by an HF diet.

Early endplate remodeling and skeletal muscle signaling events following rat hindlimb suspension

Motor endplates naturally undergo continual morphological changes that are altered in response to changes in neuromuscular activity. This study examines the consequences of acute (6-12 hr) disuse following hindlimb suspension on rat soleus muscle endplate structural stability. We identify early changes in several key signaling events including markers of protein kinase activation, AMPK phosphorylation and autophagy markers which may play a role in endplate remodeling. Acute disuse does not change endplate fragmentation, however, it decreases both the individual fragments and the total endplate area. This decrease was accompanied by an increase in the mean fluorescence intensity from the nicotinic acetylcholine receptors which compensate the endplate area loss. Muscle disuse decreased phosphorylation of AMPK and its substrate ACC, and stimulated mTOR controlled protein synthesis pathway and stimulated autophagy. Our findings provide evidence that changes in endplate stability are accompanied by reduced AMPK phosphorylation and an increase in autophagy markers, and these changes are evident within hours of onset of skeletal muscle disuse.

Anticancer Effect of Silibinin B on Colorectal Cancer Cell Line

Colorectal cancer is the third leading cause of cancer‐related mortality in developed countries. The most common treatment for colon and rectal cancers is surgical resection followed by therapy with anti‐neoplastic drugs, which has been shown to have toxicity and negative side effects. The purpose of this study was to identify anticancer effects of silibinin B in colorectal cancer cells. Silibinin B, the major bioactive component of Silybum marianum (milk thistle), has been used as a protective supplement against liver disease and is hypothesized to have anticancer properties. Silibinin B is known to play a key role in cell cycle arrest, apoptosis, and autophagy. We investigated the anticancer effect of silibinin B on a colorectal cancer cell line (DLD‐1). In DLD‐1 cells, viability decreased at higher concentrations of silibinin B as measured by Crystal Violet method. The cell death caused by silibinin B was mediated by apoptosis and autophagy. Western blot analysis of treated cells compared to the control exhibited an increase in autophagy markers, beclin‐1 and LC3, and apoptotic markers, cleaved caspase 3 and PARP. Future studies will focus on molecular pathways that mediate apoptosis and autophagy.Support or Funding InformationMH was supported by DeNardo Education and Research Foundation Fellowship.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cigarette smoke extract and isoprene resulted in the induction of apoptosis and autophagy in human placenta choriocarcinoma JEG-3 cells.

In this study, the effects of cigarette smoke (CS) on the induction of apoptosis via reactive oxygen species (ROS) production and endoplasmic reticulum stress (ER stress) of JEG-3 human choriocarcinoma cells were examined to confirm the relationship between CS and placenta development. Upon TUNEL assay, CS extract (3R4F; 0.3 and 2.1 μM) increased JEG-3 apoptosis. Western blot assay revealed that the protein expressions of p53, Bax, and CCAAT-enhancer-binding protein homologous protein (CHOP) increased, while the levels of Bcl-2 were reduced following CS extract treatment. Moreover, 2',7'-dichlorofluorescein diacetate (DCFH-DA) assay revealed increased ROS production. Upon 3-(4-5-dimethylthiazol-2-yl)-2.5-dyhphenyltetrazolium bromide (MTT) assay, isoprene (IP), one of ingredients of CS, deceased JEG-3 cell viability (10-11 to 10-6 M). After based on the MTT assay, two IP concentrations of 10-11 and 10-8 M were selected and the protein expressions of cyclin D1, cyclin E1, p21, and p27 decreased in response to IP. Furthermore, IP showed the greatest increase in autophagy at 24 hours and further induction of cell death at 72 hours upon monodansylacadaverine and TUNEL assay. Western blot analysis confirmed the increase in autophagy markers, LC3β and p62, as well as the increase or decrease of apoptosis markers p53, Bax, CHOP, and Bcl-2 in response to its treatments. In addition to confirming increases in ROS through DCFH-DA, we also confirmed the expression of Nrf2, an antioxidant marker, and the expression of Kelch-like ECH-associated protein 1 (KEAP1), which specifically degrades Nrf2, by Western blot. Taken together, these results indicate that CS and IP may inhibit the development of placenta via activation of ROS by inducing apoptosis and autophagy by affecting the expression of KEAP1, which regulates Nrf2 expression.

ER stress disturbs SR/ER-mitochondria Ca2 + transfer: Implications in Duchenne muscular dystrophy

Besides its role in calcium (Ca2+) homeostasis, the sarco-endoplamic reticulum (SR/ER) controls protein folding and is tethered to mitochondria. Under pathophysiological conditions the unfolded protein response (UPR) is associated with disturbance in SR/ER-mitochondria crosstalk. Here, we investigated whether ER stress altered SR/ER-mitochondria links, Ca2+ handling and muscle damage in WT (Wild Type) and mdx mice, the murine model of Duchenne Muscular Dystrophy (DMD). In WT mice, the SR/ER-mitochondria links were decreased in isolated FDB muscle fibers after injection of ER stress activator tunicamycin (TM). Ca2+ imaging revealed an increase of cytosolic Ca2+ transient and a decrease of mitochondrial Ca2+ uptake. The force generating capacity of muscle dropped after TM. This impaired contractile function was accompanied by an increase in autophagy markers and calpain-1 activation. Conversely, ER stress inhibitors restored SR/ER-mitochondria links, mitochondrial Ca2+ uptake and improved diaphragm contractility in mdx mice. Our findings demonstrated that ER stress-altered SR/ER-mitochondria links, disturbed Ca2+ handling and muscle function in WT and mdx mice. Thus, ER stress may open up a prospect of new therapeutic targets in DMD.

