Bulk Degradation Mechanism Research Articles

Myocardial injury: where inflammation and autophagy meet.

Autophagy is a highly conserved bulk degradation mechanism that degrades damaged organelles, aged proteins and intracellular contents to maintain the homeostasis of the intracellular microenvironment. Activation of autophagy can be observed during myocardial injury, during which inflammatory responses are strongly triggered. Autophagy can inhibit the inflammatory response and regulate the inflammatory microenvironment by removing invading pathogens and damaged mitochondria. In addition, autophagy may enhance the clearance of apoptotic and necrotic cells to promote the repair of damaged tissue. In this paper, we briefly review the role of autophagy in different cell types in the inflammatory microenvironment of myocardial injury and discuss the molecular mechanism of autophagy in regulating the inflammatory response in a series of myocardial injury conditions, including myocardial ischemia, ischemia/reperfusion injury and sepsis cardiomyopathy.

Potential Biotechnological Applications of Autophagy for Agriculture.

Autophagy is a genetically regulated, eukaryotic cellular degradation system that sequestrates cytoplasmic materials in specialised vesicles, termed autophagosomes, for delivery and breakdown in the lysosome or vacuole. In plants, autophagy plays essential roles in development (e.g., senescence) and responses to abiotic (e.g., nutrient starvation, drought and oxidative stress) and biotic stresses (e.g., hypersensitive response). Initially, autophagy was considered a non-selective bulk degradation mechanism that provides energy and building blocks for homeostatic balance during stress. Recent studies, however, reveal that autophagy may be more subtle and selectively target ubiquitylated protein aggregates, protein complexes and even organelles for degradation to regulate vital cellular processes even during favourable conditions. The selective nature of autophagy lends itself to potential manipulation and exploitation as part of designer protein turnover machinery for the development of stress-tolerant and disease-resistant crops, crops with increased yield potential and agricultural efficiency and reduced post-harvest losses. Here, we discuss our current understanding of autophagy and speculate its potential manipulation for improved agricultural performance.

Membrane Binding and Homodimerization of Atg16 Via Two Distinct Protein Regions is Essential for Autophagy in Yeast

Macroautophagy is a bulk degradation mechanism in eukaryotic cells. Efficiency of an essential step of this process in yeast, Atg8 lipidation, relies on the presence of Atg16, a subunit of the Atg12–Atg5-Atg16 complex acting as the E3-like enzyme in the ubiquitination-like reaction. A current view on the functional structure of Atg16 in the yeast S. cerevisiae comes from the two crystal structures that reveal the Atg5-interacting α-helix linked via a flexible linker to another α-helix of Atg16, which then assembles into a homodimer. This view does not explain the results of previous in vitro studies revealing Atg16-dependent deformations of membranes and liposome-binding of the Atg12–Atg5 conjugate upon addition of Atg16. Here we show that Atg16 acts as both a homodimerizing and peripheral membrane-binding polypeptide. These two characteristics are imposed by the two distinct regions that are disordered in the nascent protein. Atg16 binds to membranes in vivo via the amphipathic α-helix (amino acid residues 113–131) that has a coiled-coil-like propensity and a strong hydrophobic face for insertion into the membrane. The other protein region (residues 64–99) possesses a coiled-coil propensity, but not amphipathicity, and is dispensable for membrane anchoring of Atg16. This region acts as a Leu-zipper essential for formation of the Atg16 homodimer. Mutagenic disruption in either of these two distinct domains renders Atg16 proteins that, in contrast to wild type, completely fail to rescue the autophagy-defective phenotype of atg16Δ cells. Together, the results of this study yield a model for the molecular mechanism of Atg16 function in macroautophagy.

