Role of Cell Death in Toxicology: Does It Matter How Cells Die?

My research activity started with studies on drug metabolism in rat liver microsomes in the early 1960s. The CO-binding pigment (cytochrome P450) had been discovered a few years earlier and was subsequently found to be involved in steroid hydroxylation in adrenal cortex microsomes. Our early studies suggested that it also participated in the oxidative demethylation of drugs catalyzed by liver microsomes, and that prior treatment of the animals with phenobarbital caused increased levels of the hemoprotein in the liver, and similarly enhanced rates of drug metabolism. Subsequent studies of cytochrome P450-mediated metabolism of toxic drugs in freshly isolated rat hepatocytes characterized critical cellular defense systems and identified mechanisms by which accumulating toxic metabolites could damage and kill the cells. These studies revealed that multiple types of cell death could result from the toxic injury, and that it is important to know which type of cell death results from the toxic injury.

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.

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.

Reactive oxygen species generated in different compartments induce cell death, survival, or senescence.

Although reactive oxygen species (ROS) are well-established mediators of oxidative damage and cell demise, the mechanisms by which they trigger specific cell death modalities and the temporal/spatial requirements underlying this phenomenon are largely unknown. Yet, it is well established that most anticancer therapies depend on ROS production for efficient tumor eradication. Using several non-small-cell lung cancer cell lines, we have dissected how the site of ROS production and accumulation in various cell compartments affect cell fate. We demonstrate that high levels of exogenously generated H2O2 induce extensive DNA damage, ATP depletion, and severe cytotoxicity. Although these effects were independent of caspase activity, they could-at least in part-be prevented by RIP1 kinase inhibition. In contrast, low levels of exogenously produced H2O2 triggered a modest drop in ATP level, delayed toxicity, G2/M arrest, and cell senescence. Mitochondrially produced H2O2 induced a reversible ATP drop without affecting cell viability. Instead, the cells accumulated in the G1/S phase of the cell cycle and became senescent. Concomitant inhibition of glycolysis was found to markedly sensitize cells to death in the presence of otherwise nontoxic concentrations of H2O2, presumably by the inhibition of ATP-restoring mechanisms. Combined, our data provide evidence that ROS might dictate different cellular consequences depending on their overall concentration at steady-state levels and on their site of generation.

Targeting hepatoma using nitric oxide donor strategies.

AIMS: The study evaluated the role of increased intracellular nitric oxide (NO) concentration using NO donors or stably NO synthase-3 (NOS-3) overexpression during CD95-dependent cell death in hepatoma cells. The expression of cell death receptors and caspase activation, RhoA kinase activity, NOS-3 expression/activity, oxidative/nitrosative stress, and p53 expression were analyzed. The antitumoral activity of NO was also evaluated in the subcutaneous implantation of NOS-3-overexpressing hepatoma cells, as well NO donor injection into wild-type hepatoma-derived tumors implanted in xenograft mouse models. RESULTS: NO donor increased CD95 expression and activation of caspase-8 and 3 in HepG2, Huh7, and Hep3B cells. NOS-3 overexpression increased oxidative/nitrosative stress, p53 and CD95 expression, cellular Fas-associated death domain (FADD)-like IL-1beta converting enzyme (FLICE) inhibitory protein long (cFLIP(L)) and its short isoform (cFLIP(S)) shift, and cell death in HepG2 (4TO-NOS) cells. The inhibition of RhoA kinase and p53 knockdown using RNA interference reduced cell death in 4TO-NOS cells. The supplementation with hydrogen peroxide (H(2)O(2)) increased NOS-3 activity and cell death in 4TO-NOS cells. NOS-3 overexpression or NO donor injection into hepatoma-derived tumors reduced the size and increased p53 and cell death receptor expression in nude mice. INNOVATION AND CONCLUSIONS: The increase of intracellular NO concentration promoted oxidative and nitrosative stress, Rho kinase activity, p53 and CD95 expression, and cell death in cultured hepatoma cells. NOS-3-overexpressed HepG2 cells or intratumoral NO donor administration reduced tumor cell growth and increased the expression of p53 and cell death receptors in tumors developed in a xenograft mouse model.

Autophagy in toxicology: cause or consequence?

Research on autophagy and its effects on cell metabolism and physiology has increased dramatically during recent years. Multiple forms of autophagy have been characterized, and many of the genes involved in the regulation of this process have been identified. The importance of autophagy for embryonic development and maintenance of tissue homeostasis in the adult organism has been demonstrated convincingly, and several human diseases have been linked to deficiencies in autophagy. Most often, autophagy serves as a protective mechanism, but persistent activation of autophagy can result in cell death. This is true for many toxic agents. In fact, there are ample examples of cross talk between autophagy and other modes of cell death after exposure to toxicants. However, the relative contribution of autophagy to the overall toxicity of these compounds is not always clear, and further research is needed to clarify the toxicological significance of this process.

