As2O3-induced Cell Death Research Articles

Arsenic trioxide promotes ERK1/2-mediated phosphorylation and degradation of BIMEL to attenuate apoptosis in BEAS-2B cells

Inorganic arsenic is highly toxic, widely distributed in the human environment and may result in multisystem diseases and several types of cancers. The BCL-2-interacting mediator of cell death protein (BIM) is a key modulator of the intrinsic apoptosis pathway. Interestingly, in the present study, we found that arsenic trioxide (As2O3) decreased BIMEL levels in human bronchial epithelial cell line BEAS-2B and increased BIMEL levels in human lung carcinoma cell line A549 and mouse Sertoli cell line TM4. Mechanismly, the 26S proteasome inhibitors MG132 and bortezomib could effectively inhibit BIMEL degradation induced by As2O3 in BEAS-2B cells. As2O3 activated extracellular signal-regulated kinase (ERK) 1/2, c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) signaling pathways, but only the ERK1/2 MAPK inhibitor PD98059 blocked BIMEL degradation induced by As2O3. Furthermore, As2O3 induced-phosphorylation of BIMEL at multiple sites was inhibited by ERK1/2 MAPK inhibitor PD98059. Inhibition of As2O3-induced ERK1/2 MAPK phosphorylation increased the levels of BIMEL and cleaved-caspase-3 proteins and decreased BEAS-2B cell viability. As2O3 also markedly mitigated tunicamycin-induced apoptosis of BEAS-2B cells by increasing ERK1/2 phosphorylation and BIMEL degradation. Our results suggest that As2O3-induced activation of the ERK1/2 MAPK pathway increases phosphorylation of BIMEL and promotes BIMEL degradation, thereby alleviating the role of apoptosis in As2O3-induced cell death. This study provides new insights into how to maintain the survival of BEAS-2B cells before malignant transformation induced by high doses of As2O3.

Taurine attenuates arsenic-induced pyroptosis and nonalcoholic steatohepatitis by inhibiting the autophagic-inflammasomal pathway

Arsenic exposure causes nonalcoholic steatohepatitis (NASH). Inflammation is a key contributor to the pathology of nonalcoholic fatty liver disease (NAFLD), including NASH. However, it is unclear how arsenic induces inflammation. In mouse livers, we show that arsenic trioxide (As2O3) induced NASH, increased autophagy and NLRP3 inflammasome activation, increased lipid accumulation, and resulted in dysregulation of lipid-related genes. Supplemented with taurine (Tau) attenuated the inflammation and autophagy caused by As2O3. In HepG2 cells, we found that As2O3-induced pyroptotic cell death was dependent upon the activation of NLRP3 inflammasome, which was CTSB-dependent. In addition, inhibiting autophagy alleviated the As2O3-induced increase of cytosolic CTSB expression and subsequent release of LDH, activation of the NLRP3 inflammasome, and pyroptosis. Moreover, we found that Tau alleviated As2O3-induced elevation of autophagy, CTSB expression, and activation of the NLRP3 inflammasome, and reduced the release of LDH, pyroptotic cell death, and inflammation. Interestingly, As2O3-induced lipid accumulation could not be alleviated by either inhibition of autophagy nor by inhibition of CTSB. Additionally, neither inhibition of the NLRP3 inflammasome or Tau treatment could alleviate lipid accumulation. These results demonstrated that As2O3-induced pyroptosis involves autophagy, CTSB, and the NLRP3 inflammasome cascade, and that Tau alleviates As2O3-induced liver inflammation by inhibiting the autophagic-CTSB-NLRP3 inflammasomal pathway rather than decreasing lipid accumulation. These findings give insight into the association of autophagy, inflammation, pyroptosis, and NASH induced by As2O3.