Bio-Based Poly(butylene furandicarboxylate-co-glycolate) Copolyesters: Synthesis, Properties, and Hydrolysis in Different Aquatic Environments for Water Degradation Application

In an environment of high salinity, low temperature, and the presence of a few bacteria in seawater, improving the non-enzymatic hydrolysis of polyesters is one of the effective methods for developing seawater degradable materials. In this research, poly(butylene furandicarboxylate-co-glycolate) (PBFGA) copolyesters were synthesized via melt polycondensation. The PBFGA copolymer possessed excellent mechanical properties and thermal stability. Its tensile strength was 15–50 MPa and the elongation at break was more than 120% from the tensile tests. GA units improved the flexibility of PBF segments, and the PBFGA copolymer exhibited an amorphous structure. The PBFGA copolymer possessed good hydrolysis properties and the weight loss of PBFGA50 was more than 30% after 84 days of degradation in deionized water. Lipase catalyzed, and the salt inhibited the hydrolysis of the ester bond. PBFGA exhibited a bulk degradation mechanism, and sponge pores were formed on the surface due to preferential degradation of the PGA component, which was conducive to the further degradation of the internal groups. Some butanediol and glycolic acid terminated oligomers were produced during the degradation process, and rearrangement crystallization occurred in the chain segment. Thus, PBFGA copolyesters have the potential to serve as promising seawater degradable materials with excellent tensile and non-enzymatic degradable properties.

Hydrolytic degradation of star-shaped poly(ε-caprolactone)s with different number of arms and their cytotoxic effects

Star-shaped polymers of biodegradable aliphatic polyester, poly( ε-caprolactone), PCL, with different number of arms (three, four, and six) were synthesized by ring-opening polymerization initiated by multifunctional alcohols used as cores. As potential biomaterials, synthesized star-shaped poly( ε-caprolactone)s, sPCL, were thoroughly characterized in terms of their degradation under different pH conditions and in respect to their cytotoxicity. The in vitro degradation was performed in phosphate buffer (pH 7.4) and hydrochloric acid solution (pH 1.0) over 5 weeks. Degradation of sPCL films was followed by the weight loss measurements, GPC, FTIR, and AFM analysis. While the most of the samples were stable against the abiotic hydrolysis at pH 7.4 after 5 weeks of degradation, degradation was significantly accelerated in the acidic medium. Degradation rate of polymer films was affected by the polymer architecture and molecular weight. The molecular weight profiles during the degradation revealed random chain scission of the ester bonds indicating bulk degradation mechanism of hydrolysis at pH 7.4, while acidic hydrolysis proceeded through the bulk degradation associated with surface erosion, confirmed by AFM. The in vitro toxicity tests, cytotoxicity applying normal human fibroblasts (MRC5) and embryotoxicity assessment (using zebra fish model, Danio rerio), suggested those polymeric materials as suitable for biomedical application.

Degradation and biocompatibility of photoembossed PLGA-acrylate blend for improved cell adhesion.

We have shown previously that PMMA-acrylate photopolymers are biocomopatible and can exhibit improved cell adhesion compared to PMMA, due to an increase in negative surface charge caused by UV radiation PLGA has been used widely in soft tissue regeneration due to its high biocompatibility and cell adhesion. This polymer is also biodegradable and can be utilised in the field of vascular regeneration. In this study, PLGA is blended with a triacrylate monomer (TPETA) to create a degradable photopolymer blend. Surface relief structures are formed on this PLGA-TPETA by photoembossing. An optimum height of 950 nm was achieved for a 10 µm pitch with the height of these relief structures being controlled by changing UV intensity, processing temperature and time. Degradation studies of this blend revealed a bulk degradation mechanism with PLGA-TPETA degrading slower compared to pure PLGA. We also evaluated the adhesion of human umbilical vein endothelial cells (HUVECs) on both smooth and textured PLGA-TPETA films. Embossed PLGA-TPETA films showed improved cell adhesion compared to smooth substrates. Furthermore, HUVECs proliferated faster on the embossed surface compared to their smooth counterparts. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 163-171, 2018.

