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.

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.

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.

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.

Receptor-Mediated Mitophagy Rescues Cancer Cells under Hypoxic Conditions.

Targeting mitochondria with thenoyltrifluoroacetone (TTFA), an inhibitor of Complex II in the respiratory chain, stimulated cisplatin-induced apoptosis in various cell lines in normoxia but not in hypoxia. This can be explained by the elimination of mitochondria involved in triggering apoptotic cell death by mitophagy, either Parkin-dependent or receptor-mediated. Treatment with TTFA alone or in combination with cisplatin did not cause accumulation of PINK1, meaning that under hypoxic conditions cells survive through activation of a receptor-mediated pathway. Hypoxia triggers the accumulation of BNIP3 and BNIP3L (also known as NIX), key participants in receptor-mediated mitophagy. Under hypoxic conditions, stimulation of autophagy, as assessed by the accumulation of lipidated form of LC3 (LC3II), was observed. To exclude the contribution of canonical macroautophagy in LC3II accumulation, experiments were performed using U1810 cells lacking ATG13, a key enzyme of macroautophagy. Despite the absence of ATG13, hypoxia-mediated accumulation of LC3II was not affected, underlying the importance of the receptor-mediated pathway. In order to prove the protective role of BNIP3 against cisplatin-induced apoptosis, BNIP3-deficient A549 cells were used. Surprisingly, a BNIP3 knockout did not abolish hypoxia-induced protection; however, in cells lacking BNIP3, a compensatory upregulation of BNIP3L was detected. Thus, in the absence of BNIP3, mitophagy could be maintained by BNIP3L and lead to cell death suppression due to the elimination of proapoptotic mitochondria. When both BNIP3 and BNIP3L were knocked out, the inhibitory effect of hypoxia on apoptosis was diminished, although not abolished completely. Undoubtedly, receptor-mediated mitophagy is likely to be one of the mechanisms responsible for cell death suppression under hypoxic conditions.

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.

Modeling hypoxia facilitates cancer cell survival through downregulation of p53 expression.

A hypoxic environment of rapidly growing tumor cells makes them resistant to antitumor drugs. Mimicking hypoxia with iron chelator deferoxamine, suppressed cell death induced by widely used anticancer drugs doxorubicin or cisplatin. Deferoxamine decreased the number of dead (detached) cells, the size of SubG1 population, the release of cytochrome c, and the processing of caspase-3 in HCT116 colon carcinoma cells treated with cisplatin or doxorubicin. Deferoxamine-mediated suppression of apoptosis correlated with the level of pro-apoptotic Bcl-2 family proteins Bax, Bid, and Puma, which stimulate mitochondrial apoptotic pathway through permeabilization of the outer mitochondrial membrane and cytochrome c release. Here we show that one of the reasons for apoptosis suppression is downregulation of p53 expression under hypoxic conditions, and, as a result, attenuation of the expression of pro-apoptotic Bcl-2 family proteins. Indeed, p53 knock-out did not affect the stabilization of hypoxia-inducible factor but made undetectable the expression of pro-apoptotic proteins.

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.

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.

Dephosphorylation of Fas-ligand and caveolin-1 is a prerequisite step in Fas-ligand - caveolin-1 complex formation and cell death stimulation.

Fas-ligand/CD178 belongs to the TNF family proteins and is the well-characterized inducer of cell death. We showed previously that the interaction of Fas-ligand and caveolin-1 is necessary for Fas-ligand translocation to rafts, and the subsequent induction of Fas-ligand-dependent cell death. Both molecules can undergo phosphorylation, however the role of the phosphorylation state of Fas-ligand and caveolin-1 in their physical association, and consequently in of Fas - mediated cell death induction is currently unknown. In this study, we show that in control cells Fas-ligand interaction with caveolin-1 is not observed, and both molecules are phosphorylated. The intracellular part of Fas-ligand was shown to form a complex with p59Fyn-kinase. Upon cell death activation, the expression and activity of p59Fyn-kinase decreases substantially, leading to the disruption of Fas-ligand - p59Fyn-kinase association, dephosphorylation of Fas-ligand and caveolin-1, and formation of a complex between them (Fas-ligand - caveolin-1). The analysis of the effects of kinase and phosphatase inhibitors revealed that phosphorylation of Fas-ligand and caveolin-1 at tyrosine residues suppressed Fas-mediated cell death. Thus, dephosphorylation of Fas-ligand and caveolin-1 is critical for triggering Fas-ligand-mediated apoptotic pathway and cell death execution.

Desmin mutations result in mitochondrial dysfunction regardless of their aggregation properties.

