Ionizing radiation but not anticancer drugs causes cell cycle arrest and failure to activate the mitochondrial death pathway in MCF-7 breast carcinoma cells

There is considerable evidence that ionizing radiation (IR) and chemotherapeutic drugs mediate apoptosis through the intrinsic death pathway via the release of mitochondrial cytochrome c and activation of caspases -9 and -3. Here we show that MCF-7 cells that lack caspase-3 undergo a caspase-dependent apoptotic cell death in the absence of DNA fragmentation and alpha-fodrin cleavage following treatment with etoposide or doxorubicin, but not after exposure to IR. Re-expression of caspase-3 restored DNA fragmentation and alpha-fodrin cleavage following drug treatment, but it did not alter the radiation-resistant phenotype of these cells. In contrast to the anticancer drugs, IR failed to induce the intrinsic death pathway in MCF-7/casp-3 cells, an event readily observed in IR-induced apoptosis of HeLa cells. Although IR-induced DNA double-strand breaks were repaired with similar efficiencies in all cell lines, cell cycle analyses revealed a persistent G2/M arrest in the two MCF-7 cell lines, but not in HeLa cells. Together, our data demonstrate that caspase-3 is required for DNA fragmentation and alpha-fodrin cleavage in drug-induced apoptosis and that the intrinsic death pathway is fully functional in MCF-7 cells. Furthermore, they show that the radiation-resistant phenotype of MCF-7 cells is not due to the lack of caspase-3, but is caused by the failure of IR to activate the intrinsic death pathway. We propose (1) different signaling pathways are induced by anticancer drugs and IR, and (2) IR-induced G2/M arrest prevents the generation of an apoptotic signal required for the activation of the intrinsic death pathway.     

Alterations in the apoptotic machinery and their potential role in anticancer drug resistance

Anticancer drugs can potentially kill cells in two fundamentally different ways, by interfering with cellular processes that are essential for maintenance of viability or by triggering an endogenous physiological cell death mechanism. Apoptosis is a form of physiological cell death mediated by caspases, a unique family of intracellular cysteine proteases. Zymogen forms of these proteases are found in virtually all somatic cells, but remain latent until their activation is induced by ligation of specific cell surface receptors (the so-called "death receptors"), by mitochondrial alterations that allow release of cytochrome c and other intermembrane components, or possibly by other mechanisms. Most anticancer drugs activate the mitochondrial pathway. This apoptotic pathway is regulated by pro- and antiapoptotic members of the Bcl-2 family of proteins. Once activated, certain caspases might also be controlled by the inhibitor of apoptosis (IAP) proteins. Alterations in apoptotic pathway components or their regulators have been detected in a variety of cancers, suggesting that loss of the ability of cells to undergo apoptosis might contribute to carcinogenesis. Because cancer therapies such as radiation, glucocorticoids, and chemotherapeutic drugs exert their beneficial effects, at least in part, by inducing apoptosis of cancer cells, the same alterations in apoptotic pathways would be predicted to contribute to resistance. A key issue is whether the direct toxic activity of these treatments is of benefit when neoplastic cells contain changes that diminish their ability to undergo apoptosis.     

Mitochondrion as a novel target of anticancer chemotherapy

Mitochondrial membrane permeabilization is a critical event in the process leading to physiologic or chemotherapy-induced apoptosis (programmed cell death). This permeabilization event is, at least in part, under the control of the permeability transition pore complex (PTPC). Oncoproteins from the Bcl-2 family and tumor suppressor proteins from the Bax family interact with PTPC to inhibit or facilitate membrane permeabilization, respectively. Conventional chemotherapeutic agents elicit mitochondrial permeabilization in an indirect fashion by induction of endogenous effectors that are involved in the physiologic control of apoptosis. However, an increasing number of experimental anticancer drugs, including lonidamine, arsenite, betulinic acid, CD437, and several amphipathic cationic alpha-helical peptides, act directly on mitochondrial membranes and/or on the PTPC. Such agents may induce apoptosis in circumstances in which conventional drugs fail to act because endogenous apoptosis induction pathways, such as those involving p53, death receptors, or apical caspase activation, are disrupted. However, stabilization of the mitochondrial membrane by antiapoptotic Bcl-2-like proteins reduces the cytotoxic potential of most of these drugs. Targeting of specific PTPC components may overcome this Bcl-2-mediated apoptosis inhibition. One strategy involves cross-linking of critical redox-sensitive thiol groups within the PTPC; another involves the use of ligands to the mitochondrial benzodiazepine receptor. Thus, the design of mitochondrion-targeted cytotoxic drugs may constitute a novel strategy for overcoming apoptosis resistance.     