Enzymatic action as switch of bulk to surface degradation of clicked gelatin‐based networks

Polymer degradation occurs under physiological conditions in vitro and in vivo, especially when bonds susceptible to hydrolysis are present in the polymer. Understanding of the degradation mechanism, changes of material properties over time, and overall rate of degradation is a necessary prerequisite for the knowledge‐based design of polymers with applications in biomedicine. Here, hydrolytic degradation studies of gelatin‐based networks synthesized by copper‐catalyzed azide‐alkyne cycloaddition reaction are reported, which were performed with or without addition of an enzyme. In all cases, networks with a stilbene as crosslinker proofed to be more resistant to degradation than when an octyl diazide was used. Without addition of an enzyme, the rate of degradation was ruled by the crosslinking density of the network and proceeded via a bulk degradation mechanism. Addition of Clostridium histolyticum collagenase resulted in a much enhanced rate of degradation, which furthermore occurred via surface erosion. The mesh size of the hydrogels (>7 nm) was in all cases larger than the hydrodynamic radius of the enzyme (4.5 nm) so that even in very hydrophilic networks with large mesh size enzymes may be used to induce a fast surface degradation mechanism. This observation is of general interest when designing hydrogels to be applied in the presence of enzymes, as the degradation mechanism and material performance are closely interlinked. Copyright © 2016 John Wiley & Sons, Ltd.

Aging and Autophagy in the Heart.

The aging population is increasing in developed countries. Because the incidence of cardiac disease increases dramatically with age, it is important to understand the molecular mechanisms through which the heart becomes either more or less susceptible to stress. Cardiac aging is characterized by the presence of hypertrophy, fibrosis, and accumulation of misfolded proteins and dysfunctional mitochondria. Macroautophagy (hereafter referred to as autophagy) is a lysosome-dependent bulk degradation mechanism that is essential for intracellular protein and organelle quality control. Autophagy and autophagic flux are generally decreased in aging hearts, and murine autophagy loss-of-function models develop exacerbated cardiac dysfunction that is accompanied by the accumulation of misfolded proteins and dysfunctional organelles. On the contrary, stimulation of autophagy generally improves cardiac function in mouse models of protein aggregation by removing accumulated misfolded proteins, dysfunctional mitochondria, and damaged DNA, thereby improving the overall cellular environment and alleviating aging-associated pathology in the heart. Increasing lines of evidence suggest that autophagy is required for many mechanisms that mediate lifespan extension, such as caloric restriction, in various organisms. These results raise the exciting possibility that autophagy may play an important role in combating the adverse effects of aging in the heart. In this review, we discuss the role of autophagy in the heart during aging, how autophagy alleviates age-dependent changes in the heart, and how the level of autophagy in the aging heart can be restored.

Modular Cross-Linking of Gelatin-Based Thiol-Norbornene Hydrogels for in Vitro 3D Culture of Hepatocellular Carcinoma Cells.

Gelatin-based hydrogels are increasingly used to promote cell fate processes in 3D. Here, we report the use of orthogonal thiol-norbornene photochemistry to prepare modularly cross-linked gelatin-based hydrogels for studying the influence of independent matrix properties on hepatocellular carcinoma cell fate in vitro. In addition to demonstrating the ability to independently tune the mechanical and biological properties of modular gelatin-norbornene (GelNB) hydrogels, we also determined that network cross-linking density plays a key role in the mechanisms of proteolytic gel degradation. During in vitro degradation studies, GelNB hydrogels with lower cross-linking density degraded faster and followed a surface erosion mechanism, whereas dense GelNB hydrogels degraded in a bulk degradation mechanism. Hepatocellular carcinoma cells, Huh7, were encapsulated and grown in GelNB hydrogels with modularly tuned stiffness, bioactive motifs, and heparin content. We systematically evaluated the effect of matrix properties on cell viability and functions in vitro, including CYP3A4 activity and urea secretion. We found that encapsulated Huh7 cells exhibited higher cellular metabolic activity when encapsulated in modular GelNB hydrogels composed of higher gelatin contents or gels with lower stiffness. Interestingly, altering gelatin content and matrix stiffness did not significantly affect hepatocyte-specific cellular functions. To improve cellular function, we prepared norbornene and heparin dual-functionalized gelatin through a two-step synthesis protocol. Heparin-functionalized GelNB (i.e., GelNB-Hep) hydrogels were able to sequester and slowly release hepatocyte growth factor (HGF) in vitro. Finally, the conjugation of heparin on GelNB led to suppressed Huh7 cell metabolic activity and improved CYP3A4 activity and urea secretion.