Differential mechanistic investigation of protective effects from imperatorin and sec-O-glucosylhamaudol against arsenic trioxide-induced cytotoxicity in vitro

Background and purposeThe clinical use of arsenic trioxide (As2O3) for treating acute promyelocytic leukemia (APL) is limited due to its severe cardiotoxicity. The possible mechanisms of As2O3-induced cardiotoxicity include DNA fragmentation, reactive oxygen species (ROS) generation, cardiac ion channel changes and apoptosis. The present study is designed to investigate the protective effects of imperatorin and sec-O-glucosylhamaudol and to explore their mechanistic involvement in As2O3-induced cytotoxicity. Experimental methodsCell viability assay, Lactate dehydrogenase (LDH) release, Acridine orange/ethidium bromide (AO/EB) double staining, Caspase-3 activity assay, ROS generation, cellular calcium levels, mRNA expression levels by qRT-PCR and protein expression levels by Western blotting were measured in H9c2 cells in combination with As2O3 and imperatorin or sec-O-glucosylhamaudol. Key resultsWe observed that H9c2 cells treated with imperatorin or sec-O-glucosylhamaudol were more resistant to As2O3-induced cell death. Both imperatorin and sec-O-glucosylhamaudol reduced H9c2 cell apoptosis, but both imperatorin and sec-O-glucosylhamaudol had no effects on Caspase-3 activity and intracellular calcium accumulation. Furthermore, imperatorin was capable of suppressing ROS generation, while sec-O-glucosylhamaudol did not show this effect. Moreover, imperatorin and sec-O-glucosylhamaudol triggered Nrf2 activation, which resulted in upregulation of downstream phase II metabolic enzymes and antioxidant protein/enzyme, probably offering cellular protection to As2O3-induced cardiotoxicity via the Nrf2 signal pathway. Conclusions and implicationsImperatorin and sec-O-glucosylhamaudol can ameliorate As2O3-induced cytotoxicity and apoptosis in H9c2 cells, the mechanisms probably related to antioxidation. As2O3 in combination with imperatorin or sec-O-glucosylhamaudol could be considered as a novel strategy to expand the clinical application of As2O3.

Abstract 590: Enhancing effect of resveratrol on the antileukemic effects of arsenic trioxide.

Abstract Arsenic trioxide (As2O3) has been approved by the FDA for the treatment of APL, in which it has major clinical activity. However, the requirement of high doses to generate antileukemic effects has been a limitation for As2O3‘s potential use in other subtypes of AML. Given As2O3‘s potent antileukemic properties in vitro, approaches to enhance its effects are of clinical and translational relevance. Resveratrol (RSV), a phytoalexin found naturally in grapes and other plants, has wide-ranging health benefits, and in leukemias, has been shown to induce apoptosis and act synergistically with other agents. In studies to examine the influence of RSV on As2O3‘s antileukemic effects, we found that RSV enhances As2O3-induced cell death in KT1 cells as shown by flow cytometric and MTT analyses to assess apoptosis and cell viability. Our studies demonstrate that combined As2O3 and RSV treatment significantly reduces leukemic progenitor colony formation in a dose dependent manner by K562, KT1, and U937 cell lines. It also results in enhanced suppressessive effects on primary leukemic progenitors from patients with myeloid leukemias. Altogether, our results suggest that use of RSV may provide an approach to enhance the antileukemic effects of As2O3 treatment by potentiating the effects of lower As2O3 doses. Citation Format: Edward J. Wu, Dennis J. Goussetis, Leonidas C. Platanias. Enhancing effect of resveratrol on the antileukemic effects of arsenic trioxide. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 590. doi:10.1158/1538-7445.AM2013-590

Carbonyl Reductase 1 Offers a Novel Therapeutic Target to Enhance Leukemia Treatment by Arsenic Trioxide

Arsenic trioxide (As2O3) is used, in current practice, as an effective chemotherapeutic agent for acute promyelocytic leukemia (APL). However, the side effects and relatively low efficacy of As2O3 in treating other leukemias have limited its wider use in therapeutic applications. In the present study, we found that the expression of carbonyl reductase 1 (CBR1) affects the resistance to As2O3 in leukemias, including APL; As2O3 upregulated CBR1 expression at the transcriptional level by stimulating the activity of the transcription factor activator protein-1. Moreover, CBR1 overexpression was sufficient to protect cells against As2O3 through modulation of the generation of reactive oxygen species, whereas the attenuation of CBR1 was sufficient to sensitize cells to As2O3. A combination treatment with the specific CBR1 inhibitor hydroxy-PP-Me remarkably increased As2O3-induced apoptotic cell death compared with As2O3 alone, both in vitro and in vivo. These results were confirmed in primary cultured human acute and chronic myeloid leukemia cells, with no significant cell death observed in normal leukocytes. Taken together, our findings indicate that CBR1 contributes to the low efficacy of As2O3 and, therefore, is a rational target for the development of combination chemotherapy with As2O3 in diverse leukemias including APL.