Upregulation of Bcl-2 is involved in the mediation of chemotherapy resistance in human small cell lung cancer cell lines.

Chemotherapeutic drugs eliminate cancer cells by induction of apoptosis. Resistance to chemotherapy is partly due to a decreased apoptosis rate. Here we investigated resistance to anticancer drugs in 9 small cell lung cancer (SCLC) cell lines. Apoptosis was induced by cisplatin, doxorubicin and etoposide and was found to be independent of caspase-8 expression. Since caspase-8 is essential for signal transduction of death receptor-mediated apoptosis, all known death receptor systems are thus not required for drug-induced apoptosis in SCLC. Furthermore, we found that anticancer drugs could activate the mitochondrial pathway of apoptosis without involvement of upstream caspases. Finally, by culturing 3 sensitive cell lines in subtherapeutic concentrations of etoposide, resistant cells were generated that exhibit cross-resistance to cisplatin and doxorubicin. Drug resistance was paralleled by strong upregulation of Bcl-2, which diminished apoptosis by inhibiting the loss of the mitochondrial transmembrane potential and the release of cytochrome c. The role of bcl-2 in these processes was supported by bcl-2 transfection and antisense inhibition. These results indicate that Bcl-2 contributes to drug resistance in SCLC, a finding that has profound therapeutic implications.     

Chemotherapy enhances TNF-related apoptosis-inducing ligand DISC assembly in HT29 human colon cancer cells

Cytokines such as Fas-ligand (Fas-L) and Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL) can induce human colon cancer cell apoptosis through engagement of their death domain receptors. All the cancer cells are not sensitive to these cytokines. We have shown recently that low doses of cytotoxic drugs could restore TRAIL-induced cell death in resistant colon cancer cell lines. The present work further explores the death pathway triggered by the cytotoxic drug/TRAIL combination in HT-29 colon cancer cells (www.alexis-corp.com). Clinically relevant concentrations of cisplatin, doxorubicin and 5-fluorouracil synergize with TRAIL to trigger HT-29 cell death. Activation of this pathway leads to apoptosis that involves both caspases and the mitochondria. An increased recruitment of Fas-associated death domain (FADD) and procaspase-8 to the TRAIL-induced death-inducing signaling complex (DISC) was shown in cells exposed to anticancer drugs. Following caspase-8 activation at the DISC level, the mitochondria-dependent death pathway is activated, as demonstrated by the cleavage of Bid, the dissipation of DeltaPsi(m), the release of mitochondrial proteins in the cytosol and the inhibitory effect of Bcl-2 expression. Importantly, besides mitochondrial potentiation, we show here that cytotoxic drugs sensitize HT-29 colon cancer cells to TRAIL-induced cell death by enhancing FADD and procaspase-8 recruitment to the DISC, a novel mechanism whose efficacy could depend partly on Bcl-2 expression level.     

Caspase-8/FLICE functions as an executioner caspase in anticancer drug-induced apoptosis

Caspase-8 plays an essential role in apoptosis triggered by death receptors. Through the cleavage of Bid, a proapoptotic Bcl-2 member, it further activates the mitochondrial cytochrome c/Apaf-1 pathway. Because caspase-8 can be processed also by anticancer drugs independently of death receptors, we investigated its exact role and order in the caspase cascade. We show that in Jurkat cells either deficient for caspase-8 or overexpressing its inhibitor c-FLIP apoptosis mediated by CD95, but not by anticancer drugs was inhibited. In the absence of active caspase-8, anticancer drugs still induced the processing of caspase-9, -3 and Bid, indicating that Bid cleavage does not require caspase-8. Overexpression of Bcl-x(L) prevented the processing of caspase-8 as well as caspase-9, -6 and Bid in response to drugs, but was less effective in CD95-induced apoptosis. Similar responses were observed by overexpression of a dominant-negative caspase-9 mutant. To further determine the order of caspase-8 activation, we employed MCF7 cells lacking caspase-3. In contrast to caspase-9 that was cleaved in these cells, anticancer drugs induced caspase-8 activation only in caspase-3 transfected MCF7 cells. Thus, our data indicate that, unlike its proximal role in receptor signaling, in the mitochondrial pathway caspase-8 rather functions as an amplifying executioner caspase.     

