Arsenic trioxide promotes mitochondrial DNA mutation and cell apoptosis in primary APL cells and NB4 cell line

This study aimed to investigate the effects of arsenic trioxide (As₂O₃) on the mitochondrial DNA (mtDNA) of acute promyelocytic leukemia (APL) cells. The NB4 cell line was treated with 2.0 μmol/L As₂O₃ in vitro, and the primary APL cells were treated with 2.0 μmol/L As₂O₃ in vitro and 0.16 mg kg⁻¹ d⁻¹ As₂O₃ in vivo. The mitochondrial DNA of all the cells above was amplified by PCR, directly sequenced and analyzed by Sequence Navigatore and Factura software. The apoptosis rates were assayed by flow cytometry. Mitochondrial DNA mutation in the D-loop region was found in NB4 and APL cells before As₂O₃ use, but the mutation spots were remarkably increased after As₂O₃ treatment, which was positively correlated to the rates of cellular apoptosis, the correlation coefficient: r _NB4-As2O3=0.973818, and r _APL-As2O3=0.934703. The mutation types include transition, transversion, codon insertion or deletion, and the mutation spots in all samples were not constant and regular. It is revealed that As₂O₃ aggravates mtDNA mutation in the D-loop region of acute promyelocytic leukemia cells both in vitro and in vivo. Mitochondrial DNA might be one of the targets of As₂O₃ in APL treatment.     

Tetraarsenictetrasulfide and Arsenic Trioxide Exert Synergistic Effects on Induction of Apoptosis and Differentiation in Acute Promyelocytic Leukemia Cells

Since arsenic trioxide (As³⁺) has been successfully used in the treatment of acute promyelocytic leukemia (APL), its adverse effects on patients have been problematic and required a solution. Considering the good therapeutic potency and low toxicity of tetraarsenictetrasulfide (As₄S₄) in the treatment of APL, we investigated the effects of combining As₄S₄ and As³⁺ on the apoptosis and differentiation of NB4 and primary APL cells. As₄S₄, acting similarly to As³⁺, arrested the G₁/S transition, induced the accumulation of cellular reactive oxygen species, and promoted apoptosis. Additionally, low concentrations of As₄S₄ (0.1–0.4 μM) induced differentiation of NB4 and primary APL cells. Compared with the As₄S₄- or As³⁺-treated groups, the combination of As₄S₄ and As³⁺ obviously promoted apoptosis and differentiation of NB4 and primary APL cells. Mechanistic studies suggested that As₄S₄ acted synergistically with As³⁺ to down-regulate Bcl-2 and nuclear factor-κB expression, up-regulate Bax and p53 expression, and induce activation of caspase-12 and caspase-3. Moreover, the combination of low concentrations of As₄S₄ and As³⁺ enhanced degradation of the promyelocytic leukemia-retinoic acid receptor α oncoprotein. In summary, As₄S₄ and As³⁺ synergistically induce the apoptosis and differentiation of NB4 and primary APL cells.     

Various tolerances to arsenic trioxide between human cortical neurons and leukemic cells

Arsenic trioxide (As2O3) is very effective for treatment of acute promyelocytic leukaemia (APL) but little can pass through the blood-brain-barrier (BBB),which limits its use in the prevention and treatment of central nervous system leukaemia (CNSL). Before creating a non-invasive method to help As2O3 ’s access,the safe and effective therapeutic concentration of As2O3 in the CNS ought to be known. The changes of apoptosis biomarkers,[Ca2 ]i and PKC activity of both leukaemia cells and human cortical neurons,were monitored before and after being treated with As2O3 in vitro with laser confocal microscopy and Western blot. NSE concentration,the neuron invasive biomarker,was monitored by enzyme immunoassay (NSE-EIA). This study revealed that cortical neuron was more tolerable to As2O3 compared to NB4. 1.0 μmol / L As2O3 showed little influence on cortical neuron but effectively promoted apoptosis and induced differentiation of NB4.     

Various tolerances to arsenic trioxide between human cortical neurons and leukemic cells

Arsenic trioxide (As₂O₃) is very effective for treatment of acute promyelocytic leukaemia (APL) but little can pass through the blood-brain-barrier (BBB), which limits its use in the prevention and treatment of central nervous system leukaemia (CNSL). Before creating a non-invasive method to help As₂O₃’s access, the safe and effective therapeutic concentration of As₂O₃ in the CNS ought to be known. The changes of apoptosis biomarkers, [Ca²⁺]_i and PKC activity of both leukaemia cells and human cortical neurons, were monitored before and after being treated with As₂O₃ in vitro with laser confocal microscopy and Western blot. NSE concentration, the neuron invasive biomarker, was monitored by enzyme immunoassay (NSE-EIA). This study revealed that cortical neuron was more tolerable to As₂O₃ compared to NB4. 1.0 μmol / L As₂O₃ showed little influence on cortical neuron but effectively promoted apoptosis and induced differentiation of NB4.     