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.

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.

Calcium and cell death mechanisms: a perspective from the cell death community.

Research during the past several decades has provided convincing evidence for a crucial role of the Ca(2+) ion in cell signaling. Hence, intracellular Ca(2+) transients have been implicated in most aspects of cell physiology, including gene transcription, cell cycle regulation and cell proliferation. Further, the Ca(2+) ion has been found to also play an important role in cell death regulation. Thus, necrotic cell death was early associated with intracellular Ca(2+) overload, and multiple functions in the apoptotic process have subsequently been found to be governed by Ca(2+) signaling. More recently, other modes of cell death, notably anoikis and autophagic cell death, have been demonstrated to also be modulated by Ca(2+) transients. Characteristics, interrelationship and mechanisms involved in Ca(2+) regulation of these cell death modalities are discussed in this review.

Cell death mechanisms and their implications in toxicology.

Necrotic cell death was long regarded as the ultimate consequence of chemical toxicity and was thought to result from simple cell failure because of toxic interference with vital cell functions. Introduction of the novel concept of programmed cell death (PCD), or apoptosis, has changed this view dramatically. This development has been further stimulated by the characterization of several other genetically PCD modalities, such as autophagy and pyroptosis. Like apoptosis, these modes of cell death are governed by complex signaling networks, containing "switches" responsible for cross talk between them. Recruitment or repression of these cell death signaling networks by foreign chemicals can lead to acute as well as chronic toxicity. In many instances, such effects of toxicants are mediated by disruption/modulation of cellular Ca(2+) homeostasis or increased generation of reactive oxygen species in the mitochondria or other intracellular compartments. Caspases, calpains, lysosomal proteases, and endonucleases are the main executioners of cell death, and they often co-operate during the execution stage of apoptosis. Finally, dead or dying cells are recognized and engulfed by phagocytes to prevent inflammation and associated tissue damage. Defective macrophage engulfment and degradation of cell corpses may also result from toxicity and can contribute to both the inflammatory response and dysregulation of tissue homeostasis. Hence, the cell death and phagocytosis regulatory networks offer a multitude of targets for toxic chemicals.

Critical role for hyperpolarization-activated cyclic nucleotide-gated channel 2 in the AIF-mediated apoptosis.

Cellular calcium uptake is a controlled physiological process mediated by multiple ion channels. The exposure of cells to either one of the protein kinase C (PKC) inhibitors, staurosporine (STS) or PKC412, can trigger Ca²(+) influx leading to cell death. The precise molecular mechanisms regulating these events remain elusive. In this study, we report that the PKC inhibitors induce a prolonged Ca²(+) import through hyperpolarization-activated cyclic nucleotide-gated channel 2 (HCN2) in lung carcinoma cells and in primary culture of cortical neurons, sufficient to trigger apoptosis-inducing factor (AIF)-mediated apoptosis. Downregulation of HCN2 prevented the drug-induced Ca²(+) increase and subsequent apoptosis. Importantly, the PKC inhibitors did not cause Ca²(+) entry into HEK293 cells, which do not express the HCN channels. However, introduction of HCN2 sensitized them to STS/PKC412-induced apoptosis. Mutagenesis of putative PKC phosphorylation sites within the C-terminal domain of HCN2 revealed that dephosphorylation of Thr549 was critical for the prolonged Ca²(+) entry required for AIF-mediated apoptosis. Our findings demonstrate a novel role for the HCN2 channel by providing evidence that it can act as an upstream regulator of cell death triggered by PKC inhibitors.

Mitochondrial regulation of cell death: processing of apoptosis-inducing factor (AIF).

Apoptosis might proceed through the activation of both caspase-dependent and -independent pathways. Apoptosis-inducing factor (AIF) was discovered as the first protein that mediated caspase-independent cell death. Initially, it was regarded as a soluble protein residing in the intermembrane space of mitochondria, from where it could be exported to the nucleus to participate in large-scale DNA fragmentation and chromatin condensation. However, later it was demonstrated that AIF is N-terminally anchored to the inner mitochondrial membrane. Hence, AIF must be liberated from its membrane anchor prior to being released into the cytosol. The current knowledge about the molecular mechanisms regulating the processing and release of AIF from the mitochondria will be summarized and discussed in this review.

Cell death mechanisms: cross-talk and role in disease.