The histone deacetylase inhibitor trichostatin A induces cell cycle arrest and apoptosis in colorectal cancer cells via p53-dependent and -independent pathways

Many chemotherapeutic agents induce apoptosis via a p53-dependent pathway. However, up to 50% of human cancers have p53 mutation and loss of p53 function. Histone deacetylase inhibitors (HDACIs) are emerging as a potentially important new class of anticancer agents. Here, we report that, Trichostatin A (TSA), a pan-HDAC inhibitor, could induce G2/M cell cycle arrest and apoptosis in both colorectal cancer cell lines with wild-type p53 (HT116 cells) and mutant p53 (HT29 cells), although HCT116 cells had more apoptotic cells than HT29 cells. TSA induces apoptosis in both cell lines via the mitochondrial pathway as indicated by decrease of the mitochondrial membrane potential (MMP) and activation of caspase-3. Additionally, TSA induces expression of the pro-apoptotic protein Bax and decreases the expression of the anti-apoptotic proteins Bcl-2 and Bcl-xL in both cell lines. Bax knockdown by siRNA significantly impaired TSA-induced apoptosis in both cell lines. These data suggest that TSA induces G2/M cell cycle arrest and Bax-dependent apoptosis in colorectal cancer cells (HCT116 cells and HT29 cells) by both p53-dependent and -independent mechanisms. However, cells with normal p53 function are more sensitive to TSA-induced apoptosis.     

The clinical trail of TRAIL

The naturally occurring tumour necrosis factor related apoptosis-inducing ligand (TRAIL) induces apoptosis through two death receptors, death receptor 4 (DR4) and death receptor 5 (DR5), that are expressed on the cell membrane. Binding of the ligand to the death receptors leads to activation of the extrinsic apoptosis pathway. Chemotherapy on the other hand stimulates the intrinsic apoptosis pathway via activation of p53 in response to cellular damage. Many cancer cells have mutations in p53 causing resistance to chemotherapy-induced apoptosis. Concomitant signalling through the extrinsic pathway may overcome this resistance. Moreover, enthusiasm for TRAIL as an anticancer agent is based on the demonstration of rhTRAIL-induced selective cell death in tumour cells and not in normal cells. In this review, we provide an overview of the TRAIL pathway, the physiological role of TRAIL and the factors regulating TRAIL sensitivity. We also discuss the clinical development of novel agents, i.e. rhTRAIL and agonistic antibodies, that activate the death receptors.     

Induction of apoptosis by enediyne antibiotic calicheamicin thetaII proceeds through a caspase-mediated mitochondrial amplification loop in an entirely Bax-dependent manner

Calicheamicin thetaII is a member of the enediyne class of antitumor antibiotics that bind to DNA and induce apoptosis. These compounds differ, however, from conventional anticancer drugs as they bind in a sequence-specific manner noncovalently to DNA and cause sequence-selective oxidation of deoxyriboses and bending of the DNA helix. Calicheamicin is clinically employed as immunoconjugate to antibodies directed against, for example, CD33 in the case of gemtuzumab ozogamicin. Here, we show by the use of the unconjugated drug that calicheamicin-induced apoptosis is independent from death-receptor/FADD-mediated signals. Moreover, calicheamicin triggers apoptosis in a p53-independent manner as shown by the use of p53 knockout cells. Cell death proceeds via activation of mitochondrial permeability transition, cytochrome c release and activation of caspase-9 and -3. The overexpression of Bcl-x(L) or Bcl-2 strongly inhibited calicheamicin-induced apoptosis. Knockout of Bax abrogated cell death after calicheamicin treatment. Thus, the activation of mitochondria and execution of cell death occur through a fully Bax-dependent mechanism. Interestingly, caspase inhibition by the pancaspase-inhibitor zVAD-fmk interfered with mitochondrial activation by calicheamicin. This places caspase activation upstream of the mitochondria and indicates that calicheamicin-triggered apoptosis is enhanced through death receptor-independent activation of the caspase cascade, that is, an amplification loop that is required for full activation of the mitochondrial pathway.     