Arsenic trioxide inhibits the growth of human glioma stem cells through activating the JNK pathway

Malignant gliomas are the most common and aggressive primary brain tumor. A major barrier to the treatment of glioma is the drug resistance. The existence of glioma stem cells may be responsible for drug resistance. Recent clinical data show that arsenic trioxide (As2O3) causes remission in patients with acute promyelocytic leukemia without severe side effects. However, it is still unknown that whether As2O3 could inhibit the growth of glioma stem cells. Glioma stem cells are also called glioma stem/progenitor cells (GSPCs). The GSPCs are a population of cells capable of extensive self-renewal, differentiation into multiple lineages, initiated the original tumor in immunodeficient mice and often co-expressed differentiation surface marker with the stem/progenitor cell marker nestin. In this study, we cultivated GSPCs, which were established from the excised tumor tissue of a patient, and showed that As2O3 could inhibit the proliferation of GSPCs. Treatment of GSPCs for 24 h, 48 h and 72 h with As2O3 induced cell death in a dose- and timedependent manner. Subsequently, we demonstrated that the As2O3-induced GSPCs death was due to cell cycle arrest at the G0/G1 transition and was associated with activation of c-jun N-terminal kinase (JNK) pathway. The findings underscore the potential of As2O3 to inhibit the proliferation of GSPCs through the JNK pathway and suggest its application to the effective therapy for patients with glioma.

The role of p38 MAPK and JNK in Arsenic trioxide‐induced mitochondrial cell death in human cervical cancer cells

Previously, we have shown that the release of AIF from mitochondria is required for As2O3-induced cell death in human cervical cancer cells, and that reactive oxygen species (ROS) is necessary for AIF release from mitochondria. In this study, we further investigated the role of MAPKs in ROS-mediated mitochondrial apoptotic cell death triggered by As2O3. As2O3-induced apoptotic cell death in HeLa cells was associated with activation and mitochondrial translocation of Bax, a marked phosphorylation of Bcl-2, reduction of Bcl-2 and Bax interaction, dissipation of mitochondrial membrane potential. Using small interfering RNA, reduced Bax expression effectively attenuated As2O3-induced mitochondrial membrane potential loss and apoptotic cell death. Moreover, the phosphorylation of Bcl-2 induced by As2O3 diminished its ability to bind to Bax. Treatment of cells with As2O3 activated both the p38 MAPK and JNK pathways. Mitochondrial translocation of Bax was completely suppressed in the presence of p38 MAPK inhibitor PD169316 or si-p38 MAPK. The As2O3-induced Bcl-2 phosphorylation was attenuated largely by JNK inhibition using SP600125 or si-JNK and to some extent by p38 MAPK inhibition with PD169316 or si-p38 MAPK. In addition, N-acetyl-L-cystein (NAC), a thiol-containing anti-oxidant, completely blocked As2O3-induced p38 MAPK and JNK activations, mitochondria translocation of Bax, and phosphorylation of Bcl-2. These results support a notion that ROS-mediated activations of p38 MAPK and JNK in response to As2O3 treatment signals activation of Bax and phosphorylation of Bcl-2, resulting in mitochondrial apoptotic cell death in human cervical cancer cells.

Catalase activity and arsenic sensitivity in acute leukemia

Arsenic trioxide (As2O3) is currently employed as a treatment for relapsed acute promyelocytic leukemia (APL), where it can induce remission in greater than 90% of patients, but is ineffective in patients with non-APL acute myeloid leukemia (AML). As2O3 induces apoptosis in APL cells through mechanisms dependent and independent of the PML-RARα fusion protein. Through PML-RARα fusion-independent mechanisms, As2O3 increases H2O2 production via its effects on glutathione and glutathione peroxidase. Catalase is an alternative mechanism to convert H2O2 to water. Therefore, we explored the relationship between catalase activity and As2O3 sensitivity. In AML and APL cell lines, but not primary patient samples, basal catalase levels matched sensitivity to As2O3. However, the chemical inhibition of catalase did not enhance As2O3-induced cell death. Failure of catalase inhibition to sensitise cells to As2O3 was due to a failure of catalase inhibition to increased levels of reactive oxygen species. Therefore, other strategies should be explored to enhance the cytotoxicity of As2O3 in AML.

