To Eat or to Die: Deciphering Selective Forms of Autophagy.

Autophagy is an evolutionarily conserved process whereby damaged and redundant components of the cell are degraded in structures called autophagolysosomes. Currently, three main types of autophagy are recognized: macroautophagy, microautophagy, and chaperone-mediated autophagy (CMA). However, we still know little about some specific types of autophagy that are linked to various intracellular compartments and their roles in the physiology of the whole organism and connections to various diseases. Here, we aim to shed light on the latest insights on and mechanisms of several selective forms of autophagy.

Apoptosis is not conserved in plants as revealed by critical examination of a model for plant apoptosis-like cell death.

BACKGROUND: Animals and plants diverged over one billion years ago and evolved unique mechanisms for many cellular processes, including cell death. One of the most well-studied cell death programmes in animals, apoptosis, involves gradual cell dismantling and engulfment of cellular fragments, apoptotic bodies, through phagocytosis. However, rigid cell walls prevent plant cell fragmentation and thus apoptosis is not applicable for executing cell death in plants. Furthermore, plants are devoid of the key components of apoptotic machinery, including phagocytosis as well as caspases and Bcl-2 family proteins. Nevertheless, the concept of plant "apoptosis-like programmed cell death" (AL-PCD) is widespread. This is largely due to superficial morphological resemblances between plant cell death and apoptosis, and in particular between protoplast shrinkage in plant cells killed by various stimuli and animal cell volume decrease preceding fragmentation into apoptotic bodies. RESULTS: Here, we provide a comprehensive spatio-temporal analysis of cytological and biochemical events occurring in plant cells subjected to heat shock at 40-55 °C and 85 °C, the experimental conditions typically used to trigger AL-PCD and necrotic cell death, respectively. We show that cell death under both conditions was not accompanied by membrane blebbing or formation of apoptotic bodies, as would be expected during apoptosis. Instead, we observed instant and irreversible permeabilization of the plasma membrane and ATP depletion. These processes did not depend on mitochondrial functionality or the presence of Ca2+ and could not be prevented by an inhibitor of ferroptosis. We further reveal that the lack of protoplast shrinkage at 85 °C, the only striking morphological difference between cell deaths induced by 40-55 °C or 85 °C heat shock, is a consequence of the fixative effect of the high temperature on intracellular contents. CONCLUSIONS: We conclude that heat shock-induced cell death is an energy-independent process best matching definition of necrosis. Although the initial steps of this necrotic cell death could be genetically regulated, classifying it as apoptosis or AL-PCD is a terminological misnomer. Our work supports the viewpoint that apoptosis is not conserved across animal and plant kingdoms and demonstrates the importance of focusing on plant-specific aspects of cell death pathways.

Distinct effects of etoposide on glutamine-addicted neuroblastoma.

The majority of anticancer drugs are DNA-damaging agents, and whether or not they may directly target mitochondria remains unclear. In addition, tumors such as neuroblastoma exhibit addiction to glutamine in spite of it being a nonessential amino acid. Our aim was to evaluate the direct effect of widely used anticancer drugs on mitochondrial activity in combination with glutamine withdrawal, and possible apoptotic effects of such interaction. Our results revealed that etoposide inhibits mitochondrial respiratory chain Complex I causing the leakage of electrons and the superoxide radical formation. However, it was not sufficient to induce apoptosis, and apoptotic manifestation was detectable only alongside the withdrawal of glutamine, a precursor for antioxidant glutathione. Thus, the simultaneous depletion of glutathione and destabilization of mitochondria by ROS can compromise the barrier properties of the mitochondrial membrane, leading to cytochrome c release and the activation of the mitochondrial apoptotic pathway. Thus, the depletion of antioxidants or the inhibition of the pathways responsible for cellular antioxidant response can enhance mitochondrial targeting and strengthen antitumor therapy.

Involvement of mitophagy in cisplatin-induced cell death regulation.

