Ozlem Oral

Cleavage of Atg3 protein by caspase-8 regulates autophagy during receptor-activated cell death.

Autophagy is an evolutionarily conserved mechanism contributing to cell survival under stress conditions including nutrient and growth factor deprivation. Connections and cross-talk between cell death mechanisms and autophagy is under investigation. Here, we describe Atg3, an essential regulatory component of autophagosome biogenesis, as a new substrate of caspase-8 during receptor-mediated cell death. Both, tumor necrosis factor α and tumor necrosis factor-related apoptosis inducing ligand induced cell death was accompanied by Atg3 cleavage and this event was inhibited by a pan-caspase inhibitor (zVAD) or a caspase-8-specific inhibitor (zIETD). Indeed, caspase-8 overexpression led to Atg3 degradation and this event depended on caspase-8 enzymatic activity. Mutation of the caspase-8 cleavage site on Atg3 abolished its cleavage both in vitro and in vivo, demonstrating that Atg3 was a direct target of caspase-8. Autophagy was inactive during apoptosis and blockage of caspases or overexpression of a non-cleavable Atg3 protein reestablished autophagic activity upon death receptor stimulation. In this system, autophagy was important for cell survival since inhibition of autophagy increased cell death. Therefore, Atg3 provides a novel link between apoptosis and autophagy during receptor-activated cell death.

Physiological and pathological significance of the molecular cross-talk between autophagy and apoptosis.

Autophagy and apoptosis are two important molecular mechanisms that maintain cellular homeostasis under stress conditions. Autophagy represents an intracellular mechanism responsible for turnover of organelles and long-lived proteins through a lysosome-dependent degradation pathway. Cell death signals or sustained stress might trigger programmed cell death pathways, and among them, apoptosis is the most extensively studied one. Recent studies indicate the presence of a complex interplay between autophagy and apoptosis. Physiological relevance of autophagy-apoptosis crosstalk was mainly shown in vitro. However, in vivo consequences possibly exist both during health and disease. In this review, we will summarize the current knowledge about molecular mechanisms connecting autophagy and apoptosis, and about the significance of this crosstalk for human health.

Bubbly Cavitating Flow Generation and Investigation of Its Erosional Nature for Biomedical Applications

This paper presents a study that investigates the destructive energy output resulting from hydrodynamic bubbly cavitation in microchannels and its potential use in biomedical applications. The research performed in this study includes results from bubbly cavitation experiments and findings showing the destructive effects of bubbly cavitating flow on selected solid specimens and live cells. The bubbles generated by hydrodynamic cavitation are highly destructive at the surfaces of the target medium on which they are carefully focused. The resulting destructive energy output could be effectively used for biomedical treatments, such as destroying kidney stones (renal calculi) or killing cancer cells. Motivated by this potential, the cavitation damage to cancerous cells and material removal from chalk pieces (which possess similar material properties as some kidney stones) was investigated. Our results showed that cavitation could induce damage both on chalk pieces and leukemia/lymphoma cells. We discovered that hydrodynamic cavitation exposure had early and delayed effects on cancer cell survival. Hence, the potential of hydrodynamic bubbly cavitation generated at the microscale for biomedical treatments was revealed using the microchannel configuration as a microorifice (with an inner diameter of 147 μm and a length of 1.52 cm), which acts as the source of bubbly cavitating flows.

Hydrodynamic cavitation kills prostate cells and ablates benign prostatic hyperplasia tissue

Hydrodynamic cavitation is a physical phenomenon characterized by vaporization and bubble formation in liquids under low local pressures, and their implosion following their release to a higher pressure environment. Collapse of the bubbles releases high energy and may cause damage to exposed surfaces. We recently designed a set-up to exploit the destructive nature of hydrodynamic cavitation for biomedical purposes. We have previously shown that hydrodynamic cavitation could kill leukemia cells and erode kidney stones. In this study, we analyzed the effects of cavitation on prostate cells and benign prostatic hyperplasia (BPH) tissue. We showed that hydrodynamic cavitation could kill prostate cells in a pressure- and time-dependent manner. Cavitation did not lead to programmed cell death, i.e. classical apoptosis or autophagy activation. Following the application of cavitation, we observed no prominent DNA damage and cells did not arrest in the cell cycle. Hence, we concluded that cavitation forces directly damaged the cells, leading to their pulverization. Upon application to BPH tissues from patients, cavitation could lead to a significant level of tissue destruction. Therefore similar to ultrasonic cavitation, we propose that hydrodynamic cavitation has the potential to be exploited and developed as an approach for the ablation of aberrant pathological tissues, including BPH.