Arsenic Trioxide (As2O3) Mediated Cell Death in Hodgkin Lymphoma (HL) and Non-Hodgkin Lymphoma (NHL): Investigation of Reactive Oxygen Species (ROS), Caspase, and MAP Kinase Signaling Pathways.

The cytotoxic activity of As2O3 in leukemia and myeloma has been shown to be mediated in part by ROS. We determined the mechanism of cytotoxicity of As2O3 in HL and NHL cell lines. Ramos, HF-1, SUDHL4 (NHL lines), and L428 (HL line) cells were cultured with increasing clinically relevant As2O3 concentrations (1.0mm-10mm) at 24-72 hours with and without the oxidizing agent, buthionine sulfoxime (BSO, 100mm), and with and without caspase inhibitors Z-VAD-FMK and Z-IETD-FMK. Apoptosis was measured by Annexin-V/propidium iodide (PI) staining and detected by flow cytometry (FACS). ROS was measured by oxidation of 2′7′ dichlorofluorescein diacetate (H2DCFDA) to Dichlorofluorescein (DCF) and analyzed by FACS. Immunoblots were performed for bcl-2, PARP, cleaved caspases 3, 8, 9, and mitogen activated protein kinase (MAPK) pathways (ERK, JNK, and p38 activation). As2O3 alone at 10mM resulted in dose- and time-dependent apoptosis in all cell lines (HL and NHL) with >75% annexin+/PI+ between 48 and 72 hours. In Ramos and L428 cells, a combination of As2O3 (2 mM) and BSO 100mM resulted in >80% annexinV+/PI+, while either agent alone resulted in <15% apoptosis (p=0.001). A four-fold increase in ROS was seen in all four cell lines with As2O3/BSO, but not with As2O3 alone. Both ROS production and apoptosis induced by As2O3/BSO and As2O3 alone were reversible with the anti-oxidant N-acetylcysteine (NAC) (10mM). Furthermore, As2O3/BSO induced PARP cleavage and caspase-3 activation in all NHL cell lines, but not in L428. In Ramos, Z-VAD-FMK and Z-IETD-FMK blocked As2O3-induced apoptosis (suggesting a caspase-dependent mechanism), but had no effect on As2O3/BSO-induced cell death (caspase-independent). To investigate the signaling pathways involved, we performed western blot analysis for phospho-p38 (p-p38), phospho-JNK and phospho-ERK (p-ERK) in Ramos and L428 cells following As2O3 alone and As2O3/BSO. We found up-regulation of p-p38 in Ramos cells, while in L428, p-ERK was activated following As2O3 and As2O3/BSO. Inhibitors of p-38 in Ramos cells resulted in up-regulation of ERK. In summary, As2O3 induced dose- and time-dependent apoptosis in HL and NHL cell lines was significantly enhanced with the addition of BSO, and inhibited by NAC, suggesting that apoptosis was in part ROS-dependent. In Ramos, As2O3 alone resulted in caspase-dependent apoptosis, while addition of BSO resulted in caspase-independent apoptosis. As2O3 alone and combined with BSO resulted in activation of MAPK pathways in HL and NHL lines. These data provide a basis and a mechanism for As2O3-induced cell death in HL and NHL and have therapeutic implications.