Mitophagy, the selective degradation of mitochondria via the autophagic pathway, is a vital mechanism of mitochondrial quality control in cells. The removal of malfunctioning or damaged mitochondria is essential for normal cellular physiology and tissue development. Stimulation of mitochondrial permeabilization and release of proapoptotic factors from the intermembrane space is an essential step in triggering the mitochondrial pathway of cell death. In this study, we analyzed the extent to which mitophagy interferes with cell death, attenuating the efficiency of cancer therapy. We show that stimulation of mitophagy suppressed cisplatin-induced apoptosis, while mitophagy inhibition stimulates apoptosis and autophagy. Suppression of mitophagy involved production of reactive oxygen species, and the fate of cell was dependent on the interplay between endoplasmic reticulum stress and autophagy.

Contrasting effects of glutamine deprivation on apoptosis induced by conventionally used anticancer drugs.

Tumor cells dependence on glutamine offers a rationale for their elimination via targeting of glutamine metabolism. The aim of this work was to investigate how glutamine deprivation affects the cellular response to conventionally used anticancer drugs. To answer this question, neuroblastoma cells were pre-incubated in a glutamine-free medium and treated with cisplatin or etoposide. Obtained results revealed that glutamine withdrawal affected cellular response to therapeutic drugs in a different manner. Glutamine deprivation suppressed etoposide-induced, but markedly stimulated cisplatin-induced apoptosis. Suppression of etoposide-induced cell death correlated with a downregulation of p53 expression, which, among other functions, regulates the expression of death receptor 5, one of the activators of caspase-8. In contrast, stimulation of cisplatin-induced cell death involved reactive oxygen species-mediated downregulation of FLIP-S, an inhibitor of caspase-8. As a result, the activity of caspase-8 was stimulated causing cleavage of the pro-apoptotic protein Bid, which is involved in the permeabilization of the outer mitochondrial membrane and the release of pro-apoptotic factors, such as cytochrome c from mitochondria. Thus, suppression of glutamine metabolism can sensitize tumor cells to treatment and could be utilized for anti-cancer therapy. However, it should be done cautiously, since adverse effects may occur when combined with an inappropriate therapeutic drug.

Targeting succinate:ubiquinone reductase potentiates the efficacy of anticancer therapy.

Mitochondria play a pivotal role in apoptosis: permeabilization of the outer mitochondrial membrane and the release of pro-apoptotic proteins from the intermembrane space of mitochondria are regarded as the key event in apoptosis induction. Here we demonstrate how non-toxic doses of the mitochondrial Complex II inhibitor thenoyltrifluoroacetone (TTFA), which specifically inhibits the ubiquinone-binding site of succinate dehydrogenase (SDH), synergistically stimulated cell death, induced by harmless doses of cisplatin in a panel of chemoresistant neuroblastoma cell lines. Apoptotic cell death was confirmed by cytochrome c release from the mitochondria, cleavage of poly ADP-ribose polymerase, processing of caspase-3, which is an important executive enzyme in apoptosis, and caspase-3-like activity. Methyl malonate, an inhibitor of the SDHA subunit partially reversed apoptosis stimulated by TTFA in SK-N-BE(2) neuroblastoma cells (NB), indicating that sensitization requires oxidation of succinate. In contrast, in IMR-32 NB cells, the same concentrations of TTFA markedly suppressed cisplatin-induced apoptosis. Comparison of oxygen consumption in cisplatin-resistant SK-N-BE(2) and cisplatin-sensitive IMR-32 cells clearly demonstrated impaired Complex II activity in IMR-32 cells. We also found that in SK-N-BE(2) cells co-treatment with cisplatin and TTFA markedly stimulated formation of reactive oxygen species (ROS), whereas in IMR cells, cisplatin-mediated ROS production was attenuated by TTFA, which explains apoptosis suppression in these cells. Thus, functionally active SDH is a prerequisite for the ROS-mediated sensitization to treatment by TTFA.

Mitophagy: Link to cancer development and therapy.

