Spencer B. Gibson

Oxidative stress induces autophagic cell death independent of apoptosis in transformed and cancer Cells

Autophagy is a self-digestion process that degrades intracellular structures in response to stresses leading to cell survival. When autophagy is prolonged, this could lead to cell death. Generation of reactive oxygen species (ROS) through oxidative stress causes cell death. The role of autophagy in oxidative stress-induced cell death is unknown. In this study, we report that two ROS-generating agents, hydrogen peroxide (H2O2) and 2-methoxyestradiol (2-ME), induced autophagy in the transformed cell line HEK293 and the cancer cell lines U87 and HeLa. Blocking this autophagy response using inhibitor 3-methyladenine or small interfering RNAs against autophagy genes, beclin-1, atg-5 and atg-7 inhibited H2O2 or 2-ME-induced cell death. H2O2 and 2-ME also induced apoptosis but blocking apoptosis using the caspase inhibitor zVAD-fmk (benzyloxycarbonyl-Val-Ala-Asp fluoromethylketone) failed to inhibit autophagy and cell death suggesting that autophagy-induced cell death occurred independent of apoptosis. Blocking ROS production induced by H2O2 or 2-ME through overexpression of manganese-superoxide dismutase or using ROS scavenger 4,5-dihydroxy-1,3-benzene disulfonic acid-disodium salt decreased autophagy and cell death. Blocking autophagy did not affect H2O2- or 2-ME-induced ROS generation, suggesting that ROS generation occurs upstream of autophagy. In contrast, H2O2 or 2-ME failed to significantly increase autophagy in mouse astrocytes. Taken together, ROS induced autophagic cell death in transformed and cancer cells but failed to induce autophagic cell death in non-transformed cells.

Superoxide is the major reactive oxygen species regulating autophagy.

Autophagy is involved in human diseases and is regulated by reactive oxygen species (ROS) including superoxide (O2•−) and hydrogen peroxide (H2O2). However, the relative functions of O2•− and H2O2 in regulating autophagy are unknown. In this study, autophagy was induced by starvation, mitochondrial electron transport inhibitors, and exogenous H2O2. We found that O2•− was selectively induced by starvation of glucose, L-glutamine, pyruvate, and serum (GP) whereas starvation of amino acids and serum (AA) induced O2•− and H2O2. Both types of starvation induced autophagy and autophagy was inhibited by overexpression of SOD2 (manganese superoxide dismutase, Mn-SOD), which reduced O2•− levels but increased H2O2 levels. Starvation-induced autophagy was also inhibited by the addition of catalase, which reduced both O2•− and H2O2 levels. Starvation of GP or AA also induced cell death that was increased following treatment with autophagy inhibitors 3-methyladenine, and wortamannin. Mitochondrial electron transport chain (mETC) inhibitors in combination with the SOD inhibitor 2-methoxyestradiol (2-ME) increased O2•− levels, lowered H2O2 levels, and increased autophagy. In contrast to starvation, cell death induced by mETC inhibitors was increased by 2-ME. Finally, adding exogenous H2O2 induced autophagy and increased intracellular O2•− but failed to increase intracellular H2O2. Taken together, these findings indicate that O2•− is the major ROS-regulating autophagy.

Mitochondrial electron-transport-chain inhibitors of complexes I and II induce autophagic cell death mediated by reactive oxygen species

Autophagy is a self-digestion process important for cell survival during starvation. It has also been described as a form of programmed cell death. Mitochondria are important regulators of autophagy-induced cell death and damaged mitochondria are often degraded by autophagosomes. Inhibition of the mitochondrial electron transport chain (mETC) induces cell death through generating reactive oxygen species (ROS). The role of mETC inhibitors in autophagy-induced cell death is unknown. Herein, we determined that inhibitors of complex I (rotenone) and complex II (TTFA) induce cell death and autophagy in the transformed cell line HEK 293, and in cancer cell lines U87 and HeLa. Blocking the expression of autophagic genes (beclin 1 and ATG5) by siRNAs or using the autophagy inhibitor 3-methyladenine (3-MA) decreased cell death that was induced by rotenone or TTFA. Rotenone and TTFA induce ROS production, and the ROS scavenger tiron decreased autophagy and cell death induced by rotenone and TTFA. Overexpression of manganese-superoxide dismutase (SOD2) in HeLa cells decreased autophagy and cell death induced by rotenone and TTFA. Furthermore, blocking SOD2 expression by siRNA in HeLa cells increased ROS generation, autophagy and cell death induced by rotenone and TTFA. Rotenone- and TTFA-induced ROS generation was not affected by 3-MA, or by beclin 1 and ATG5 siRNAs. By contrast, treatment of non-transformed primary mouse astrocytes with rotenone or TTFA failed to significantly increase levels of ROS or autophagy. These results indicate that targeting mETC complex I and II selectively induces autophagic cell death through a ROS-mediated mechanism.