Key factors for tuning hydrolytic degradation of polylactide/zinc oxide nanocomposites

The hydrolytic degradation of thin films of polylactide/surface treated zinc oxide [poly(lactic acid) (PLA)/ZnOs] nanocomposites was investigated in phosphate buffer solution at the temperature of 37°C for more than 10 months. To produce PLA/ZnOs nanocomposites, the previously silanized metal oxide nanofiller has been dispersed into PLA by melt blending using twin screw extruders and the resulting dried pellets were shaped into thin films of about 70 μm thickness. For sake of comparison, pristine PLA was processed and investigated under similar conditions. The evolution of molecular weights of the PLA matrix, as well as of crystallinity and thermal parameters of interest, with the hydrolysis time, has been recorded by size exclusion chromatography (SEC) and differential scanning calorimetry (DSC), respectively. Accordingly, at longer hydrolysis time, the nanocomposites revealed better resistence to the hydrolytic degradation (lower weight loss, smaller decrease of molecular mass, no dramatic increase in dispersity), data that were also associated with the changes in the morphology of specimens over time as evidenced by visual analysis or by microscopy. The results show the possibility to tune the hydrolytic degradation and prolonging the service life of PLA throught the incorporation of a small amount of hydrophobic silanized nanofiller (ZnOs). A bulk degradation mechanism was assumed, whereas the delayed degradation of nanocomposites was ascribed to a slowdown of the water diffusion into PLA matrix thanks to important increases of the crystallinity and especially to the hydrophobic properties of ZnO nanofiller treated with ∼3 wt-% triethoxycaprylylsilane. Accordingly, the rate of hydrolytic degradation of PLA/ZnOs nanocomposite films can be reduced by increasing the loading of nanofiller and PLA crystallinity.

Secretory function of autophagy in innate immune cells.

Eukaryotic cells utilize two main secretory pathways to transport proteins to the extracellular space. Proteins with a leader signal sequence often undergo co-translational transport into the endoplasmic reticulum (ER), and then to the Golgi apparatus before they reach their destination. This pathway is called the conventional secretory pathway. Proteins without signal peptides can bypass this ER-Golgi system and are secreted by a variety of mechanisms collectively called the unconventional secretory pathway. The molecular mechanisms of unconventional secretion are emerging. Autophagy is a conserved bulk degradation mechanism that regulates many intracellular functions. Recent evidence implicates autophagy in the secretory pathway. This review focuses on potential secretory roles of autophagy and how they could modulate the functions of innate immune cells that secrete a wide range of mediators in response to environmental and biological stimuli. We provide a brief overview of the secretory pathways, enumerate the potential mechanistic themes by which autophagy interacts with these pathways and describe their relevance in the context of innate immune cell function.

WIPI-dependent autophagy during neutrophil differentiation of NB4 acute promyelocytic leukemia cells.