IBMPFD Disease-Causing Mutant VCP/p97 Proteins Are Targets of Autophagic-Lysosomal Degradation.

The ubiquitin-proteasome system (UPS) degrades soluble proteins and small aggregates, whereas macroautophagy (autophagy herein) eliminates larger protein aggregates, tangles and even whole organelles in a lysosome-dependent manner. VCP/p97 was implicated in both pathways. VCP/p97 mutations cause a rare multisystem disease called IBMPFD (Inclusion Body Myopathy with Paget’s Disease and Frontotemporal Dementia). Here, we studied the role IBMPFD-related mutants of VCP/p97 in autophagy. In contrast with the wild-type VCP/p97 protein or R155C or R191Q mutants, the P137L mutant was aggregate-prone. We showed that, unlike commonly studied R155C or R191Q mutants, the P137L mutant protein stimulated both autophagosome and autolysosome formation. Moreover, P137L mutant protein itself was a substrate of autophagy. Starvation- and mTOR inhibition-induced autophagy led to the degradation of the P137L mutant protein, while preserving the wild-type and functional VCP/p97. Strikingly, similar to the P137L mutant, other IBMPFD-related VCP/p97 mutants, namely R93C and G157R mutants induced autophagosome and autolysosome formation; and G157R mutant formed aggregates that could be cleared by autophagy. Therefore, cellular phenotypes caused by P137L mutant expression were not isolated observations, and some other IBMPFD disease-related VCP/p97 mutations could lead to similar outcomes. Our results indicate that cellular mechanisms leading to IBMPFD disease may be various, and underline the importance of studying different disease-associated mutations in order to better understand human pathologies and tailor mutation-specific treatment strategies.

RACK1 Is an Interaction Partner of ATG5 and a Novel Regulator of Autophagy.

Autophagy is biological mechanism allowing recycling of long-lived proteins, abnormal protein aggregates, and damaged organelles under cellular stress conditions. Following sequestration in double- or multimembrane autophagic vesicles, the cargo is delivered to lysosomes for degradation. ATG5 is a key component of an E3-like ATG12-ATG5-ATG16 protein complex that catalyzes conjugation of the MAP1LC3 protein to lipids, thus controlling autophagic vesicle formation and expansion. Accumulating data indicate that ATG5 is a convergence point for autophagy regulation. Here, we describe the scaffold protein RACK1 (receptor activated C-kinase 1, GNB2L1) as a novel ATG5 interactor and an autophagy protein. Using several independent techniques, we showed that RACK1 interacted with ATG5. Importantly, classical autophagy inducers (starvation or mammalian target of rapamycin blockage) stimulated RACK1-ATG5 interaction. Knockdown of RACK1 or prevention of its binding to ATG5 using mutagenesis blocked autophagy activation. Therefore, the scaffold protein RACK1 is a new ATG5-interacting protein and an important and novel component of the autophagy pathways.

Review on Lithotripsy and Cavitation in Urinary Stone Therapy

Cavitation is the sudden formation of vapor bubbles or voids in liquid media and occurs after rapid changes in pressure as a consequence of mechanical forces. It is mostly an undesirable phenomenon. Although the elimination of cavitation is a major topic in the study of fluid dynamics, its destructive nature could be exploited for therapeutic applications. Ultrasonic and hydrodynamic sources are two main origins for generating cavitation. The purpose of this review is to give the reader a general idea about the formation of cavitation phenomenon and existing biomedical applications of ultrasonic and hydrodynamic cavitation. Because of the high number of the studies on ultrasound cavitation in the literature, the main focus of this review is placed on the lithotripsy techniques, which have been widely used for the treatment of urinary stones. Accordingly, cavitation phenomenon and its basic concepts are presented in Section II. The significance of the ultrasound cavitation in the urinary stone treatment is discussed in Section III in detail and hydrodynamic cavitation as an important alternative for the ultrasound cavitation is included in Section IV. Finally, side effects of using both ultrasound and hydrodynamic cavitation in biomedical applications are presented in Section V.