The PML and PML/RARα Domains: From Autoimmunity to Molecular Oncology and from Retinoic Acid to Arsenic

Acute promyelocytic leukemia (APL) is specifically associated to a t(15; 17) translocation which fuses a gene encoding a nuclear receptor for retinoic acid, RARα, to a previously unknown gene PML. The PML protein is localized in the nucleus on a specific domain of unknown function (PML nuclear bodies, NB) previously detected with autoimmune sera from patients with primary biliary cirrhosis (PBC). These bodies are nuclear matrix-associated and all of their identified components (PML, Sp100, and NDP52) are sharply upregulated by interferons. We show that autoantibodies against both PML and Sp100 are usually associated in sera with multiple nuclear dot anti-nuclear antibodies and demonstrate that PML is an autoantigen, not only in PBC, but also in other autoimmune diseases. In APL, the PML/RARα fusion interferes with both the retinoic acid (RA) response and PML localization on nuclear bodies, but the respective contribution of each defect to leukemogenesis is unclear. RA induces the terminal differentiation of APL blasts, yielding to complete remissions, and corrects the localization of NB antigens. Arsenic trioxide (As₂O₃) also induces remissions in APL, seemingly through induction of apoptosis. We show that in APL, As₂O₃leads to the rapid reformation of PML bodies. Thus, both agents correct the defect in NB antigen localization, stressing the role of nuclear bodies in the pathogenesis of APL.     

Combination of arsenic trioxide and BCNU synergistically triggers redox-mediated autophagic cell death in human solid tumors

Arsenic trioxide (As₂O₃) is an effective treatment for relapsed or refractory acute promyelocytic leukemia (APL). After the discovery of As₂O₃ as a promising treatment for APL, several studies investigated the use of As₂O₃ as a single agent in the treatment of solid tumors; however, its therapeutic efficacy is limited. Thus, the systematic study of the combination of As₂O₃ with other clinically used chemotherapeutic drugs to improve its therapeutic efficacy in treating human solid tumors is merited. In this study, we demonstrate for the first time, using isobologram analysis, that As₂O₃ exhibits a synergistic interaction with N,N′-bis(2-chloroethyl)-N-nitrosourea (BCNU). The synergistic augmentation of the cytotoxicity of As₂O₃ with BCNU is in part through the autophagic cell death machinery in human solid tumor cells. As₂O₃ and BCNU in combination produce enhanced cytotoxicity via the depletion of reduced glutathione (GSH) and augmentation of reaction oxygen species (ROS) production. Further analysis indicated that the extension of GSH depletion by this combined regimen occurs through the inhibition of the catalytic activity of glutathione reductase. Blocking ROS production with antioxidants or ROS scavengers effectively inhibits cell death and autophagy formation, indicating that redox-mediated autophagic cell death involves the synergism of As₂O₃ with BCNU. Taken together, this is the first evidence that BCNU could help to extend the therapeutic spectrum of As₂O₃. These findings will be useful in designing future clinical trials of combination chemotherapy with As₂O₃ and BCNU, with the potential for broad use against a variety of solid tumors.     

Arsenic Sulfide Promotes Apoptosis in Retinoid Acid Resistant Human Acute Promyelocytic Leukemic NB4-R1 Cells through Downregulation of SET Protein

Tetra-arsenic tetra-sulfide (As₄S₄) is an arsenic compound with anti-tumor activity, especially in acute promyelocytic leukemia (APL) that are resistant to retinoic acid (RA). Although recent studies revealed that the therapeutic action of As₄S₄ is closely associated with the induction of cellular apoptosis, the exact molecular mechanism of action of As₄S₄ in RA-resistant APL remains to be clarified. In this study, we found that As₄S₄-induced apoptosis was accompanied by reduced mRNA and protein expression of SET gene in RA-resistant NB4-R1 cells. Moreover, RNAi knockdown of SET gene further promoted As₄S₄-induced apoptosis, while SET over-expression inhibited it, suggesting that As₄S₄ induces apoptosis through the reduction of SET protein in NB4-R1 cells. We also demonstrated that the knockdown of SET gene resulted in the upregulation of protein phosphatase 2 (PP2A) expression and the downregulation of promyelocytic leukemia and retinoic acid receptor α fusion gene (PML-RARα) expression, which were enhanced by As₄S₄ treatments. By contrast, over-expression of SET gene resulted in PP2A downregulation and PML-RARα upregulation, which were abolished by As₄S₄ pretreatment. Since PP2A is a pro-apoptotic factor and PMLRARα is an anti-apoptotic factor, our results suggest that As₄S₄-induced apoptosis in NB4-R1 cells is through the downregulation of SET protein expression, which in turn increases PP2A and reduces PML-RARα expressions to lead to cell apoptosis.     