Protection Against Arsenic Trioxide-Induced Autophagic Cell Death in U118 Human Glioma Cells by Use of Lipoic Acid

P-698 Introduction: Exposure to toxic heavy metals has become an increasingly recognized source of illness worldwide. Arsenic is an environmental toxicant found naturally in ground water. Neurotoxic effects have been reported in clinical cases and animal experiments with chronic exposure to arsenic. Epidemiological studies also have suggested a correlation between arsenic exposure and potential neurotoxicity. Lipoic acid is a thiol-compound naturally occurring in plants and animals, which is thought to be a strong antioxidant and possess neuroprotective effects. The objective of this study was to determine if the AS2O3-induced neurotoxic effects could be prevented by lipoic acid. The U118 human glioma cell was selected as a research model. Methods: In this study, U118 cells were treated with As2O3 alone or in combination with lipoic acid, and the viability of the cells was determined with MTT assay. DNA fragmentation assay and flow cytometric analysis were performed to confirm the apoptotic cells with Annexin V-PI staining. Supravital cell staining with acridine orange was performed to detect the autophagic cell death. The regulatory proteins which were related to apoptosis was determined by immunoblotting analysis, using antibodies including HSP70, p53, p21/Cip1, Bax, and Bcl2. Results: The viability assays revealed that 50% mortality of the AS2O3-treated cells that died within 24 hrs was reversed by lipoic acid. The lipoic acid treatment promoted long-term survival in cells treated with AS2O3 and provided more than 80% protection during days 2–3. We also found that autophagic, but not apoptotic cell death was significantly induced by AS2O3 in U118 cells, and that AS2O3-mediated autophagic cell death was nearly completely attenuated by lipoic acid. The down-regulation of p53 and Bax proteins and the up-regulation of Bcl-2 and HSP-70 proteins were involved in AS2O3-mediated autophagic cell death. Discussion and Conclusions: The present study demonstrated for the first time that AS2O3-induced neurotoxicity could be completely attenuated by lipoic acid in U118 cells. Apoptotic neuronal cell death was suggested as a general mechanism underlying arsenic neurotoxicity. However, when U118 cells were exposed to AS2O3, autophagy but not apoptosis was induced. Such results implied that U118 cells response to AS2O3 must be regulated by different signaling mechanisms rather than the general apoptotic mechanism. Our results provide the molecular basis for the lipoic acid prevention of AS2O3-induced cell death in U118 cells; such observations may have significance in clinical application.

Identification and Characterization of AIFsh2, a Mitochondrial Apoptosis-inducing Factor (AIF) Isoform with NADH Oxidase Activity

Apoptosis-inducing factor (AIF) is a bifunctional NADH oxidase involved in mitochondrial respiration and caspase-independent apoptosis. Three alternatively spliced mRNA isoforms of AIF have been identified previously: AIF, AIF-exB, and AIFsh. Here, we report the cloning and the biochemical characterization of a new isoform named AIF short 2 (AIFsh2). AIFsh2 transcript includes a previously unknown exon placed between exons 9 and 10 of AIF. The resulting AIFsh2 protein, which localizes in mitochondria, corresponds to the oxidoreductase domain of AIF. In this way, AIFsh2 exhibits similar NADH oxidase activity to AIF and generates reactive oxygen species. Like AIF, AIFsh2 is released from mitochondria to cytosol after an apoptotic insult in a calpain or cathepsin-dependent manner. However, in contrast to AIF, AIFsh2 does not induce nuclear apoptosis. Thus, it seems that the reactive oxygen species produced by the oxidoreductase domain of AIF/AIFsh2 are not important for AIF-dependent nuclear apoptosis. In addition, we demonstrate that the AIFsh2 mRNA is absent in normal brain tissue, whereas it is expressed in neuroblastoma-derived cells, suggesting a different regulation in normal and transformed cells from the brain lineage. Together, our results reveal that AIF yields an original and independent genetic regulation of the two AIF functions. This is an important issue to understand the physiological role of this protein.

Inhibition of mitogen‐activated protein kinase kinase enhances apoptosis induced by arsenic trioxide in human breast cancer MCF‐7 cells