Induction of Autophagic Cell Death by Anticancer Agents

Autophagy is a cellular degradation and recycling pathway contributing to cell survival under stressful conditions (e.g., starvation and growth factor deprivation). Depending on the cellular context, excessive autophagy might sometimes lead to a non-apoptotic programmed death, namely autophagic cell death. Studies using various pharmacological agents and substances with anticancer properties revealed a role for autophagy in cancer cell elimination. In this chapter, we will document and analyze the contribution of autophagy to cell death activated by various anticancer agents. The chapter will be limited to cases where blockage of autophagy using chemical inhibitors or genetic approaches resulted in the survival of cancer cells during drug treatment – hence, to examples of autophagy-related or autophagic cell death (ACD). In other words, in studies that will be discussed here, autophagy seemed to play a rate-limiting role in cellular demise, irrespective of the downstream executionary event. Paradoxically, autophagy was reported to play a prosurvival role (i.e., inhibition of autophagy potentiated cell death) in other cancer cell types treated with similar drugs, underlining the cellular context dependence of ACD. Usage of drugs downregulating anti-autophagic pathways (e.g., mTORC1 and AKT pathways) or stimulating dissociation of the key autophagy protein BECN1 (Beclin 1 protein) from an inhibitory complex with BCL-2 proteins as monotherapy or combination treatment seemed to strongly activate ACD and to overcome the death resistance of cancer cells in several cases. Yet death execution mechanisms downstream of autophagy have been revealed to be multiple. In addition to lysosomal degradation-mediated mechanisms, apoptosis, necroptosis, or lysosomal membrane permeabilization were involved in autophagy-related cell death. Nevertheless, the study and exploitation of autophagy and ACD certainly has the potential to give rise to novel and effective treatment strategies, especially in cancer types that are refractory to conventional chemotherapy and associated with poor prognosis.

Effect of Varying Magnetic Fields on Targeted Gene Delivery of Nucleic Acid-Based Molecules

Several physical methods have been developed to introduce nucleic acid expression vectors into mammalian cells. Magnetic transfection (magnetofection) is one such transfection method, and it involves binding of nucleic acids such as DNA, RNA or siRNA to magnetic nanoparticles followed by subsequent exposure to external magnetic fields. However, the challenge between high efficiency of nucleic acid uptake by cells and toxicity was not totally resolved. Delivery of nucleic acids and their transport to the target cells require carefully designed and controlled systems. In this study, we introduced a novel magnetic system design providing varying magnet turn speeds and magnetic field directions. The system was tested in the magnetofection of human breast (MCF-7), prostate (DU-145, PC-3) and bladder (RT-4) cancer cell lines using green fluorescent protein DNA as a reporter. Polyethylenimine coated superparamagnetic iron oxide nanoparticles (SPIONs) were used as nucleic acid carriers. Adsorption of PEI on SPION improved the cytocompatibility dramatically. Application of external magnetic field increased intracellular uptake of nanoparticles and transfection efficiency without any additional cytotoxicity. We introduce our novel magnetism-based method as a promising tool for enhanced nucleic acid delivery into mammalian cells.

Involvement of autophagy in T cell biology

Autophagy is an essential cellular pathway that sequesters various cytoplasmic components, including accumulated proteins, damaged organelles or invading microorganisms and delivers them to lysosomes for degradation. The function of autophagy has been reported in various tissues and systems, including its role in the regulation of cellular immunity. Autophagy plays a fundamental role at various stages of T cell maturation. It regulates the thymocyte selection and the generation of T cell repertoire by presenting intracellular antigens to MHC class molecules. Autophagy is crucial for metabolic regulation of T cells, and therefore supports cell survival and homeostasis, particularly in activated mature T cells. Furthermore, deletion of specific autophagy-related genes induces several immunological alterations including differentiation of activated T cells into regulatory, memory or natural killer T cells. In this review, we emphasize the impact of autophagy on T cell development, activation and differentiation, which is pivotal for the adaptive immune system.