Publications by authors named "Sten Orrenius"

64 Publications

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

Authors:
Sten Orrenius

Annu Rev Pharmacol Toxicol 2019 01 25;59:1-14. Epub 2018 Jul 25.

Institute of Environmental Medicine, Karolinska Institutet, SE-171 77 Stockholm, Sweden; email:

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.
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http://dx.doi.org/10.1146/annurev-pharmtox-010818-021725DOI Listing
January 2019

Calcium and mitochondria in the regulation of cell death.

Biochem Biophys Res Commun 2015 Apr;460(1):72-81

Institute of Environmental Medicine, Division of Toxicology, Karolinska Institutet, SE-171 77 Stockholm, Sweden; MV Lomonosov Moscow State University, 119991 Moscow, Russia.

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.
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http://dx.doi.org/10.1016/j.bbrc.2015.01.137DOI Listing
April 2015

Analysis of mitochondrial dysfunction during cell death.

Methods Mol Biol 2015 ;1264:385-93

Division of Toxicology, Institute of Environmental Medicine, Karolinska Institutet, 210, Stockholm, 171 77, Sweden,

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.
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http://dx.doi.org/10.1007/978-1-4939-2257-4_33DOI Listing
October 2015

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

Free Radic Biol Med 2013 Apr 4;57:176-87. Epub 2013 Jan 4.

Division of Toxicology, Institute of Environmental Medicine, Karolinska Institutet, SE-171 77 Stockholm, Sweden.

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.
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http://dx.doi.org/10.1016/j.freeradbiomed.2012.12.024DOI Listing
April 2013

Autophagy in toxicology: cause or consequence?

Annu Rev Pharmacol Toxicol 2013 16;53:275-97. Epub 2012 Oct 16.

Institute of Environmental Medicine, Division of Toxicology, Karolinska Institutet, SE-17177 Stockholm, Sweden.

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.
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http://dx.doi.org/10.1146/annurev-pharmtox-011112-140210DOI Listing
July 2013

Citrate kills tumor cells through activation of apical caspases.

Cell Mol Life Sci 2012 Dec 10;69(24):4229-37. Epub 2012 Oct 10.

Division of Toxicology, Institute of Environmental Medicine, Karolinska Institutet, Box 210, 171 77 Stockholm, Sweden.

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.
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http://dx.doi.org/10.1007/s00018-012-1166-3DOI Listing
December 2012

Targeting hepatoma using nitric oxide donor strategies.

Antioxid Redox Signal 2013 Feb 26;18(5):491-506. Epub 2012 Sep 26.

Department of Biochemistry and Molecular Biology, University of Córdoba, Córdoba, Spain.

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.
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http://dx.doi.org/10.1089/ars.2011.4476DOI Listing
February 2013

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

Cell Mol Life Sci 2012 Jun;69(12):2091-9

Division of Toxicology, Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden.

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.
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http://dx.doi.org/10.1007/s00018-012-0918-4DOI Listing
June 2012

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

Cell Calcium 2011 Sep 3;50(3):211-21. Epub 2011 Apr 3.

Institute of Environmental Medicine, Division of Toxicology, Karolinska Institutet, Box 210, SE-171 77 Stockholm, Sweden.

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.
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http://dx.doi.org/10.1016/j.ceca.2011.03.003DOI Listing
September 2011

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

EMBO J 2010 Nov 29;29(22):3869-78. Epub 2010 Oct 29.

Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden.

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.
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http://dx.doi.org/10.1038/emboj.2010.253DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2989107PMC
November 2010

Cell death mechanisms and their implications in toxicology.

Toxicol Sci 2011 Jan 9;119(1):3-19. Epub 2010 Sep 9.

Institute of Environmental Medicine, Division of Toxicology, Karolinska Institutet, Box 210, SE-171 77 Stockholm, Sweden.

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.
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http://dx.doi.org/10.1093/toxsci/kfq268DOI Listing
January 2011

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

Biochem Biophys Res Commun 2010 May;396(1):95-100

Institute of Environmental Medicine, Division of Toxicology Karolinska Institutet, SE-17177 Stockholm, Sweden.

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.
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http://dx.doi.org/10.1016/j.bbrc.2010.02.163DOI Listing
May 2010

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

Exp Cell Res 2010 May 6;316(8):1374-83. Epub 2010 Mar 6.

Institute of Environmental Medicine, Karolinska Institutet, Box 210, SE-171 77 Stockholm, Sweden.

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.
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http://dx.doi.org/10.1016/j.yexcr.2010.02.037DOI Listing
May 2010

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

Int J Cancer 2010 Oct;127(8):1823-32

Division of Toxicology, Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden.

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.
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http://dx.doi.org/10.1002/ijc.25204DOI Listing
October 2010

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

Free Radic Biol Med 2010 Mar 4;48(6):791-7. Epub 2010 Jan 4.

Institute of Environmental Medicine, Division of Toxicology, Karolinska Institutet, SE-171 77 Stockholm, Sweden.