Arsenic trioxide (As2O3) has recently been used to treat acute promyelocytic leukaemia and has activity in vitro against several solid tumour cell lines where the induction of differentiation and apoptosis are the prime effects. The mechanism of As2O3-induced cell death has yet to be clarified, especially in solid cancers. In the present study, the human breast cancer cell line MCF-7 was examined as a cellular model for As2O3 treatment. The involvement of extracellular signal-regulated kinase (ERK), p38 and c-Jun N-terminal kinase (JNK) was investigated in As2O3-induced cell death. 3. It was found that As2O3 activates the prosurvival mitogen-activated protein kinase kinase (MEK)/ERK pathway in MCF-7 cells, which, conversely, may compromise the efficacy of As2O3. Hence, a combination treatment of As2O3 and MEK inhibitors was investigated to determine whether this treatment could lead to enhanced growth inhibition and apoptosis in MCF-7 cells. 4. Inhibition of MEK/ERK with the pharmacological inhibitors U0126 (10 micromol/L) or PD98059 (20 micromol/L) together with As2O3 (2 and 5 micromol/L) resulted in a significant enhancement of growth inhibition in breast cancer MCF-7 cells as determined by the 3-(4,5-dimethyl-2 thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide assay and [Methyl-3H]-thymidine incorporation. Furthermore, the results demonstrated that combined treatment with As2O3 and the MEK1/2 inhibitor U0126 could augment breast cancer MCF-7 cell apoptosis approximately twofold compared with the effects of the two drugs alone, as determined by Hoechst 33258 or annexin V/propidium iodide (PI) staining and flow cytometry. 5. In addition, As2O3 activated p38 in a dose-dependent manner, but had no effect on JNK1/2. Treatment with a p38 inhibitor did not prevent As2O3-induced apoptosis. 6. In conclusion, the results of the present study showed that enhanced apoptosis is detected in breast cancer MCF-7 cells in the presence of As2O3 and an MEK inhibitor, which may be a new promising adjuvant to current breast cancer treatments.

Arsenic Trioxide Induces Regulated, Death Receptor- and Caspase-Independent Cell Death through the Mitochondrial Death Pathway.

Arsenic trioxide (As2O3, arsenite) efficiently kills cells from various hematologic malignancies and has successfully been employed especially for the treatment of acute promyelocytic leukemia. There, and in lymphoid cells we demonstrated that arsenite induces cell death in a caspase-2 and -9-independent fashion. Here, we address a potential role of death receptor signaling through the FADD/caspase-8 death inducing signaling complex in arsenite-induced cell death. In detail, we demonstrate that arsenite induces cell death independently of caspase-8 or FADD and cannot be blocked by disruption of CD95/Fas receptor ligand interaction. Unlike in death receptor ligation-induced apoptosis, As2O3-induced cell death was not blocked by the broad spectrum caspase inhibitor z-VAD-fmk or the caspase-8-specific inhibitor z-IETD-fmk. Nevertheless, arsenite-induced cell death occurred in a regulated manner and was abrogated upon Bcl-2 overexpression. In contrast, arsenite-induced cell demise was neither blocked by the caspase-9 inhibitor z-LEHD-fmk nor substantially inhibited through the expression of a dominant negative caspase-9 mutant. Altogether our data demonstrate that As2O3-induced cell death occurs independently of the extrinsic death receptor pathway of apoptosis. Cell death proceeds entirely via an intrinsic, Bcl-2 controlled mitochondrial pathway that does, however, not rely on caspase-9.

Opposite effect of ERK1/2 and JNK on p53-independent p21WAF1/CIP1 activation involved in the arsenic trioxide-induced human epidermoid carcinoma A431 cellular cytotoxicity

While arsenic trioxide (As2O3) is an infamous carcinogen, it is also an effective chemotherapeutic agent for acute promyelocytic leukemia and some solid tumors. In human epidermoid carcinoma A431 cells, we found that As2O3 induced cell death in time- and dose-dependent manners. Similarly, dependent regulation of the p21WAF1/CIP1 (p21) promoter, mRNA synthesis, and resultant protein expression was also observed. Additionally, transfection of a small interfering RNA of p21 could block the As2O3-induced cell growth arrest. The As2O3-induced p21 activation was attenuated by inhibitors of EGFR and MEK in a dose-dependent manner. Using a reporter assay, we demonstrated the involvement of the EGFR-Ras-Raf-ERK1/2 pathway in the promoter activation. In contrast, JNK inhibitor enhanced the As2O3-induced p21 activation, also in a dose-dependent fashion. Over-expression of a dominant negative JNK plasmid likewise also enhanced this activation. Furthermore, MEK inhibitor attenuated the anti-tumor effect of As2O3. In contrast, in combination with JNK inhibitor and As2O3 enhanced cellular cytotoxicity. Therefore, we conclude that in A431 cells the ERK1/2 and JNK pathways might differentially contribute to As2O3-induced p21 expression and then due to cellular cytotoxicity.

