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 degrees C and 85 degrees 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 degrees C, the only striking morphological difference between cell deaths induced by 40-55 degrees C or 85 degrees 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

Autocrine Prostaglandin E2 Signaling Promotes Tumor Cell Survival and Proliferation in Childhood Neuroblastoma

Background: Prostaglandin E2 (PGE2) is an important mediator in tumor-promoting inflammation. High expression of cyclooxygenase-2 (COX-2) has been detected in the embryonic childhood tumor neuroblastoma, and treatment with COX inhibitors significantly reduces tumor growth. Here, we have investigated the significance of a high COX-2 expression in neuroblastoma by analysis of PGE2 production, the expression pattern and localization of PGE2 receptors and intracellular signal transduction pathways activated by PGE2. Principal Findings: A high expression of the PGE2 receptors, EP1, EP2, EP3 and EP4 in primary neuroblastomas, independent of biological and clinical characteristics, was detected using immunohistochemistry. In addition, mRNA and protein corresponding to each of the receptors were detected in neuroblastoma cell lines. Immunofluorescent staining revealed localization of the receptors to the cellular membrane, in the cytoplasm, and in the nuclear compartment. Neuroblastoma cells produced PGE2 and stimulation of serum-starved neuroblastoma cells with PGE2 increased the intracellular concentration of calcium and cyclic AMP with subsequent phosphorylation of Akt. Addition of 16,16-dimethyl PGE 2 (dmPGE2) increased cell viability in a time, dose- and cell line-dependent manner. Treatment of neuroblastoma cells with a COX-2 inhibitor resulted in a diminished cell growth and viability that was reversed by the addition of dmPGE2. Similarly, PGE 2 receptor antagonists caused a decrease in neuroblastoma cell viability in a dose-dependent manner

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

Mitochondrial cytochrome c release may occur by volume-dependent mechanisms not involving permeability transition.

The mechanisms regulating mitochondrial outer-membrane permeabilization and the release of cytochrome c during apoptosis remain controversial. In the present study, we show in an in vitro model system that the release of cytochrome c may occur via moderate modulation of mitochondrial volume, irrespective of the mechanism leading to the mitochondrial swelling. In contrast with mitochondrial permeability transition-dependent release of cytochrome c, in the present study mitochondria remain intact and functionally active

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

A folding variant of human alpha-lactalbumin induces mitochondrial permeability transition in isolated mitochondria

A human milk fraction containing multimeric alpha-lactalbumin (MAL) is able to kill cells via apoptosis. MAL is a protein complex of a folding variant of alpha-lactalbumin and lipids. Previous results have shown that upon treatment of transformed cells, MAL localizes to the mitochondria and cytochrome c is released into the cytosol. This is followed by activation of the caspase cascade. In this study, we further investigated the involvement of mitochondria in apoptosis induced by the folding variant of alpha-lactalbumin. Addition of MAL to isolated rat liver mitochondria induced a loss of the mitochondrial membrane potential (Delta Psi(m)), mitochondrial swelling and the release of cytochrome c. These changes were Ca(2+)-dependent and were prevented by cyclosporin A, an inhibitor of mitochondrial permeability transition. MAL also increased the rate of state 4 respiration in isolated mitochondria by exerting an uncoupling effect. This effect was due to the presence of fatty acids in the MAL complex because it was abolished completely by BSA. BSA delayed, but failed to prevent, mitochondrial swelling as well as dissipation of Delta Psi(m), indicating that the fatty acid content of MAL facilitated, rather than caused, these effects. Similar results were obtained with HAMLET (human alpha-lactalbumin made lethal to tumour cells), which is native alpha-lactalbumin converted in vitro to the apoptosis-inducing folding variant of the protein in complex with oleic acid. Our findings demonstrate that a folding variant of alpha-lactalbumin induces mitochondrial permeability transition with subsequent cytochrome c release, which in transformed cells may lead to activation of the caspase cascade and apoptotic death

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