Human Immunodeficiency Virus Type 1 Nef Inhibits Autophagy through Transcription Factor EB Sequestration

HIV Nef acts as an anti-autophagic maturation factor through interaction with beclin-1 (BECN1). We report that exposure of macrophages to infectious or non-infectious purified HIV induces toll-like receptor 8 (TLR8) and BECN1 dependent dephosphorylation and nuclear translocation of TFEB and that this correlates with an increase in autophagy markers. RNA interference for ATG13, TFEB, TLR8, or BECN1 inhibits this HIV-induced autophagy. However, once HIV establishes a productive infection, TFEB phosphorylation and cytoplasmic sequestration are increased resulting in decreased autophagy markers. Moreover, by 7 d post-infection, autophagy levels are similar to mock infected controls. Conversely, although Nef deleted HIV similarly induces TFEB dephosphorylation and nuclear localization, and increases autophagy, these levels remain elevated during continued productive infection. Thus, the interaction between HIV and TLR8 serves as a signal for autophagy induction that is dependent upon the dephosphorylation and nuclear translocation of TFEB. During permissive infection, Nef binds BECN1 resulting in mammalian target of rapamycin (MTOR) activation, TFEB phosphorylation and cytosolic sequestration, and the inhibition of autophagy. To our knowledge, this is the first report of a virus modulating TFEB localization and helps to explain how HIV modulates autophagy to promote its own replication and cell survival.

The combination of cannabidiol and Δ9-tetrahydrocannabinol enhances the anticancer effects of radiation in an orthotopic murine glioma model.

High-grade glioma is one of the most aggressive cancers in adult humans and long-term survival rates are very low as standard treatments for glioma remain largely unsuccessful. Cannabinoids have been shown to specifically inhibit glioma growth as well as neutralize oncogenic processes such as angiogenesis. In an attempt to improve treatment outcome, we have investigated the effect of Δ(9)-tetrahydrocannabinol (THC) and cannabidiol (CBD) both alone and in combination with radiotherapy in a number of glioma cell lines (T98G, U87MG, and GL261). Cannabinoids were used in two forms, pure (P) and as a botanical drug substance (BDS). Results demonstrated a duration- and dose-dependent reduction in cell viability with each cannabinoid and suggested that THC-BDS was more efficacious than THC-P, whereas, conversely, CBD-P was more efficacious than CBD-BDS. Median effect analysis revealed all combinations to be hyperadditive [T98G 48-hour combination index (CI) at FU50, 0.77-1.09]. Similarly, pretreating cells with THC-P and CBD-P together for 4 hours before irradiation increased their radiosensitivity when compared with pretreating with either of the cannabinoids individually. The increase in radiosensitivity was associated with an increase in markers of autophagy and apoptosis. These in vitro results were recapitulated in an orthotopic murine model for glioma, which showed dramatic reductions in tumor volumes when both cannabinoids were used with irradiation (day 21: 5.5 ± 2.2 mm(3) vs. 48.7 ± 24.9 mm(3) in the control group; P < 0.01). Taken together, our data highlight the possibility that these cannabinoids can prime glioma cells to respond better to ionizing radiation, and suggest a potential clinical benefit for glioma patients by using these two treatment modalities.

Environmental Stress Affects the Activity of Metabolic and Growth Factor Signaling Networks and Induces Autophagy Markers in MCF7 Breast Cancer Cells

Phosphoproteomic techniques are contributing to our understanding of how signaling pathways interact and regulate biological processes. This technology is also being used to characterize how signaling networks are remodeled during disease progression and to identify biomarkers of signaling pathway activity and of responses to cancer therapy. A potential caveat in these studies is that phosphorylation is a very dynamic modification that can substantially change during the course of an experiment or the retrieval and processing of cellular samples. Here, we investigated how exposure of cells to ambient conditions modulates phosphorylation and signaling pathway activity in the MCF7 breast cancer cell line. About 1.5% of 3,500 sites measured showed a significant change in phosphorylation extent upon exposure of cells to ambient conditions for 15 min. The effects of this perturbation in modifying phosphorylation patterns did not involve random changes due to stochastic activation of kinases and phosphatases. Instead, exposure of cells to ambient conditions elicited an environmental stress reaction that involved a coordinated response to a metabolic stress situation, which included: (1) the activation of AMPK; (2) the inhibition of PI3K, AKT, and ERK; (3) an increase in markers of protein synthesis inhibition at the level of translation elongation; and (4) an increase in autophagy markers. We also observed that maintaining cells in ice modified but did not completely abolish this metabolic stress response. In summary, exposure of cells to ambient conditions affects the activity of signaling networks previously implicated in metabolic and growth factor signaling. Mass spectrometry data have been deposited to the ProteomeXchange with identifier PXD000472.