Cerulenin-induced apoptosis is mediated by disrupting the interaction between AIF and hexokinase II

Fatty acid synthase (FASN) is a key enzyme that plays a critical role in numerous metabolic functions by catalyzing the synthesis for long-chain fatty acids. FASN is highly expressed in various human cancers. This preferential expression makes FASN an attractive target for anticancer therapy. Hexokinase II (HKII) is overexpressed in most cancer cells, and it generally localizes to the outer mitochondrial membrane. Recent studies have demonstrated the protective role of mitochondrial HKII in preservation of mitochondrial integrity. The association of hexokinase with mitochondria has emerged as a powerful mechanism in protecting numerous cell types against cell death. We performed this study to examine the mechanism underlying apoptosis induced by cerulenin and with specific focus on its effect on HKII in ZR-75-1 human breast cancer cells. Additionally, we sought to elucidate whether inhibition of the PI3K/Akt pathway can potentiate the anticancer effect of cerulenin. Here, we showed that cerulenin disrupts the physical association between HKII and AIF, leading to eventual cell death. In addition, LY294002, a PI3K/Akt inhibitor, sensitized ZR-75-1 breast cancer cells to cerulenin-induced apoptosis. Collectively, cerulenin induces apoptosis via disrupting the interaction between AIF and HKII and inhibition of PI3K sensitizes cells to cerulenin-induced apoptosis in ZR-75-1 cells.     

Black tea induces tumor cell apoptosis by Bax translocation, loss in mitochondrial transmembrane potential, cytochrome c release and caspase activation

Recently the anti-cancer role of black tea has gained immense importance. Nevertheless, the signaling pathways underlying black tea-induced tumor cell death are still unknown. Previously we reported that black tea induces Ehrlich's ascites carcinoma (EAC) cell apoptosis by changing the balance between pro-and anti-apoptotic proteins. It is now well accepted that many cell death pathways converge at the mitochondria to decrease mitochondrial transmembrane potential (MTP) thereby releasing apoptogenic proteins and resulting in the activation of effecter caspases responsible for the biochemical and morphological alterations associated with apoptosis. The role of pro-apoptotic protein, Bax, in initiating mitochondrial death cascade has also been established. Here we demonstrate that in culture black tea extract induces EAC apoptosis in a dose-dependent manner--with IC50 at 100 microg/ml. At this dose, intracellular Bax level increases in EAC followed by its translocation from cytosol to mitochondria resulting in loss in MTP. A search for the downstream pathway further reveals that black tea induces mitochondrial cytochrome c release and activates caspases 9 and 3 by 2 pathways, a) independent of and b) dependent on MTP loss. Interestingly, Black tea-induced death signal might probably be amplified through mitochondrial membrane depolarization via a feedback activation loop from caspase 3. All these findings indicate that black tea initiates mitochondrial death cascade in EAC cells and thereby results in EAC apoptosis.     

Sensitization for anticancer drug-induced apoptosis by betulinic Acid

We previously described that betulinic acid (BetA), a naturally occurring pentacyclic triterpenoid, induces apoptosis in tumor cells through the mitochondrial pathway. Here, for the first time, we provide evidence that BetA cooperated with anticancer drugs to induce apoptosis and to inhibit clonogenic survival of tumor cells. Combined treatment with BetA and anticancer drugs acted in concert to induce loss of mitochondrial membrane potential and the release of cytochrome c and Smac from mitochondria, resulting in activation of caspases and apoptosis. Overexpression of Bcl-2, which blocked mitochondrial perturbations, also inhibited the cooperative effect of BetA and anticancer drugs, indicating that cooperative interaction involved the mitochondrial pathway. Notably, cooperation of BetA and anticancer drugs was found for various cytotoxic compounds with different modes of action (e.g., doxorubicin, cisplatin, Taxol, VP16, or actino-mycin D). Importantly, BetA and anticancer drugs cooperated to induce apoptosis in different tumor cell lines, including p53 mutant cells, and also in primary tumor cells, but not in human fibroblasts indicating some tumor specificity. These findings indicate that using BetA as sensitizer in chemotherapy-based combination regimens may be a novel strategy to enhance the efficacy of anticancer therapy, which warrants further investigation.     

Caspase-Dependent Apoptosis Induced by Simarouba Glauca on Human Non-Small-Cell Lung Cancer,A549 Cells

Background: The leaves of Simarouba glauca (S. glauca) have been used as a potential source of anticancer agents in traditional medicine. Attempts have been made to isolate anticancer agents from the leaves of S. glauca. The objective of the present study was to demonstrate the anticancer and apoptotic effect of the leaf extract of petroleum ether (LPE) on human non-small-cell lung cancer A549 cells. Methods: MTT assay was used to investigate the effect of LPE on the viability of A-549 cells. The apoptotic effect of human lung cancer cells was evaluated using fluorescence staining, acridine orange/ ethidium bromide staining, Hoechst staining, flow cytometry analysis, annexin V staining, and caspase assay. Results: The results showed a direct correlation between the dose and the rate of cytotoxicity. Fluorescence staining revealed apoptotic features, such as blebbing and chromatin condensation. Flow cytometry analysis and annexin V staining revealed phosphatidyl serine externalization. Caspase assay confirmed that the extract inhibited cell death. Caspase 3 expressions indicated that the cell death occurred either through the mitochondrial pathway or the death receptor. Conclusions: The study revealed that the LPE induced the apoptosis of human non-small-cell lung cancer, A549 cells, either through mitochondrial or death receptor pathway.     