Mitophagy, the selective degradation of mitochondria via the autophagic pathway, is a vital mechanism of mitochondrial quality control in cells. Mitophagy is responsible for the removal of malfunctioning or damaged mitochondria, which is essential for normal cellular physiology and tissue development. Pathways involved in the regulation of mitophagy, tumorigenesis, and cell death are overlapping in many cases and may be triggered by common upstream signals, which converge at the mitochondria. The failure to properly modulate mitochondrial turnover in response to oncogenic stresses can either stimulate or suppress tumorigenesis. Thus, the analysis of crosstalk among the processes of mitophagy, cell death and tumorigenesis is important for the identification of targets responsible for the stimulation of cell death and selective elimination of cancer cells. In the present review, we analyze the mechanisms of mitophagy regulation, the pathways underlying the utilization of damaged mitochondria, and how intervention with mitophagy can affect tumor cell resistance to treatment.

Mitotic catastrophe and cancer drug resistance: A link that must to be broken.

An increased tendency of genomic alterations during the life cycle of cells leads to genomic instability, which is a major driving force for tumorigenesis. A considerable fraction of tumor cells are tetraploid or aneuploid, which renders them intrinsically susceptible to mitotic aberrations, and hence, are particularly sensitive to the induction of mitotic catastrophe. Resistance to cell death is also closely linked to genomic instability, as it enables malignant cells to expand even in a stressful environment. Currently it is known that cells can die via multiple mechanisms. Mitotic catastrophe represents a step preceding apoptosis or necrosis, depending on the expression and/or proper function of several proteins. Mitotic catastrophe was proposed to be an onco-suppressive mechanism and the evasion of mitotic catastrophe constitutes one of the gateways to cancer development. Thus, stimulation of mitotic catastrophe appears to be a promising strategy in cancer treatment. Indeed, several chemotherapeutic drugs are currently used at concentrations that induce apoptosis irrespective of the cell cycle phase, yet are very efficient at triggering mitotic catastrophe at lower doses, significantly limiting side effects. In the present review we summarize current data concerning the role of mitotic catastrophe in cancer drug resistance and discuss novel strategies to break this link.

Contrasting effects of cardiac glycosides on cisplatin- and etoposide-induced cell death.

Cardiac glycosides (CGs) or cardiotonic steroids, which constitute a group of naturally occurring compounds with a steroid-like structure, can act on Na+/K+-ATPase as a receptor and activate intracellular signaling messengers leading to a variety of cellular responses. Epidemiological studies have revealed that CGs, used for the treatment of cardiac disorders, may also be beneficial as anti-cancer agents. CGs, acting in combination with other chemotherapeutic agents, may significantly alter their efficiency in relation to cancer cell elimination, causing both sensitization and an increase in cancer cell death, and in some cases resistance to chemotherapy. Here we show the ability of CGs to modulate apoptotic response to conventionally used anti-cancer drugs. In combination with etoposide, CGs digoxin may enhance cytotoxic potential, thereby allowing the chemotherapeutic dose to be decreased and minimizing toxicity and adverse reactions. Mechanisms behind this event are discussed.

Calcium and mitochondria in the regulation of cell death.

The calcium ion has long been known to play an important role in cell death regulation. Hence, necrotic cell death was early associated with intracellular Ca(2+) overload, leading to mitochondrial permeability transition and functional collapse. Subsequent characterization of the signaling pathways in apoptosis revealed that Ca(2+)/calpain was critically involved in the processing of the mitochondrially localized, Apoptosis Inducing Factor. More recently, the calcium ion has been demonstrated to play important regulatory roles also in other cell death modalities, notably autophagic cell death and anoikis. In this review, we summarize current knowledge about the mechanisms involved in Ca(2+) regulation of these various modes of cell death with a focus on the importance of the mitochondria.

Targeting mitochondria by α-tocopheryl succinate overcomes hypoxia-mediated tumor cell resistance to treatment.