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.
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http://dx.doi.org/10.1016/j.freeradbiomed.2009.12.020DOI Listing
March 2010

The Warburg effect and mitochondrial stability in cancer cells.

Mol Aspects Med 2010 Feb 6;31(1):60-74. Epub 2009 Dec 6.

Institute of Environmental Medicine, Division of Toxicology, Karolinska Institutet, Box 210, Stockholm SE-17177, Sweden.

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.
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http://dx.doi.org/10.1016/j.mam.2009.12.004DOI Listing
February 2010

Clinical perspectives of cell death: where we are and where to go...

Apoptosis 2009 Apr;14(4):333-5

Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden.

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http://dx.doi.org/10.1007/s10495-009-0326-xDOI Listing
April 2009

Mitochondria as targets for chemotherapy.

Apoptosis 2009 Apr;14(4):624-40

Division of Toxicology, Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden.

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.
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http://dx.doi.org/10.1007/s10495-009-0323-0DOI Listing
April 2009

The Warburg Effect returns to the cancer stage.

Semin Cancer Biol 2009 Feb 31;19(1):1-3. Epub 2008 Dec 31.

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http://dx.doi.org/10.1016/j.semcancer.2008.12.003DOI Listing
February 2009

Mitochondria as targets for cancer chemotherapy.

Semin Cancer Biol 2009 Feb 3;19(1):57-66. Epub 2008 Dec 3.

Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden.

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.
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http://dx.doi.org/10.1016/j.semcancer.2008.11.007DOI Listing
February 2009

Mitochondria in cancer cells: what is so special about them?

Trends Cell Biol 2008 Apr 4;18(4):165-73. Epub 2008 Mar 4.

Institute of Environmental Medicine, Division of Toxicology, Karolinska Institutet, Box 210, Stockholm, SE-171 77, Sweden.

The past decade has revealed a new role for the mitochondria in cell metabolism--regulation of cell death pathways. Considering that most tumor cells are resistant to apoptosis, one might question whether such resistance is related to the particular properties of mitochondria in cancer cells that are distinct from those of mitochondria in non-malignant cells. This scenario was originally suggested by Otto Warburg, who put forward the hypothesis that a decrease in mitochondrial energy metabolism might lead to development of cancer. This review is devoted to the analysis of mitochondrial function in cancer cells, including the mechanisms underlying the upregulation of glycolysis, and how intervention with cellular bioenergetic pathways might make tumor cells more susceptible to anticancer treatment and induction of apoptosis.
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http://dx.doi.org/10.1016/j.tcb.2008.01.006DOI Listing
April 2008

Analysis of mitochondrial dysfunction during cell death.

Curr Protoc Cell Biol 2003 Aug;Chapter 18:Unit 18.5

Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden.

Attempts to identify a common underlying step in the apoptotic program in response to various cytotoxic stimuli have focused on the role of mitochondria in this form of cell death. This unit contains a family of protocols that can be used to assess mitochondrial functions during apoptotic responses. Protocols are included for the collection and analysis of released proteins, for detection of the mitochondrial permeability transition, for measurement of mitochondrial membrane potential, and for preparation of mitochondria from different tissue sources.
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http://dx.doi.org/10.1002/0471143030.cb1805s19DOI Listing
August 2003

Assessment of apoptosis and necrosis by DNA fragmentation and morphological criteria.

Curr Protoc Cell Biol 2001 Nov;Chapter 18:18.3.1-18.3.23

Karolinska Institute, Stockholm, Sweden.

Apoptotic cells share a number of common features, such as phosphatidylserine (PS) exposure, cell shrinkage, chromatin cleavage, nuclear condensation, and formation of pyknotic bodies of condensed chromatin. Necrotic cells exhibit nuclear swelling, chromatin flocculation, loss of nuclear basophilia, breakdown of cytoplasmic structure and organelle function, and cytolysis by swelling. This unit describes some of the techniques most commonly used to detect cell death. A number of assays are used for characterizing and distinguishing apoptosis and necrosis. Morphological assays include trypan blue exclusion, differential staining, and Hoechst staining. Methods to detect chromatin cleavage include TUNEL assays for whole cells and paraffin sections, DNA fragmentation assays using whole cells, assays of total genomic DNA, analysis of DNA fragmentation by agarose gel electrophoresis, phenol extraction of DNA for analysis of fragmentation, a quantitative assay for DNA fragmentation, and detection of DNA fragmentation by pulsed-field gel electrophoresis. A protocol is also provided for Cytospin preparations from cell suspensions.
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http://dx.doi.org/10.1002/0471143030.cb1803s12DOI Listing
November 2001

Reactive oxygen species in mitochondria-mediated cell death.

Authors:
Sten Orrenius

Drug Metab Rev 2007 ;39(2-3):443-55

Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden.