Increased Expression of Death Receptors 4 and 5 Synergizes the Apoptosis Response to Combined Treatment with Etoposide and TRAIL

ABSTRACT Chemotherapeutic genotoxins induce apoptosis in epithelial-cell-derived cancer cells. The death receptor ligand TRAIL also induces apoptosis in epithelial-cell-derived cancer cells but generally fails to induce apoptosis in nontransformed cells. We show here that the treatment of four different epithelial cell lines with the topoisomerase II inhibitor etoposide in combination with TRAIL (tumor necrosis factor [TNF]-related apoptosis-inducing ligand) induces a synergistic apoptotic response. The mechanism of the synergistic effect results from the etoposide-mediated increase in the expression of the death receptors 4 (DR4) and 5 (DR5). Inhibition of NF-κB activation by expression of kinase-inactive MEK kinase 1(MEKK1) or dominant-negative IκB (ΔIκB) blocked the increase in DR4 and DR5 expression following etoposide treatment. Addition of a soluble decoy DR4 fusion protein (DR4:Fc) to cell cultures reduced the amount of etoposide-induced apoptosis in a dose-dependent manner. The addition of a soluble TNF decoy receptor (TNFR:Fc) was without effect, demonstrating the specificity of DR4 binding ligands in the etoposide-induced apoptosis response. Thus, genotoxin treatment in combination with TRAIL is an effective inducer of epithelial-cell-derived tumor cell apoptosis relative to either treatment alone.

HYPOXIA INDUCES AUTOPHAGIC CELL DEATH IN APOPTOSIS-COMPETENT CELLS THROUGH A MECHANISM INVOLVING BNIP3

Hypoxia (lack of oxygen) is a physiological stress often associated with solid tumors. Hypoxia correlates with poor prognosis since hypoxic regions within tumors are considered apoptosis-resistant. Autophagy (cellular “self digestion”) has been associated with hypoxia during cardiac ischemia and metabolic stress as a survival mechanism. However, although autophagy is best characterized as a survival response, it can also function as a mechanism of programmed cell death. Our results show that autophagic cell death is induced by hypoxia in cancer cells with intact apoptotic machinery. We have analyzed two glioma cell lines (U87, U373), two breast cancer cell lines (MDA-MB-231, ZR75) and one embryonic cell line (HEK293) for cell death response in hypoxia (

Reovirus-Induced Apoptosis Is Mediated by TRAIL

ABSTRACT Members of the tumor necrosis factor (TNF) receptor superfamily and their activating ligands transmit apoptotic signals in a variety of systems. We now show that the binding of TNF-related, apoptosis-inducing ligand (TRAIL) to its cellular receptors DR5 (TRAILR2) and DR4 (TRAILR1) mediates reovirus-induced apoptosis. Anti-TRAIL antibody and soluble TRAIL receptors block reovirus-induced apoptosis by preventing TRAIL-receptor binding. In addition, reovirus induces both TRAIL release and an increase in the expression of DR5 and DR4 in infected cells. Reovirus-induced apoptosis is also blocked following inhibition of the death receptor-associated, apoptosis-inducing molecules FADD (for FAS-associated death domain) and caspase 8. We propose that reovirus infection promotes apoptosis via the expression of DR5 and the release of TRAIL from infected cells. Virus-induced regulation of the TRAIL apoptotic pathway defines a novel mechanism for virus-induced apoptosis.