Members of the WD-repeat protein interacting with phosphoinositides (WIPI) family are phosphatidylinositol 3-phosphate (PI3P) effectors that are essential for the formation of autophagosomes. Autophagosomes, unique double-membraned organelles, are characteristic for autophagy, a bulk degradation mechanism with cytoprotective and homeostatic function. Both, WIPI-1 and WIPI-2 are aberrantly expressed in several solid tumors, linking these genes to carcinogenesis. We now found that the expression of WIPI-1 was significantly reduced in a large cohort of 98 primary acute myeloid leukemia (AML) patient samples (complex karyotypes; t(8;21); t(15,17); inv(16)). In contrast, the expression of WIPI-2 was only reduced in acute promyelocytic leukemia (APL), a distinct subtype of AML (t(15,17)). As AML cells are blocked in their differentiation, we tested if the expression levels of WIPI-1 and WIPI-2 increase during all-trans retinoic acid (ATRA)-induced neutrophil differentiation of APL. According to the higher WIPI-1 expression in granulocytes compared with immature blast cells, WIPI-1 but not WIPI-2 expression was significantly induced during neutrophil differentiation of NB4 APL cells. Interestingly, the induction of WIPI-1 expression was dependent on the transcription factor PU.1, a master regulator of myelopoiesis, supporting our notion that WIPI-1 expression is reduced in AML patients lacking proper PU-1 activity. Further, knocking down WIPI-1 in NB4 cells markedly attenuated the autophagic flux and significantly reduced neutrophil differentiation. This result was also achieved by knocking down WIPI-2, suggesting that both WIPI-1 and WIPI-2 are functionally required and not redundant in mediating the PI3P signal at the onset of autophagy in NB4 cells. In line with these data, downregulation of PI3KC3 (hVPS34), which generates PI3P upstream of WIPIs, also inhibited neutrophil differentiation. In conclusion, we demonstrate that both WIPI-1 and WIPI-2 are required for the PI3P-dependent autophagic activity during neutrophil differentiation, and that PU.1-dependent WIPI-1 expression is significantly repressed in primary AML patient samples and that the induction of autophagic flux is associated with neutrophil differentiation of APL cells.

Autophagy Plays an Essential Role in Mediating Regression of Hypertrophy during Unloading of the Heart

Autophagy is a bulk degradation mechanism for cytosolic proteins and organelles. The heart undergoes hypertrophy in response to mechanical load but hypertrophy can regress upon unloading. We hypothesize that autophagy plays an important role in mediating regression of cardiac hypertrophy during unloading. Mice were subjected to transverse aortic constriction (TAC) for 1 week, after which the constriction was removed (DeTAC). Regression of cardiac hypertrophy was observed after DeTAC, as indicated by reduction of LVW/BW and cardiomyocyte cross-sectional area. Indicators of autophagy, including LC3-II expression, p62 degradation and GFP-LC3 dots/cell, were significantly increased after DeTAC, suggesting that autophagy is induced. Stimulation of autophagy during DeTAC was accompanied by upregulation of FoxO1. Upregulation of FoxO1 and autophagy was also observed in vitro when cultured cardiomyocytes were subjected to mechanical stretch followed by incubation without stretch (de-stretch). Transgenic mice with cardiac-specific overexpression of FoxO1 exhibited smaller hearts and upregulation of autophagy. Overexpression of FoxO1 in cultured cardiomyocytes significantly reduced cell size, an effect which was attenuated when autophagy was inhibited. To further examine the role of autophagy and FoxO1 in mediating the regression of cardiac hypertrophy, beclin1+/− mice and cultured cardiomyocytes transduced with adenoviruses harboring shRNA-beclin1 or shRNA-FoxO1 were subjected to TAC/stretch followed by DeTAC/de-stretch. Regression of cardiac hypertrophy achieved after DeTAC/de-stretch was significantly attenuated when autophagy was suppressed through downregulation of beclin1 or FoxO1. These results suggest that autophagy and FoxO1 play an essential role in mediating regression of cardiac hypertrophy during mechanical unloading.

Autophagy and Autoimmunity Crosstalks

Autophagy, initially viewed as a conserved bulk-degradation mechanism, has emerged as a central player in a multitude of immune functions. Autophagy is important in host defense against intracellular and extracellular pathogens, metabolic syndromes, immune cell homeostasis, antigen processing and presentation, and maintenance of tolerance. The observation that the above processes are implicated in triggering or exacerbating autoimmunity raises the possibility that autophagy is involved in mediating autoimmune processes, either directly or as a consequence of innate or adaptive functions mediated by the pathway. Genome-wide association studies have shown association between single nucleotide polymorphisms (SNPs) in autophagy related gene 5 (Atg5), and Atg16l1 with susceptibility to systemic lupus erythematosus (SLE) and Crohn’s disease, respectively. Enhanced expression of Atg5 was also reported in blood of mice with experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis (MS), and in T cells isolated from blood or brain tissues from patients with active relapse of MS. This review explores the roles of autophagy pathway in the innate and adaptive immune systems on regulating or mediating the onset, progression, or exacerbation of autoimmune processes.