Anticancer drugs induce increased mitochondrial cytochrome c expression that precedes cell death

Recent studies have demonstrated that cytochrome c plays an important role in cell death. In the present study, we report that teniposide and various other chemotherapeutic agents induced a dose-dependent increase in the expression of the mitochondrial respiratory chain proteins cytochrome c, subunits I and IV of cytochrome c oxidase, and the free radical scavenging enzyme manganous superoxide dismutase. The teniposide-induced increase of cytochrome c was inhibited by cycloheximide, indicating new protein synthesis. Elevated cytochrome c levels were associated with enhanced cytochrome c oxidase-dependent oxygen uptake using TMPD/ascorbate as the electron donor, suggesting that the newly synthesized proteins were functional. Cytochrome c was released into the cytoplasm only after maximal levels had been reached in the mitochondria, but there was no concomitant decrease in mitochondrial membrane potential or caspase activation. Our results suggest that the increase in mitochondrial protein expression may play a role in the early cellular defense against anticancer drugs.     

Caspase-dependent and -independent cell death pathways after DNA damage (Review)

Apoptosis is known to be an important phenomenon in exerting antitumor response to cancer therapy, which is regulated by Bcl-2 family proteins through mitochondrial permeability transition (MPT). Insertion by the activated Bax/Bak in response to DNA damage induces mitochondrial membrane permeabilization (MMP) via an anion channel, VDAC in mitochondrial outer membrane that plays a crucial role in releasing small molecules such as cytochrome c, Smac/DIABLO, Omi/HtrA2, AIF, and endonuclease G leading to cell death. The released small molecules are involved in caspase-dependent and -independent cell death pathway that is inhibited by Bcl-2/xL. Despite the fact that the pancaspase inhibitor, zVAD-fmk inhibited the caspase cascade, cell death mediated by caspase-independent pathway was not blocked. Similarly, although etoposide induced-apoptosis was inhibited in Bax(-/-)/Bak(-/-)mouse embryonic fibroblasts, autophagy was not inhibited, which was regulated by Bcl-xL. It appears that the cross-talk between caspase-dependent and -independent apoptotic cell death including autophagic cell death that was mediated by MPT affects overall tumor response to anticancer treatment. In this review, to assist a comprehensive understanding of MPT-mediated cell death pathway for exploring appropriate targets in cancer therapy, role of the caspase-dependent and -independent cell death pathway in the interaction of these pathways is discussed.     

GoldIII porphyrin 1a induced apoptosis by mitochondrial death pathways related to reactive oxygen species

Apoptosis is a tightly controlled multistep mechanism of cell death, and mitochondria are considered to play a central role in this process. Mitochondria initiate two distinct apoptosis pathways, one caspase-dependent and the other caspase-independent. In addition, mitochondrial production of reactive oxygen species (ROS) seems to play a role in cell death. Most chemotherapeutic agents induce apoptosis through at least one of these pathways. The post-initiation mechanisms of gold(III) porphyrin 1a were investigated in this study. HONE1 cells exposed to gold(III) porphyrin 1a underwent apoptosis after 24 hours. Functional proteomic studies revealed the alteration of several cytoplasmic protein expressions in HONE1 cells after treatment with the drug. These proteins include enzymes participating in energy production and proteins involved in cellular redox balance. There was a quick attenuation of mitochondrial membrane potential (DeltaPsi(m)) with the alterations of Bcl-2 family proteins, the release of cytochrome c, and apoptosis-inducing factor (AIF) following gold(III) porphyrin 1a treatment. Cytochrome c in turn activated caspase-9 and caspase-3. Cotreatment with caspase inhibitor (zVAD-fmk) showed that the activated caspases worked in conjunction with AIF-initiated apoptosis pathways. Further study showed that ROS played a part in gold(III) porphyrin 1a-induced apoptosis by regulating DeltaPsi(m). In summary, gold(III) porphyrin 1a induced apoptosis through both caspase-dependent and caspase-independent mitochondrial pathways, and intracellular oxidation affected gold(III) porphyrin 1a-induced apoptosis. These results support a role for gold(III) porphyrin 1a as a promising anticancer drug lead and as a possible novel therapeutic agent directed toward the mitochondria.     