Rapidly proliferating tumor cells easily become hypoxic. This results in acquired stability towards treatment with anticancer drugs. Here, we show that cells grown at 0.1 % oxygen are more resistant towards treatment with the conventionally used anticancer drugs doxorubicin and cisplatin. The stimulation of apoptosis, as assessed by the number of cells in the SubG1 fraction of the cell cycle, release of cytochrome c into the cytosol, activation of caspase-3, and cleavage of PARP, was markedly suppressed under low oxygen content or when hypoxia was mimicked by deferoxamine. Hypoxia or deferoxamine treatment was accompanied by stabilization of the hypoxia-inducible factor (HIF-1). The downregulation of HIF-1 using siRNA technique restored cell sensitivity to treatment under hypoxic conditions to the levels detected under normoxic conditions. In contrast to cisplatin or doxorubicin, α-tocopheryl succinate (α-TOS), a compound that targets mitochondria, stimulated cell death irrespective of the oxygen concentration. Moreover, under hypoxic condition cell death induced by α-TOS was even enhanced. Thus, α-TOS can successfully overcome resistance to treatment caused by hypoxia, which makes α-TOS an attractive candidate for antitumor therapy via mitochondrial targeting.

Permeabilization of the outer mitochondrial membrane: Mechanisms and consequences.

Permeabilization of the outer mitochondrial membrane is а physiological process that can allow certain molecules to pass through it, such as low molecular weight solutes required for cellular respiration. This process is also important for the development of various modes of cell death. Depending on the severity of this process, cells can die by autophagy, apoptosis, or necrosis/necroptosis. Distinct types of pores can be opened at the outer mitochondrial membrane depending on physiological or pathological stimuli, and different mechanisms can be activated in order to open these pores. In this comprehensive review, all these types of permeabilization, the mechanisms of their activation, and their role in various diseases are discussed.

Targeting mitochondria by α-tocopheryl succinate kills neuroblastoma cells irrespective of MycN oncogene expression.

Amplification of the MycN oncogene characterizes a subset of highly aggressive neuroblastomas, the most common extracranial solid tumor of childhood. However, the significance of MycN amplification for tumor cell survival is controversial, since down-regulation of MycN was found to decrease markedly neuroblastoma sensitivity towards conventional anticancer drugs, cisplatin, and doxorubicin. Here, we show that a redox-silent analogue of vitamin E, α-tocopheryl succinate (α-TOS), which triggers apoptotic cell death via targeting mitochondria, can kill tumor cells irrespective of their MycN expression level. In cells overexpressing MycN, as well as cells in which MycN was switched off, α-TOS stimulated rapid entry of Ca(2+) into the cytosol, compromised Ca(2+) buffering capacity of the mitochondria and sensitized them towards mitochondrial permeability transition and subsequent apoptotic cell death. Prevention of mitochondrial Ca(2+) accumulation or chelation of cytosolic Ca(2+) rescued the cells. Thus, targeting mitochondria might be advantageous for the elimination of tumor cells with otherwise dormant apoptotic pathways.

Citrate kills tumor cells through activation of apical caspases.

Most tumor cells exhibit a glycolytic phenotype. Thus, inhibition of glycolysis might be of therapeutic value in antitumor treatment. Among the agents that can suppress glycolysis is citrate, a member of the Krebs cycle and an inhibitor of phosphofructokinase. Here, we show that citrate can trigger cell death in multiple cancer cell lines. The lethal effect of citrate was found to be related to the activation of apical caspases-8 and -2, rather than to the inhibition of cellular energy metabolism. Hence, increasing concentrations of citrate induced characteristic manifestations of apoptosis, such as caspase-3 activation, and poly-ADP-ribose polymerase cleavage, as well as the release of cytochrome c. Apoptosis induction did not involve the receptor-mediated pathway, since the processing of caspase-8 was not attenuated in cells deficient in Fas-associated protein with Death Domain. We propose that the activation of apical caspases by citrate could be explained by its kosmotropic properties. Caspase-8 is activated by proximity-induced dimerization, which might be facilitated by citrate through the stabilization of intermolecular interactions between the proteins.

Induction and Detection of Mitophagy.

Mitophagy, a process of selective elimination of mitochondria by autophagy, is a mechanism of mitochondrial quality control that maintains mitochondrial network functionality. The elimination of damaged mitochondria through autophagy requires two steps: induction of general autophagy and priming of damaged mitochondria for selective autophagic recognition. Mitophagy impairment is linked to various pathologies; thus, removal of malfunctioning or even harmful mitochondria is vital to cellular physiology. Here, we describe methods that can be applied to the investigation of mitophagy.

Mitochondrial sirtuin 3 and various cell death modalities.