Comparison of abiotic and biotic degradation of PDLLA, PCL and partially miscible PDLLA/PCL blend

Important differences between the biotic and abiotic hydrolysis of neat poly(dl-lactide) (PDLLA), poly(ε-caprolactone) (PCL) and a partially miscible PDLLA/PCL blend were found in this work. In abiotic conditions, the degradation process of all the studied specimens mainly proceeded by a bulk degradation mechanism and the diffusion rate of degradation products into the medium was relatively slow after 18weeks. The hydrophobic and semi-crystalline nature of PCL matrix made this polymer very stable against abiotic hydrolysis, whereas the degradation of PDLLA proceeded much faster due to its amorphous structure. In the first stages of degradation, PDLLA/PCL blend showed that its rich continuous PDLLA phase catalyzed the hydrolysis of the PCL phase, while the low content of PCL molecules partially delayed hydrolysis of PDLLA matrix. In compost, higher levels of degradation were found for all the studied systems during the first 12weeks of degradation, indicating an important effect of enzymes from microbes in compost on the degradation of PDLLA and PCL. Neat PDLLA and PDLLA/PCL blend seemed to degrade mainly by a bulk degradation mechanism, at least during the first 12weeks, such as previously observed in abiotic conditions, whereas PCL was preferentially degraded by a surface mechanism due to a very strong catalytic effect of microorganisms on its degradation. Contrary to the abiotic degradation, it was observed that the presence of a low content of PCL molecules in the rich-PDLLA blend did not interfere with the degradation trend of PDLLA in compost, and that the continuous PDLLA phase could partially suppress the biotic degradation of PCL molecules.In general, abiotic and biotic hydrolysis of PDLLA/PCL blends seems to be a complex phenomenon depending not only on the mixing ratio of both polymers but also on their crystallinity, miscibility level of polymer phases and the preferential degradation mechanism of each polymer component. If blends of PDLLA/PCL present a similar level of miscibility, it would be possible to estimate if the presence of the PDLLA or PCL phase could have a general catalytic or delaying role in the blend during the initial degradation stages, depending on which environment they are subjected to and the content of each polymer phase in the blend.

Abstract 2263: Autophagy protects against Src tyrosine kinase inhibitor treatment in non-small cell lung cancer cells via a novel miR-106a/ULK1 pathway

Abstract Autophagy is a bulk degradation mechanism mainly involved in cell survival responses to a variety of stress stimuli. Its role in tumorigenesis is still ambiguous. Low levels of basal autophagy are crucial in order to sustain cellular homeostasis thereby also contributing to tumor prevention, whereas autophagy induction upon a variety of anti-cancer treatments is often involved in resistance mechanism to therapy. Src tyrosine kinase inhibitors (TKIs) inhibit cell migration and invasion in non-small cell lung cancer (NSCLC) cells. In clinical trials, however, they show modest activity in combination with chemotherapeutic agents. We hypothesized that cytoprotective autophagy plays a role in resistance mechanisms against Src TKIs. We found a marked induction of autophagic activity in NSCLC cell lines (A549, H460, H1299) treated with two different Src TKIs (saracatinib, dasatinib) as measured by increased LC3-I to -II conversion, increased GFP-LC3 dot formation, and decreased p62/SQSTM1 protein expression. Most importantly, the addition of pharmacological autophagy inhibitors (chloroquine, bafilomycin) in combination with Src TKI inhibitors resulted in cell death as compared to only decreased migration by Src TKI treatment alone. Next, we identified ULK1 as a key player in Src TKI-induced autophagy using a set of lentiviral vectors expressing shRNAs targeting ATG genes. Src TKIs significantly induced ULK1 mRNA and protein expression and knocking down ULK1 significantly attenuated Src TKI-induced autophagy. Furthermore, to exclude Src inhibition-independent effects of saracatinib and dasatinib on ULK1 expression, we targeted Src by RNAi and found similar induction of ULK1 as seen with the Src TKI treatment. We further investigated if micro(mi)RNAs are involved in ULK1 regulation during Src inhibitor treatment. Using miRNA target identification software and 3′-UTR luciferase reporter assays we identified miR-106a as ULK1-targeting miRNA. Incubating NSCLC cells with Src inhibitors resulted in a dose-dependent decrease of miR-106a paralleled by an increase in ULK1 expression. Moreover, ectopic expression of miR-106a and anti-miR-106a in NSCLC cell lines caused decreased and increased ULK1 expression upon Src inhibition, respectively. Importantly, similar to the pharmacological autophagy inhibition and or knocking down ULK1, expression of miR-106a enhanced the cytotoxicity of Src TKIs. Lastly, we found significantly higher miR-106a levels in human lung adenocarcinoma compared to matched normal lung tissue (n=23), whereas ULK1 mRNA expression levels were significantly lower (p<0.0002) in tumor tissue than in normal lung tissue. In conclusion, Src inhibitors induce protective autophagy in NSCLC cells that is mediated via miR-106 and its target ULK1. Combining Src and autophagy inhibitors may represent attractive treatment option for certain NSCLC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2263. doi:1538-7445.AM2012-2263