Arsenic trioxide induces regulated, death receptor-independent cell death through a Bcl-2-controlled pathway

Arsenic trioxide (As2O3, arsenite) efficiently kills cells from various hematologic malignancies and has successfully been employed especially for the treatment of acute promyelocytic leukemia. There and in lymphoid cells, we demonstrated that As2O3 induces cell death in a caspase-2- and -9-independent fashion. Here, we address a potential role of death receptor signaling through the FADD/caspase-8 death-inducing signaling complex in As2O3-induced cell death. In detail, we demonstrate that As2O3 induces cell death independently of caspase-8 or FADD and cannot be blocked by disruption of CD95/Fas receptor ligand interaction. Unlike in death receptor ligation-induced apoptosis, As2O3-induced cell death was not blocked by the broad-spectrum caspase inhibitor z-VAD-fmk or the caspase-8-specific inhibitor z-IETD-fmk. Nevertheless, As2O3-induced cell death occurred in a regulated manner and was abrogated upon Bcl-2 overexpression. In contrast, As2O3-induced cell demise was neither blocked by the caspase-9 inhibitor z-LEHD-fmk nor substantially inhibited through the expression of a dominant negative caspase-9 mutant. Altogether our data demonstrate that As2O3-induced cell death occurs independently of the extrinsic death receptor pathway of apoptosis. Cell death proceeds entirely via an intrinsic, Bcl-2-controlled mitochondrial pathway that does, however, not rely on caspase-9.

Multidrug-resistant neuroblastoma cells are responsive to arsenic trioxide at both normoxia and hypoxia

Despite intensive treatment, the outcome of high-risk neuroblastoma patients is poor with acquired multidrug resistance as an important cause. Previously, our group has shown that arsenic trioxide (As(2)O(3)) kills multidrug-resistant neuroblastoma cells in vitro and in vivo at clinically tolerable doses. Regions of tissue hypoxia often arise in aggressive solid tumors, and hypoxic tumors exhibit augmented invasiveness and metastatic ability in several malignancies. Furthermore, hypoxia may impair the treatment efficiency; therefore, we have studied the cytotoxic effect of As(2)O(3) on neuroblastoma cells grown under normoxic as well as hypoxic (1% oxygen) conditions. At both normoxia and hypoxia, 2 and 4 mumol/L As(2)O(3) induced evident cell death in the drug-sensitive SH-SY5Y and IMR-32 cells as well as in the multidrug-resistant SK-N-BE(2)c (with a mutated p53) and SK-N-FI cells after 72 hours of exposure. In contrast, the conventional chemotherapeutic drug etoposide showed lowered efficiency in hypoxic IMR-32 cells. In accordance with our previously published results, although not to the same extent as in their normoxic counterparts, Bax is proteolytically cleaved also in neuroblastoma cells exposed to As(2)O(3) at hypoxia. This suggests that similar molecular mechanisms are involved in As(2)O(3)-induced neuroblastoma cell death during hypoxia compared with normoxia. Together, our results support As(2)O(3) as a potential candidate drug as a complement to conventional treatments for high-risk neuroblastoma patients and perhaps also for patients with other multidrug-resistant solid tumors.

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.

Caspase-Independent Cell Death by Arsenic Trioxide in Human Cervical Cancer Cells