Apoptosis regulators as targets for cancer therapy

Apoptosis serves to remove excess or damaged cells and its dysregulation may lead to a number of pathological disorders including cancer. Studies during the last 20 years have unravelled much of the molecular mechanisms that control apoptosis. Whether a cell dies in response to diverse apoptotic stimuli, including DNA-damaging agents, is determined largely by interactions between proteins of the Bcl-2 family. A death signal is transmitted through the BH3-only proteins to Bax and Bak which in turn permeabilise the outer mitochondrial membrane allowing the release of apoptogenic factors, which triggers activation of cell-deathpromoting caspases. These proteolytic enzymes are tightly controlled by members of the inhibitor of apoptosis (IAP) family. Activation of the caspase cascade via cell death receptors also represents a key apoptotic pathway in both normal and tumour cells. Basic knowledge of these apoptosis regulators provides the basis for novel therapeutic strategies aimed at promoting tumour cell death or enhancing susceptibility to apoptotic inducers. This review focuses on these strategies.     

Induction of Human Hepatocellular Carcinoma HepG2 Cell Apoptosis by Naringin

Naringin, a bioflavonoid found in Citrus seeds, inhibits proliferation of cancer cells. The objectives of this study were to investigate the mode and mechanism(s) of hepatocellular carcinoma HepG2 cell death induced by naringin. The cytotoxicity of naringin towards HepG2 cells proved dosedependent, measured by MTT assay. Naringintreated HepG2 cells underwent apoptosis also in a concentration related manner, determined by annexin Vfluorescein isothiocyanate (FITC) and propidium iodide (PI) employing flow cytometry. Mitochondrial transmembrane potential (MTP) measured using 3,3dihexyloxacarbocyanine iodide (DiOC6) and flow cytometer was reduced concentrationdependently, which indicated influence on the mitochondrial signaling pathway. Caspase3, 8 and 9 activities were enhanced as evidenced by colorimetric detection of paranitroaniline tagged with a substrate for each caspase. Thus, the extrinsic and intrinsic pathways were linked in human naringintreated HepG2 cell apoptosis. The expression levels of proapoptotic Bax and Bak proteins were increased whereas that of the antiapoptotic BclxL protein was decreased, confirming the involvement of the mitochondrial pathway by immunoblotting. There was an increased expression of truncated Bid (tBid), which indicated caspase8 proteolysis activity in Bid cleavage as its substrate in the extrinsic pathway. In conclusion, naringin induces human hepatocellular carcinoma HepG2 cell apoptosis via mitochondriamediated activation of caspase9 and caspase8mediated proteolysis of Bid. Naringin anticancer activity warrants further investigation for application in medical treatment.     

Arsenic trioxide triggers a regulated form of caspase-independent necrotic cell death via the mitochondrial death pathway

Cell death is generally believed to occur either by accidental, lytic necrosis or by programmed cell death, that is, apoptosis. The initiation and execution of cell death, however, is far more complex and includes pathways like caspase-independent apoptosis or actively triggered necrosis. In this study, we investigated the mechanisms of cell death induced by arsenic trioxide (arsenite, As2O3), a clinically efficient agent in anticancer therapy. As2O3-induced cell death coincides with cytochrome c release, facilitates mitochondrial permeability transition and is sensitive to inhibition by Bcl-x(L), indicating that cell demise is regulated through the mitochondrial apoptosis pathway. Nevertheless, only little caspase-3 activation was observed and As2O3-induced cell death was only weakly obstructed by the broad spectrum caspase inhibitor z-VAD-fmk. Moreover, disruption of caspase-9 or -2 failed to decrease the amount of As2O3-mediated cell death. Interestingly, As2O3-induced cell death had a predominantly necrosis-like phenotype as assessed by Annexin-V/propidium iodide staining and LDH release. Finally, blocking glutathione synthetase by buthionine sulfoximine enhanced the As2O3-mediated necrosis-like cell death without increasing caspase-3 cleavage. As2O3 does, however, not directly inhibit caspases, but appears to interfere with caspase activation. Altogether, our data clearly delineate a mode of As2O3-triggered cell death that differs considerably from that induced by conventional anticancer drugs. These findings may explain the capability of As2O3 to efficiently kill even chemoresistant tumor cells with disturbed apoptosis signaling and caspase activation, a frequent finding in malignancy.