Inhibition of Key Autophagy Genes in Primary AML Patients and Their Functional Role in Neutrophil Differentiation of APL Cells

Abstract 2456There is accumulating evidence that autophagic mechanisms are operative in the development of different hematopoietic lineages. Autophagy may support the remodelling processes that are taking place during differentiation and maturation of different cell types. Generally, autophagy is a bulk degradation mechanism mainly involved in cell survival responses to a variety of stress stimuli. Furthermore, autophagy-inducing drugs support degradation of aggregated PML-RARA protein in acute promyelocytic leukemia (APL). We used customized 384-well micro fluidic cards to determine the expression of key autophagy genes (ATGs) in a large cohort of primary acute myeloid leukemia (AML) samples (n=122) and normal neutrophils (n=14). In general, we found significantly lower ATG mRNA levels in AML as compared to neutrophils for 21/38 autophagy genes investigated. Among the most significantly downregulated genes in AML were ATG1, ATG4D, ATG14, ATG16L2, WDFY3, BNIP3L, FIP200, GATE-16 and GABARAPL1. Given the low expression of these ATGs in AML, a disease mainly characterized by a block in differentiation, we hypothesized that these genes are implicated in myeloid differentiation. We therefore analyzed expression of selected ATGs in two APL cell line models for neutrophil differentiation. We found a significant 2- to 40-fold increase of ATG1, ATG7, WDFY3, GATE16 and GABARAPL1 in NB4 and HT93 APL cell lines upon all-trans retinoic (ATRA)-induced neutrophil differentiation. The expression of these genes did not change in ATRA-resistant NB4-R2 cells upon neutrophil differentiation indicating that their increased expression is functional in neutrophil differentiation. Next, we knocked down ATG1, WDFY3, GATE16 and GAPARAPL1 in NB4 cells using lentivirally delivered shRNAs. Inhibition of ATG1, WDFY3 and GATE16 but not GABARAPL1 significantly attenuated ATRA-induced neutrophil differentiation as measured by reduced levels of the myeloid markers CD11b, CEBPE and G-CSF-R. Analysis of the autophagic markers p62/SQSTM1 and LC3B revealed that inhibiting ATG1, WDFY3 and GATE16 not only diminished neutrophil differentiation of APL cells but also myeloid differentiation-associated autophagy (MDAA). In summary, low ATG expression is generally associated with an immature AML phenotype. Furthermore, knocking down key autophagy genes attenuates ATRA-induced neutrophil differentiation and MDAA of APL cells. Our studies indicate that autophagy is a putative tumor suppressor mechanism in AML/APL and already available autophagy-inducing drugs such as lithium chloride may be used in combination with differentiation-inducing drugs. Disclosures:No relevant conflicts of interest to declare.