Although mechanisms of arsenic trioxide (As(2)O(3))-induced cell death have been studied extensively in hematologic cancers, those in solid cancers have yet to be clearly defined. In this study, we showed that the translocation of apoptosis-inducing factor (AIF) from mitochondria to the nucleus is required for As(2)O(3)-induced cell death in human cervical cancer cells. We also showed that reactive oxygen species (ROS)-mediated poly(ADP-ribose) polymerase-1 (PARP-1) activation is necessary for AIF release from mitochondria. The treatment of human cervical cancer cells with As(2)O(3) induces dissipation of mitochondrial membrane potential (Deltapsi(m)), translocation of AIF from mitochondria to the nucleus, and subsequent cell death. Small interfering RNA targeting of AIF effectively protects cervical cancer cells against As(2)O(3)-induced cell death. As(2)O(3) also induces an increase of intracellular ROS level and a marked activation of PARP-1. N-acetyl-l-cystein, a thiol-containing antioxidant, completely blocks As(2)O(3)-induced PARP-1 activation, Deltapsi(m) loss, nuclear translocation of AIF from mitochondria, and the consequent cell death. Furthermore, pretreatment of 1,5-dihydroxyisoquinoline or 3,4-dihydro-5-[4-(1-piperidinyl)butoxy]-1(2H)-isoquinolinone, PARP-1 inhibitors, effectively attenuates the loss of Deltapsi(m), AIF release, and cell death. These data support a notion that ROS-mediated PARP-1 activation signals AIF release from mitochondria, resulting in activation of a caspase-independent pathway of cell death in solid tumor cells by As(2)O(3) treatment.

Leukemia-associated translocation products able to activate RAS modify PML and render cells sensitive to arsenic-induced apoptosis.

Since the 19th century, arsenic (As2O3) has been used in the treatment of chronic myelogenous leukemia (CML) characterized by the t(9;22) translocation. As2O3 induces complete remissions in patients with acute promyelocytic leukemia. The response to As2O3 is genetically determined by the t(15;17)-or the t(9;22)-specific fusion proteins PML/RARalpha or BCR/ABL. The PML portion of PML/RARalpha is crucial for the sensitivity to As2O3. PML is nearly entirely contained in PML/RARalpha. PML is upregulated by oncogenic RAS in primary fibroblasts. The aberrant kinase activity of BCR/ABL leads to constitutive activation of RAS. Therefore, we hypothesized that BCR/ABL could increase sensitivity to As2O2-induced apoptosis by modifying PML expression. To disclose the mechanism of As2O3-induced apoptosis in PML/RARalpha- and BCR/ABL-expressing cells, we focused on the role of PML for As2O3-induced cell death. Here we report that (i) sensitivity to As2O3-induced apoptosis of U937 cells can be increased either by overexpression of PML, or by conditional expression of activated RAS; (ii) also the expression of the t(8;21)-related AML-1/ETO increased sensitivity to As2O3-induced apoptosis; (iii) both BCR/ABL and AML-1/ETO activated RAS and modified the PML expression pattern; (iv) the expression of either BCR/ABL or AML-1/ETO rendered U937 cells sensitive to interferon alpha-induced apoptosis. In summary, these data suggest a crucial role of factors able to upregulate PML for As2O2-induced cell death.

Ascorbic acid enhances arsenic trioxide–induced cytotoxicity in multiple myeloma cells

AbstractMultiple myeloma (MM) is a clonal B-cell malignancy characterized by slow-growing plasma cells in the bone marrow (BM). Patients with MM typically respond to initial chemotherapies; however, essentially all progress to a chemoresistant state. Factors that contribute to the chemorefractory phenotype include modulation of free radical scavenging, increased expression of drug efflux pumps, and changes in gene expression that allow escape from apoptotic signaling. Recent data indicate that arsenic trioxide (As2O3) induces remission of refractory acute promyelocytic leukemia and apoptosis of cell lines overexpressing Bcl-2 family members; therefore, it was hypothesized that chemorefractory MM cells would be sensitive to As2O3. As2O3 induced apoptosis in 4 human MM cell lines: 8226/S, 8226/Dox40, U266, and U266/Bcl-xL. The addition of interleukin-6 had no effect on cell death. Glutathione (GSH) has been implicated as an inhibitor of As2O3-induced cell death either through conjugating As2O3 or by sequestering reactive oxygen induced by As2O3. Consistent with this possibility, increasing GSH levels with N-acetylcysteine attenuated As2O3 cytotoxicity. Decreases in GSH have been associated with ascorbic acid (AA) metabolism. Clinically relevant doses of AA decreased GSH levels and potentiated As2O3-mediated cell death of all 4 MM cell lines. Similar results were obtained in freshly isolated human MM cells. In contrast, normal BM cells displayed little sensitivity to As2O3 alone or in combination with AA. Together, these data suggest that As2O3 and AA may be effective antineoplastic agents in refractory MM and that AA might be a useful adjuvant in GSH-sensitive therapies.