Sirtuin 3, a member of the mammalian sirtuin family of proteins, is involved in the regulation of multiple processes in cells. It is a major mitochondrial NAD+-dependent deacetylase with a broad range of functions, such as regulation of oxidative stress, reprogramming of tumor cell energy pathways, and metabolic homeostasis. One of the intriguing functions of sirtuin 3 is the regulation of mitochondrial outer membrane permeabilization, a key step in apoptosis initiation/progression. Moreover, sirtuin 3 is involved in the execution of various cell death modalities, which makes sirtuin 3 a possible regulator of crosstalk between them. This review is focused on the role of sirtuin 3 as a target for tumor cell elimination and how mitochondria and reactive oxygen species (ROS) are implicated in this process.

Photobiomodulation in 3D tissue engineering.

SIGNIFICANCE: The method of photobiomodulation (PBM) has been used in medicine for a long time to promote anti-inflammation and pain-resolving processes in different organs and tissues. PBM triggers numerous cellular pathways including stimulation of the mitochondrial respiratory chain, alteration of the cytoskeleton, cell death prevention, increasing proliferative activity, and directing cell differentiation. The most effective wavelengths for PBM are found within the optical window (750 to 1100 nm), in which light can permeate tissues and other water-containing structures to depths of up to a few cm. PBM already finds its applications in the developing fields of tissue engineering and regenerative medicine. However, the diversity of three-dimensional (3D) systems, irradiation sources, and protocols intricate the PBM applications. AIM: We aim to discuss the PBM and 3D tissue engineered constructs to define the fields of interest for PBM applications in tissue engineering. APPROACH: First, we provide a brief overview of PBM and the timeline of its development. Then, we discuss the optical properties of 3D cultivation systems and important points of light dosimetry. Finally, we analyze the cellular pathways induced by PBM and outcomes observed in various 3D tissue-engineered constructs: hydrogels, scaffolds, spheroids, cell sheets, bioprinted structures, and organoids. RESULTS: Our summarized results demonstrate the great potential of PBM in the stimulation of the cell survival and viability in 3D conditions. The strategies to achieve different cell physiology states with particular PBM parameters are outlined. CONCLUSIONS: PBM has already proved itself as a convenient and effective tool to prevent drastic cellular events in the stress conditions. Because of the poor viability of cells in scaffolds and the convenience of PBM devices, 3D tissue engineering is a perspective field for PBM applications.

Mitochondria as targets for cancer chemotherapy.

Heterogeneity of tumors dictates an individual approach to anticancer treatment. Despite their variability, almost all cancer cells demonstrate enhanced uptake and utilization of glucose, a phenomenon known as the Warburg effect, whereas mitochondrial activity in tumor cells is suppressed. Considering the key role of mitochondria in cell death, it appears that resistance of most tumors towards treatment can be, at least in part, explained by mitochondrial silencing in cancer cells. This review is devoted to the role of mitochondria in cell death, and describes how targeting of mitochondria can make tumor cells more susceptible to anticancer treatment.

Analysis of Mitochondrial Dysfunction During Cell Death.

Mitochondria play a key role in various modes of cell death. Analysis of mitochondrial dysfunction and the release of proteins from the intermembrane space of mitochondria represent essential tools in cell death investigation. Here we describe how to evaluate release of intermembrane space proteins during apoptosis, alterations in the mitochondrial membrane potential, and oxygen consumption in apoptotic cells.

Mitophagy in carcinogenesis and cancer treatment.

In order to maintain a functional mitochondrial network, cells have developed a quality control mechanism, namely mitophagy. This process can be induced through different pathways. The most studied is the so-called PINK1/Parkin pathway, which is associated with ubiquitylation of several mitochondrial proteins that were initially found to be related to Parkinson's disease. Another type of mitophagy is known as receptor-mediated mitophagy, which includes proteins, such as BNIP3 and BNIP3L, also known as Nix. Through these two mechanisms, mitophagy fulfills its functions and maintains cellular homeostasis. Here, we summarize the current knowledge about the mechanisms of mitophagy regulation and their interplay with cancer progression as well as anticancer treatment.