Characterization of degraded hydrogels based on poly(vinyl alcohol) grafted with poly(lactide‐co‐glycolide)

AbstractPoly(vinyl alcohol) (PVA) grafted with poly(lactide‐co‐glycolide) and cross‐linked as a material of increased hydrophobicity relative to PVA was produced. The properties were examined with respect to the mass loss, water uptake, hydrophilicity, and mechanical characteristics upon hydrolytical degradation. The hydrogels investigated display water uptake increasing with degradation time because of increasing hydrophilicity. The mass loss amounts up to 15% after eight weeks of degradation. The mechanical properties of the hydrogels are within the range of those of natural tissue, the E modulus is 18 MPa, or even 100–200 MPa, depending on the structure of material. The mechanical characteristic and their dependence degradation show the most recognizable correlation with the chemical structure. Studies of the topography of degraded samples (scanning electron microscopy) and IR measurements demonstrate the degradation to occur at slow rate due to the high degree of grafting. The mass loss is rather low and a bulk degradation mechanism takes place. © 2010 Wiley Periodicals, Inc. J Appl Polym Sci, 2011

Tissue Response to, and Degradation Rate of, Photocrosslinked Trimethylene Carbonate-Based Elastomers Following Intramuscular Implantation

Cylindrical elastomers were prepared through the UV-initiated crosslinking of terminally acrylated, 8,000 Da star-poly(trimethylene carbonate-co-ε-caprolactone) and star-poly(trimethylene carbonate-co-d,l-lactide). These elastomers were implanted intramuscularly into the hind legs of male Wistar rats to determine the influence of the comonomer on the weight loss, tissue response, and change in mechanical properties of the elastomer. The elastomers exhibited only a mild inflammatory response that subsided after the first week; the response was greater for the stiffer d,l-lactide-containing elastomers. The elastomers exhibited weight loss and sol content changes consistent with a bulk degradation mechanism. The d,l-lactide-containing elastomers displayed a nearly zero-order change in Young’s modulus and stress at break over the 30 week degradation time, while the ε-caprolactone-containing elastomers exhibited little change in modulus or stress at break.

Evaluation of the in vitro degradation of macroporous hydrogels using gravimetry, confined compression testing, and microcomputed tomography.

This study investigated the in vitro degradation characteristics of macroporous hydrogels based on poly(propylene fumarate-co-ethylene glycol) (P(PF-co-EG)). Four formulations were fabricated to test the effect of porosity and cross-linking density on the degradation of the resulting macroporous hydrogels. Macroporosity was introduced by the addition of sodium bicarbonate and ascorbic acid, the precursors of the carbon dioxide porogen, in the initiation system for the hydrogel cross-linking. Macroporous hydrogels with porosities of 0.80 +/- 0.03 and 0.89 +/- 0.03 were synthesized by the addition of sodium bicarbonate of concentrations 40 and 80 mg/mL and ascorbic acid of concentrations 0.05 and 0.1 mol/L, respectively. Poly(ethylene glycol) diacrylate (PEG-DA) was utilized as a cross-linker. The molecular weight between cross-links had a significant effect on weight loss after 12 weeks, where samples with M(C) of 1,880 +/- 320 synthesized with a P(PF-co-EG):PEG-DA ratio of 3:1 had a significantly greater mass loss due to degradation than those with M(C) of 1,000 +/- 100 synthesized with a P(PF-co-EG):PEG-DA ratio of 1:1. In contrast, porosity played a minimal role in determining the weight loss. Mechanical testing of the hydrogels under confined compression showed a decrease in compressive modulus over the degradation time for all formulations. In addition, an increase in hydrogel equilibrium water content and pore wall thickness was observed with degradation time, whereas the hydrogel porosity and surface area density remained invariant. The results from microcomputed tomography corroborated with the rest of the measurements and indicated a bulk degradation mechanism of the macroporous hydrogels.