Sten Orrenius’s research while affiliated with Karolinska Institutet and other places

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. Expected final online publication date for the Annual Review of Pharmacology and Toxicology Volume 59 is January 6, 2019. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

For many years, mitochondria were regarded exclusively as cellular power plants, destined to provide the cell with energy in the form of ATP. Mitochondrial deterioration can have fatal consequences for cellular physiology and be a cause of various diseases. Subsequent studies revealed the involvement of mitochondria in the regulation and execution of multiple modalities of cell death. Analysis of the involvement of mitochondria in the control of various modes of cell death led to the conclusion that mitochondria might play a leading role as a “switch-board” between various cell death modalities.

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. Copyright © 2015 Elsevier Inc. All rights reserved.

Arsenic trioxide is a potent chemotherapeutic agent that selectively triggers apoptosis in tumor cells. Previous studies have demonstrated that arsenicals cause direct damage to mitochondria, but it is not clear that these effects initiate apoptosis. Here we used Bak-/- mouse liver mitochondria and virally immortalized Bax-/-Bak-/- mouse embryonic fibroblasts (MEFs) to investigate whether or not multidomain proapoptotic Bcl-2 family proteins were required for arsenic-induced mitochondrial damage and cell death. Near clinically achievable concentrations, arsenic stimulated cytochrome c release and apoptosis via a Bax/Bak- dependent mechanism. At higher concentrations (125 mM- 1 mM), cells died via a Bax/Bak-independent mechanism mediated by oxidative stress that resulted in necrosis. Consistent with previous reports, arsenic directly inhibited complex I of the mitochondrial electron transport chain, which resulted in mitochondrial permeability transition (MPT), the generation of reactive oxygen species (ROS), and thiol oxidation. However, these effects only occurred at concentrations of arsenic trioxide of 50 mM and higher, and the oxidative stress associated with them blocked caspase activation. Our data demonstrate for the first time that the cytochrome c release which initiates apoptosis in cells exposed to this classic mitochondrial poison occurs indirectly via the activation of Bax/Bak rather than via direct mitochondrial damage. Furthermore, the results implicate reactive oxygen species in a concentration-dependent mechanistic switch between apoptosis and necrosis.

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.

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 H(2)O(2)induce extensive DNA damage, ATP depletion and severe cytotoxicity. While 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 H(2)O(2) triggered a modest drop in ATP level, delayed toxicity, G2/M arrest and cell senescence. Mitochondrially-produced H(2)O(2) 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 H(2)O(2,) 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.

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. Expected final online publication date for the Annual Review of Pharmacology and Toxicology Volume 53 is January 06, 2013. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.

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.

Investigation of different forms of cell death has become an important area of biomedical research. Recently, several cell death modalities, in addition to necrosis and apoptosis, have been described and characterized based on morphological and biochemical criteria. In 2009, the Nomenclature Committee on Cell Death proposed unified criteria for the definition of 12 cell death modalities (Kroemer et al. 2009). Among the best characterized of these modes of cell death are apoptosis, autophagy, cornification, and necrosis. Until recently, a requirement for gene expression was documented only for apoptotic and autophagic cell death. Cornification is a special form of programmed cell death in the epidermis. To some extent, it represents an example of terminal differentiation, similar to the maturation of red blood cells or lens epithelium, although there are significant differences between them at the biochemical level. The interaction between the different forms of cell death is complex and is still a matter of debate. However, recently, the mitochondria have been demonstrated to play a crucial role in the effectuation of several cell death modalities, although the precise mechanisms of their involvement are still unclear. In this review, we focus on the mitochondrial involvement in four cell death modalities, namely, necrosis, apoptosis, autophagy, and anoikis (Figure 2.1).