Is mitochondrial generation of reactive oxygen species a trigger for autophagy

Autophagy is a conserved lysosomal degradation pathway that has been extensively studied in recent years. However, the mechanism of autophagy induction is still not clear. Mitochondria are important regulators of both apoptosis and autophagy. One of the triggers for mitochondrial mediated apoptosis is the production of reactive oxygen species (ROS). Recently, several studies have indicated that ROS may be also involved in induction of autophagy. ROS are molecules or ions that are formed by the incomplete one-electron reduction of oxygen, including superoxide (O2.-), hydrogen peroxide (H2O2), hydroxyl radical (·OH), nitric oxide (NO), and peroxynitrite (ONOO-). Our recent studies provide strong evidences for the involvement of mitochondrially-generated ROS production in the induction of autophagy as determined by the formation of autophagosomes and autolysosomes. This was accomplished through treatment with mitochondrial toxins that inhibit the electron transport chain in transformed and cancer cells. In addition, we have determined that H2O2 and 2-methoxyestradiol (inhibitor of superoxide dismutases and electron transport chain) induce autophagy leading to cell death. In contrast, normal astrocytes fail to induce autophagy following treatment with mitochondrial toxins. Herein, we discuss several important points of our studies and provide a model for mitochondrially-induced autophagic cell death mediated by ROS. Addendum to: Chen Y, McMillan-Ward E, Kong J, Israels SJ, Gibson SB. Mitochondrial Electron-Transport-Chain Inhibitors of Complexes I and II Induce Autophagic Cell Death Mediated by Reactive Oxygen Species. J Cell Sci 2007; 120:4155-66.

BNIP3 plays a role in hypoxic cell death in human epithelial cells that is inhibited by growth factors EGF and IGF

Hypoxic regions within solid tumors are often resistant to chemotherapy and radiation. BNIP3 (Bcl-2/E1B 19 kDa interacting protein) is a proapoptotic member of the Bcl-2 family that is expressed in hypoxic regions of tumors. During hypoxia, BNIP3 expression is increased in many cell types and upon forced overexpression BNIP3 induces cell death. Herein, we have demonstrated that blockage of hypoxia-induced BNIP3 expression using antisense oligonucleotides against BNIP3 or blockage of BNIP3 function through expression of a mutant form of BNIP3 inhibits hypoxia-induced cell death in human embryonic kidney 293 cells. We have also determined that hypoxia-mediated BNIP3 expression is regulated by the transcription factor, hypoxia-inducible factor-1α (HIF-1α) in human epithelial cell lines. Furthermore, HIF-1α directly binds to a consensus HIF-1α-responsive element (HRE) in the human BNIP3 promoter that upon mutation of this HRE site eliminates the hypoxic responsiveness of the promoter. Since BNIP3 is expressed in hypoxic regions of tumors but fails to induce cell death, we determined whether growth factors block BNIP3-induced cell death. Treatment of the breast cancer cell line MCF-7 cells with epidermal growth factor (EGF) or insulin-like growth factor effectively protected these cells from BNIP3-induced cell death. Furthermore, inhibiting EGF receptor signaling using antibodies against ErbB2 (Herceptin) resulted in increased hypoxia-induced cell death in MCF-7 cells. Taken together, BNIP3 plays a role in hypoxia-induced cell death in human epithelial cells that could be circumvented by growth factor signaling.

Anti-apoptotic versus pro-apoptotic signal transduction: checkpoints and stop signs along the road to death.

The activation of caspases is a final commitment step for apoptosis. It is now evident that signal transduction pathways involving specific protein kinases modulate the apoptotic response. Both pro-apoptotic and anti-apoptotic pathways integrate environmental cues that control the decision to undergo apoptosis. Pro- and anti-apoptotic signal pathways regulate the activation of the caspases. In this review we describe our current understanding of apoptotic signal transduction.

Transcription Factor NF-κB Differentially Regulates Death Receptor 5 Expression Involving Histone Deacetylase 1

ABSTRACT The transcription factor nuclear factor κB (NF-κB) regulates the expression of both antiapoptotic and proapoptotic genes. Death receptor 5 (DR5, TRAIL-R2) is a proapoptotic protein considered to be a potential target for cancer therapy, and its expression is mediated by NF-κB. The mechanism of NF-κB-induced DR5 expression is, however, unknown. Herein, we determined that etoposide-induced DR5 expression requires the first intronic region of the DR5 gene. Mutation of a putative NF-κB binding site in this intron eliminates DR5 promoter activity, as do mutations in the p53 binding site in this region. Reduction in p53 expression also blocks p65 binding to the intronic region of the DR5 gene, indicating cooperation between p53 and p65 in DR5 expression. In contrast, the antiapoptotic stimulus, epidermal growth factor (EGF), fails to increase DR5 expression but effectively activates NF-κB and induces p65 binding to the DR5 gene. EGF, however, induces the association of histone deacetylase 1 (HDAC1) with the DR5 gene, whereas etoposide treatment fails to induce this association. Indeed, HDAC inhibitors activate NF-κB and p53 and upregulate DR5 expression. Blockage of DR5 activation decreased HDAC inhibitor-induced apoptosis, and a combination of HDAC inhibitors and TRAIL increased apoptosis. This provides a mechanism for regulating NF-κB-mediated DR5 expression and could explain the differential roles NF-κB plays in regulating apoptosis.