Abstract A18: Autophagy as a mechanism implicated in osteosarcoma resistance to gemcitabine.

Abstract The purpose of this study is to demonstrate the role of autophagy in osteosarcoma (OS) lung metastases resistance to aerosol Gemcitabine (GCB). Despite many advances in chemotherapy and successful control of the primary tumor, osteosarcoma overall survival has remained the same in the last 15 years. Pulmonary metastases remain the main cause of death in patients with OS underscoring the need for new therapeutic strategies. Using a human OS mouse model, we had previously demonstrated that aerosol Gemcitabine (GCB) has a significant therapeutic effect in OS lung metastases. Nevertheless, a subset of cells failss to respond to GCB treatment and persist as small isolated lung metastases in vivo. Resistance to GCB is also evidenced by survival of OS cells after treatment in vitro. Therefore, understanding the underlying mechanism of OS resistance to GCB will allow the implementation of new therapeutic strategies. Autophagy has been suggested as a mechanism induced by a variety tumor cells after chemotherapy treatment. It is a process involved in the degradation of organelles and damaged proteins, providing the cells amino acids to sustain survival under stressful conditions. Recently, it has also been associated with tumor cells resistance to chemotherapy. GCB-induced autophagy in OS primary and metastatic disease has not been studied. Therefore, we characterized the role of autophagy in OS cells after treatment with GCB. We first demonstrated GCB-induced autophagy in LM7 OS cells as indicated by an increase in autophagy markers: microtubule-associated light chain 3 (LC3I/LC3II) conversion, Beclin 1 (BECN1) expression and development of acidic vesicular organelles. GCB also caused a decrease in p62/SQSTM1 and Blc-2 expression, further indicating autophagy induction. Blockage of autophagy with the pharmacologic inhibitor Hydroxychroloquine (HCQ) coupled with GCB treatment decreased cell viability of LM7 OS cells. Furthermore, knockdown of BECN1 in LM7 cells coupled with GCB treatment also decreased cell viability of LM7 OS cells, indicating that autophagy plays an important role in OS cells resistance to GCB treatment. In conclusion, we demonstrated that autophagy is one of the mechanisms implicated in the resistance of OS cells to GCB. Autophagy modulation may shed new light towards metastatic OS therapy, possibly by implementing the use of combination therapy HCQ-aerosol GCB in the adjuvant setting. Current studies are underway to test our hypothesis in vivo and to address these results using other OS cells in vitro. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A18. Citation Format: Janice M. Santiago-O'Farrill, Mario G. Hollomon, Eugenie S. Kleinerman, Nancy Gordon. Autophagy as a mechanism implicated in osteosarcoma resistance to gemcitabine. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A18.

Abstract 151: A Novel Role for DDiT4L in Regulation of mTOR and Autophagy in the Heart

Introduction: DDiT4L is a known negative regulator of mTOR signaling in skeletal muscle; however its role in the heart is unknown. We have recently showed increased DDiT4L mRNA in a murine transgenic model of pathological but not physiological hypertrophy. Here we test the hypothesis that DDiT4L is a regulator of mTOR signaling in the heart and may play a role in pathological hypertrophy and heart failure. Methods: We investigated the regulation of DDiT4L in murine models of hypertrophy and in cultured neonatal rat ventricular cardiomyocytes (NRVMs). Loss and gain of function of DDiT4L in mTOR regulation and autophagy was investigated using confocal imaging, immunoblotting, and qRT-PCR in NRVMs. Results: DDiT4L gene and protein expression was increased four-fold in pressure overload hypertrophy (n = 4-6, p&lt;0.001), but not in a swim model of physiological hypertrophy. DDiT4L gene expression also significantly increased in a genetic model of dilated cardiomyopathy model (n = 4, p&lt;0.001). In NRVMs, DDiT4L was induced by cardiac stressors such as pathological stretch, hypoxia, and glucose deprivation (n = 3-5 in duplicate, p&lt;0.05-0.01). Increased DDiT4L expression correlated with inhibition of mTOR signaling, and an increase in autophagy markers. siRNA ablation of DDiT4L revealed that inhibition of mTOR signaling by DDiT4L was necessary for glucose deprivation induced autophagy, as determined by imaging of GFP-LC3 autophagosomes (n = 3 in duplicate, p&lt;0.01), and immunoblotting of autophagy markers. Conversely, adenoviral-driven overexpression of DDiT4L inhibited mTOR signaling and significantly increased basal autophagy (n = 3 in duplicate, p&lt;0.05). In TAC mice, the increase in DDiT4L protein expression correlated to inhibition of mTOR signaling, increases in autophagy markers (p&lt;0.01), and preceded the transition to LV dilation and HF. Conclusion: Our data suggests that DDiT4L expression is altered in diverse models of pathological hypertrophy and precedes the development of LV dilatation and overt heart failure. DDiT4L inhibition of mTOR and modulation of autophagy may play a role in the progression to heart failure. DDiT4L may represent a novel therapeutic target to prevent this transition.