Multiple cell death mechanisms operate in both uni- and multicellular organisms. Hence, research during the past forty years has revealed that apoptosis is not the only cell death program involved in the regulation of tissue homeostasis and the removal of unwanted cells in biological organisms. While the molecular pathways of apoptosis and necrosis are now relatively well established, the precise mechanisms of other cell death modalities, and their cross-talk, require additional study. This is particularly important, since many human disorders can be attributed, directly or indirectly, to defective cell death mechanisms. In this review we shall discuss the characteristics and cross-talk between various modes of cell death and their role in cell death-related disorders, notably, neurodegenerative disease and cancer.

Involvement of Ca2+ and ROS in alpha-tocopheryl succinate-induced mitochondrial permeabilization.

Release of mitochondrial proteins such as cytochrome c, AIF, Smac/Diablo etc., plays a crucial role in apoptosis induction. A redox-silent analog of vitamin E, alpha-tocopheryl succinate (alpha-TOS), was shown to stimulate cytochrome c release via production of reactive oxygen species (ROS) and Bax-mediated permeabilization of the outer mitochondrial membrane. Here we show that alpha-TOS facilitates mitochondrial permeability transition (MPT) in isolated rat liver mitochondria, Tet21N neuroblastoma cells and Jurkat T-lymphocytes. In particular, in addition to ROS production, alpha-TOS stimulates rapid Ca(2+) entry into the cells with subsequent accumulation of Ca(2+) in mitochondria-a prerequisite step for MPT induction. Alteration of mitochondrial Ca(2+) buffering capacity was observed as early as 8 hr after incubation with alpha-TOS, when no activation of Bax was yet detected. Ca(2+) accumulation in mitochondria was important for apoptosis progression, since inhibition of mitochondrial Ca(2+) uptake significantly mitigated the apoptotic response. Importantly, Ca(2+)-induced mitochondrial destabilization might cooperate with Bax-mediated mitochondrial outer membrane permeabilization to induce cytochrome c release from mitochondria.

Oxidative modification sensitizes mitochondrial apoptosis-inducing factor to calpain-mediated processing.

Although processing of mitochondrial apoptosis-inducing factor (AIF) is essential for its function during apoptosis in most cell types, the detailed mechanisms of AIF cleavage remain elusive. Recent findings indicate that the proteolytic process is Ca(2+)-dependent and that it is mediated by a calpain located in the mitochondrial intermembrane space. We can now report that, in addition to a sustained intracellular Ca(2+) elevation, enhanced formation of reactive oxygen species (ROS) is a prerequisite step for AIF to be cleaved and released from mitochondria in staurosporine-treated cells. These events occurred independent of the redox state of the mitochondria and were not influenced by binding of pyridine nucleotides to AIF. Chelation of cytosolic Ca(2+) by BAPTA/AM suppressed the elevation of both Ca(2+) and ROS, suggesting that the Ca(2+) rise was the most upstream signal required for AIF processing. We could further show that the stimulated ROS production leads to oxidative modification (carbonylation) of AIF, which markedly increases its rate of cleavage by calpain. Accordingly, pretreatment of the cells with antioxidants blocked AIF carbonylation, as well as its subsequent cleavage and release from the mitochondria. Combined, our data provide evidence that ROS-mediated, posttranslational modification of AIF is critical for its cleavage by calpain and thus for AIF-mediated cell death.

The Warburg effect and mitochondrial stability in cancer cells.

The last decade has witnessed a renaissance of Otto Warburg's fundamental hypothesis, which he put forward more than 80 years ago, that mitochondrial malfunction and subsequent stimulation of cellular glucose utilization lead to the development of cancer. Since most tumor cells demonstrate a remarkable resistance to drugs that kill non-malignant cells, the question has arisen whether such resistance might be a consequence of the abnormalities in tumor mitochondria predicted by Warburg. The present review discusses potential mechanisms underlying the upregulation of glycolysis and silencing of mitochondrial activity in cancer cells, and how pharmaceutical intervention in cellular energy metabolism might make tumor cells more susceptible to anti-cancer treatment.

Mitochondria as targets for chemotherapy.

Mitochondrial malfunctioning is implicated in the pathogenesis of a variety of disorders, including cancer and multiple neurodegenerative diseases, such as Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis, and Huntington's disease. Disturbance of mitochondrial vital functions, e.g., production of ATP, calcium buffering capacity, and generation of reactive oxygen species, can be potentially involved in disease pathogenesis. Neurological disorders caused by mitochondrial deterioration are often associated with cell loss within specific brain regions. In contrast, mitochondrial alterations in tumor cells and the "Warburg effect" might lead to cell survival and resistance of tumor cells to chemotherapy. This review is devoted to the role of mitochondria in neurodegeneration and tumor formation, and describes how targeting of mitochondria can be beneficial in the therapy of these diseases, which affect a large human population.