In addition to the well-established role of the mitochondria in energy metabolism, regulation of cell death has recently emerged as a second major function of these organelles. This, in turn, seems to be intimately linked to their role as the major intracellular source of reactive oxygen species (ROS) which are mainly, generated at Complex I and III of the respiratory chain. Excessive ROS production can lead to oxidation of macromolecules and has been implicated in mtDNA mutations, ageing, and cell death. Although mitochondrial dysfunction can cause ATP depletion and necrosis, these organelles are also involved in the regulation of apoptotic cell death by mechanisms, which have been conserved through evolution. Thus, many lethal agents target the mitochondria and cause release of cytochrome c and other pro-apoptotic proteins, which can trigger caspase activation and apoptosis. Taken together, these findings have placed the mitochondria in the focus of current cell death research.
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http://dx.doi.org/10.1080/03602530701468516DOI Listing
October 2007

The mitochondrial TOM complex is required for tBid/Bax-induced cytochrome c release.

J Biol Chem 2007 Sep 16;282(38):27633-9. Epub 2007 Jul 16.

Institute of Environmental Medicine, Division of Toxicology, Karolinska Institutet, SE-171 77 Stockholm, Sweden.

Cytochrome c release from mitochondria is a key event in apoptosis signaling that is regulated by Bcl-2 family proteins. Cleavage of the BH3-only protein Bid by multiple proteases leads to the formation of truncated Bid (tBid), which, in turn, promotes the oligomerization/insertion of Bax into the mitochondrial outer membrane and the resultant release of proteins residing in the intermembrane space. Bax, a monomeric protein in the cytosol, is targeted by a yet unknown mechanism to the mitochondria. Several hypotheses have been put forward to explain this targeting specificity. Using mitochondria isolated from different mutants of the yeast Saccharomyces cerevisiae and recombinant proteins, we have now investigated components of the mitochondrial outer membrane that might be required for tBid/Bax-induced cytochrome c release. Here, we show that the protein translocase of the outer mitochondrial membrane is required for Bax insertion and cytochrome c release.
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http://dx.doi.org/10.1074/jbc.M703155200DOI Listing
September 2007

Mitochondria, oxidative stress and cell death.

Apoptosis 2007 May;12(5):913-22

Institute of Environmental Medicine, Karolinska Institutet, S-171 77 Stockholm, Sweden.

In addition to the well-established role of the mitochondria in energy metabolism, regulation of cell death has recently emerged as a second major function of these organelles. This, in turn, seems to be intimately linked to their role as the major intracellular source of reactive oxygen species (ROS), which are mainly generated at Complex I and III of the respiratory chain. Excessive ROS production can lead to oxidation of macromolecules and has been implicated in mtDNA mutations, ageing, and cell death. Mitochondria-generated ROS play an important role in the release of cytochrome c and other pro-apoptotic proteins, which can trigger caspase activation and apoptosis. Cytochrome c release occurs by a two-step process that is initiated by the dissociation of the hemoprotein from its binding to cardiolipin, which anchors it to the inner mitochondrial membrane. Oxidation of cardiolipin reduces cytochrome c binding and results in an increased level of "free" cytochrome c in the intermembrane space. Conversely, mitochondrial antioxidant enzymes protect from apoptosis. Hence, there is accumulating evidence supporting a direct link between mitochondria, oxidative stress and cell death.
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http://dx.doi.org/10.1007/s10495-007-0756-2DOI Listing
May 2007

HAMLET, protein folding, and tumor cell death.

Biochem Biophys Res Commun 2007 Mar 29;354(1):1-7. Epub 2006 Dec 29.

Trinity College, School of Biochemistry and Immunology, University of Dublin, Dublin 2, Ireland.

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http://dx.doi.org/10.1016/j.bbrc.2006.12.167DOI Listing
March 2007

Mitochondrial oxidative stress: implications for cell death.

Annu Rev Pharmacol Toxicol 2007 ;47:143-83

Institute of Environmental Medicine, Karolinska Institutet, S-171 77 Stockholm, Sweden.

In addition to the established role of the mitochondria in energy metabolism, regulation of cell death has emerged as a second major function of these organelles. This seems to be intimately linked to their generation of reactive oxygen species (ROS), which have been implicated in mtDNA mutations, aging, and cell death. Mitochondrial regulation of apoptosis occurs by mechanisms, which have been conserved through evolution. Thus, many lethal agents target the mitochondria and cause release of cytochrome c and other pro-apoptotic proteins into the cytoplasm. Cytochrome c release is initiated by the dissociation of the hemoprotein from its binding to the inner mitochondrial membrane. Oxidation of cardiolipin reduces cytochrome c binding and increases the level of soluble cytochrome c in the intermembrane space. Subsequent release of the hemoprotein occurs by pore formation mediated by pro-apoptotic Bcl-2 family proteins, or by Ca(2+) and ROS-triggered mitochondrial permeability transition, although the latter pathway might be more closely associated with necrosis. Taken together, these findings have placed the mitochondria in the focus of current cell death research.
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http://dx.doi.org/10.1146/annurev.pharmtox.47.120505.105122DOI Listing
April 2007

The future of toxicology--does it matter how cells die?

Chem Res Toxicol 2006 Jun;19(6):729-33

Institute of Environmental Medicine, Division of Toxicology, Karolinska Institutet, Stockholm, Sweden.

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http://dx.doi.org/10.1021/tx0600651DOI Listing
June 2006