Desmin, being a major intermediate filament of muscle cells, contributes to stabilization and positioning of mitochondria. Desmin mutations have been reported in conjunction with skeletal myopathies accompanied by mitochondrial dysfunction. Depending on the ability to promote intracellular aggregates formation, mutations can be considered aggregate-prone or non-aggregate-prone. The aim of the present study was to describe how expression of different desmin mutant isoforms effects mitochondria and contributes to the development of myocyte dysfunction. To achieve this goal, two non-aggregate-prone (Des S12F and Des A213V) and four aggregate-prone (Des L345P, Des A357P, Des L370P, Des D399Y) desmin mutations were expressed in skeletal muscle cells. We showed that all evaluated mutations affected the morphology of mitochondrial network, suppressed parameters of mitochondrial respiration, diminished mitochondrial membrane potential, increased ADP/ATP ratio, and enhanced mitochondrial DNA (mtDNA) release. mtDNA was partially secreted through exosomes as demonstrated by GW4869 treatment. Dysfunction of mitochondria was observed regardless the type of mutation: aggregate-prone or non-aggregate-prone. However, expression of aggregate-prone mutations resulted in more prominent phenotype. Thus, in this comparative study of six pathogenic desmin mutations that cause skeletal myopathy development, we confirmed a role of mitochondrial dysfunction and mtDNA release in the pathogenesis of desmin myopathies, regardless of the aggregation capacity of the mutated desmin.

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.

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.

Cationic gold nanoparticles elicit mitochondrial dysfunction: a multi-omics study.

Systems biology is increasingly being applied in nanosafety research for observing and predicting the biological perturbations inflicted by exposure to nanoparticles (NPs). In the present study, we used a combined transcriptomics and proteomics approach to assess the responses of human monocytic cells to Au-NPs of two different sizes with three different surface functional groups, i.e., alkyl ammonium bromide, alkyl sodium carboxylate, or poly(ethylene glycol) (PEG)-terminated Au-NPs. Cytotoxicity screening using THP-1 cells revealed a pronounced cytotoxicity for the ammonium-terminated Au-NPs, while no cell death was seen after exposure to the carboxylated or PEG-modified Au-NPs. Moreover, Au-NR3+ NPs, but not the Au-COOH NPs, were found to trigger dose-dependent lethality in vivo in the model organism, Caenorhabditis elegans. RNA sequencing combined with mass spectrometry-based proteomics predicted that the ammonium-modified Au-NPs elicited mitochondrial dysfunction. The latter results were validated by using an array of assays to monitor mitochondrial function. Au-NR3+ NPs were localized in mitochondria of THP-1 cells. Moreover, the cationic Au-NPs triggered autophagy in macrophage-like RFP-GFP-LC3 reporter cells, and cell death was aggravated upon inhibition of autophagy. Taken together, these studies have disclosed mitochondria-dependent effects of cationic Au-NPs resulting in the rapid demise of the cells.

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.

2-Deoxy-D-glucose has distinct and cell line-specific effects on the survival of different cancer cells upon antitumor drug treatment.

The dependence of tumors on glycolysis for ATP generation offers a rationale for therapeutic strategies aimed at selective inhibition of the glycolytic pathway. Analysis of tumor cell responses to anticancer drugs revealed that inhibition of glycolysis by 2-deoxy-D-glucose (2-DG) generally augmented the apoptotic response; however, in HCT116 human colon carcinoma cells, apoptosis was suppressed. A comparison of neuroblastoma SK-N-BE(2) and HCT116 cells revealed, that in contrast to HCT116, in SK-N-BE(2) cells 2-DG alone was able to induce cell death. In SK-N-BE(2) cells the decrease in ATP levels upon treatment with 2-DG was more prominent because in HCT116 cells mitochondria compensated for the loss of ATP caused by glycolysis suppression. In both cells lines 2-DG triggered endoplasmic reticulum (ER) stress, assessed by the accumulation of the marker protein GRP78/BiP. Suppression of ER stress by mannose attenuated the 2-DG-induced apoptotic response in SK-N-BE(2) cells, implying that apoptosis in these cells is a consequence of ER stress induction. In HCT116 cells, ER stress stimulated autophagy, assessed by the accumulation of the lipidated form of LC3. The inhibitor of ER stress mannose attenuated autophagy and reversed 2-DG-mediated suppression of cisplatin-induced apoptosis. When autophagy in HCT116 cells was suppressed by bafilomycin, cisplatin-induced apoptosis was decreased. At the same time, stimulation of autophagy in SK-N-BE(2) cells suppressed cell death. Thus, successful treatment of tumors with conventionally used anticancer drugs should be combined with targeting metabolic pathways involved in the regulation of apoptosis, autophagy, and cellular bioenergetics.

Cell death-based treatment of neuroblastoma.

Neuroblastoma (NB) is the most common solid childhood tumor outside the brain and causes 15% of childhood cancer-related mortality. The main drivers of NB formation are neural crest cell-derived sympathoadrenal cells that undergo abnormal genetic arrangements. Moreover, NB is a complex disease that has high heterogeneity and is therefore difficult to target for successful therapy. Thus, a better understanding of NB development helps to improve treatment and increase the survival rate. One of the major causes of sporadic NB is known to be MYCN amplification and mutations in ALK (anaplastic lymphoma kinase) are responsible for familial NB. Many other genetic abnormalities can be found; however, they are not considered as driver mutations, rather they support tumor aggressiveness. Tumor cell elimination via cell death is widely accepted as a successful technique. Therefore, in this review, we provide a thorough overview of how different modes of cell death and treatment strategies, such as immunotherapy or spontaneous regression, are or can be applied for NB elimination. In addition, several currently used and innovative approaches and their suitability for clinical testing and usage will be discussed. Moreover, significant attention will be given to combined therapies that show more effective results with fewer side effects than drugs targeting only one specific protein or pathway.

Impairment of Fas-ligand-caveolin-1 interaction inhibits Fas-ligand translocation to rafts and Fas-ligand-induced cell death.

Fas-ligand/CD178 belongs to the TNF family proteins and can induce apoptosis through death receptor Fas/CD95. The important requirement for Fas-ligand-dependent cell death induction is its localization to rafts, cholesterol- and sphingolipid-enriched micro-domains of membrane, involved in regulation of different signaling complexes. Here, we demonstrate that Fas-ligand physically associates with caveolin-1, the main protein component of rafts. Experiments with cells overexpressing Fas-ligand revealed a FasL N-terminal pre-prolin-rich region, which is essential for the association with caveolin-1. We found that the N-terminal domain of Fas-ligand bears two caveolin-binding sites. The first caveolin-binding site binds the N-terminal domain of caveolin-1, whereas the second one appears to interact with the C-terminal domain of caveolin-1. The deletion of both caveolin-binding sites in Fas-ligand impairs its distribution between cellular membranes, and attenuates a Fas-ligand-induced cytotoxicity. These results demonstrate that the interaction of Fas-ligand and caveolin-1 represents a molecular basis for Fas-ligand translocation to rafts, and the subsequent induction of Fas-ligand-dependent cell death. A possibility of a similar association between other TNF family members and caveolin-1 is discussed.

Suppressed translation as a mechanism of initiation of CASP8 (caspase 8)-dependent apoptosis in autophagy-deficient NSCLC cells under nutrient limitation.

Macroautophagy/autophagy inhibition under stress conditions is often associated with increased cell death. We found that under nutrient limitation, activation of CASP8/caspase-8 was significantly increased in autophagy-deficient lung cancer cells, which precedes mitochondria outer membrane permeabilization (MOMP), CYCS/cytochrome c release, and activation of CASP9/caspase-9, indicating that under such conditions the activation of CASP8 is a primary event in the initiation of apoptosis as well as essential to reduce clonogenic survival of autophagy-deficient cells. Starvation leads to suppression of CFLAR proteosynthesis and accumulation of CASP8 in SQSTM1 puncta. Overexpression of CFLARs reduces CASP8 activation and apoptosis during starvation, while its silencing promotes efficient activation of CASP8 and apoptosis in autophagy-deficient U1810 lung cancer cells even under nutrient-rich conditions. Similar to starvation, inhibition of protein translation leads to efficient activation of CASP8 and cell death in autophagy-deficient lung cancer cells. Thus, here for the first time we report that suppressed translation leads to activation of CASP8-dependent apoptosis in autophagy-deficient NSCLC cells under conditions of nutrient limitation. Our data suggest that targeting translational machinery can be beneficial for elimination of autophagy-deficient cells via the CASP8-dependent apoptotic pathway.

Involvement of autophagy in the outcome of mitotic catastrophe.

Evading cell death is a major driving force for tumor progression that is one of the main problems in current cancer research. Mitotic catastrophe (MC) represents attractive platform compromising tumor resistance to current therapeutic modalities. MC appeared as onco-suppressive mechanism and is defined as a stage driving the cell to an irreversible destiny, i.e. cell death via apoptosis or necrosis. Our study highlights that MC induction in colorectal carcinoma cell lines ultimately leads to the autophagy followed by apoptosis. We show that autophagy suppression in Atg 13 knockout non-small cell lung carcinoma cells lead to the dramatic decrease of MC rate. Furthermore, mitochondria-linked anti-apoptotic proteins Mcl-1 and Bcl-xL play a crucial role in the duration of MC and a cross-talk between autophagy and apoptosis. Thus, the suppression of apoptosis by overexpression of Mcl-1 or Bcl-xL affected MC and lead to a significant induction of autophagy in HCT116 wt and HCT116 14-3-3σ-/- cells. Our data demonstrate that MC induction is a critical stage, in which a cell decides how to die, while mitochondria are responsible for the maintaining the balance between MC - autophagy - apoptosis.