Targeting Catalase but Not Peroxiredoxins Enhances Arsenic Trioxide-Induced Apoptosis in K562 Cells

Despite considerable efficacy of arsenic trioxide (As₂O₃) in acute promyelocytic leukemia (APL) treatment, other non-APL leukemias, such as chronic myeloid leukemia (CML), are less sensitive to As₂O₃ treatment. However, the underlying mechanism is not well understood. Here we show that relative As₂O₃-resistant K562 cells have significantly lower ROS levels than As₂O₃-sensitive NB4 cells. We compared the expression of several antioxidant enzymes in these two cell lines and found that peroxiredoxin 1/2/6 and catalase are expressed at high levels in K562 cells. We further investigated the possible role of peroxirdoxin 1/2/6 and catalase in determining the cellular sensitivity to As₂O₃. Interestingly, knockdown of peroxiredoxin 1/2/6 did not increase the susceptibility of K562 cells to As₂O₃. On the contrary, knockdown of catalase markedly enhanced As₂O₃-induced apoptosis. In addition, we provide evidence that overexpression of BCR/ABL cannot increase the expression of PRDX 1/2/6 and catalase. The current study reveals that the functional role of antioxidant enzymes is cellular context and treatment agents dependent; targeting catalase may represent a novel strategy to improve the efficacy of As₂O₃ in CML treatment.     

The inherent cellular level of reactive oxygen species: One of the mechanisms determining apoptotic susceptibility of leukemic cells to arsenic trioxide

Though reactive oxygen species (ROS) has been noticed to be involved in arsenic trioxide (As₂O₃)-induced apoptosis of tumor cells, its role in apoptosis signaling remained to be elucidated. The objective of this work was to explore the association of the inherent cellular ROS level with the susceptibility of the tumor cells to apoptosis induction by As₂O₃. Low concentration of As₂O₃ was administered to cultured leukemic cell lines NB4, U937, HL60 and K562. The difference in apoptotic sensitivity was displayed among four cell types. ROS probes were incubated with the cells in the absence of As₂O₃, and ROS was thus quantified relatively by flow cytometry. We manifested, in four cell types, the inherently existed difference in whole ROS quantity, and a positive correlation between the inherent ROS level and their apoptotic sensitivity to As₂O₃. Furthermore, by interference using a ROS producer, we demonstrated that an elevation of ROS level would sensitize the cells to As₂O₃-induced apoptosis. The results of the present work suggested that the inherent ROS level might be determinative in tumor cells for their apoptotic susceptibility to As₂O₃.     

Enhancement of esculetin on arsenic trioxide-provoked apoptosis in human leukemia U937 cells

In order to overcome chemotherapy resistance, many laboratories are searching for agents that increase the sensitivity of cancer cells to anticancer drugs. Arsenic trioxide (As₂O₃) is widely used in treating human acute polymyelocytic leukemia (APL). However, solid tumors and other leukemia cells such as U937 promonocytic leukemia cells are insensitive to As₂O₃. Esculetin, a coumarin derivative, has previously induced cell cycle arrest and apoptosis of HL-60 cells as well as enhanced taxol-induced apoptosis in HepG2 cells, thereby displaying anticancer potential. In this study, esculetin inhibited proliferation and mitogen activated protein kinases (MAPKs) activation in human leukemia U937 cells. Since inhibitors of MAPKs have modulated the GSH-redox state and enhanced the sensitivity of leukemia cells to As₂O₃-provoked apoptosis, we monitored the effect of combining esculetin and As₂O₃ (2.5 μM) on the GSH level. Our study showed that esculetin, PD98059 (MEK/ERK inhibitor), and SP600125 (JNK inhibitor) similarly enhanced the As₂O₃-induced GSH depletion. We found that the As₂O₃ (2.5 μM) treatment slightly induced apoptosis and the pretreatment of esculetin enhanced the As₂O₃-provoked apoptosis significantly. In addition, esculetin enhanced the effect of As₂O₃ on caspase activation in U937 cells. We compared the combined esculetin and As₂O₃ treatment to the As₂O₃ treated alone. The combined esculetin and As₂O₃ treatment increased Bid cleavage, Bax conformation change and cytochrome C release. The study also indicated that esculetin enhanced the As₂O₃-induced lysosomal leakage and apoptosis. Furthermore, pretreatment with N-acetylcysteine (NAC) reduced these enhanced effects. Based on these studies, esculetin enhances the As₂O₃-provoked apoptosis by modulating the MEK/ERK and JNK pathways and reducing intracellular GSH levels. GSH depletion led to higher oxidative stress which activated lysosomal-mitochondrial pathway of apoptosis.     

Resveratrol,a polyphenol phytoalexin,protects against doxorubicin‐induced cardiotoxicity

Doxorubicin is the mainstay of treatment for various haematological malignancies and solid tumours. However, its clinical application may be hampered by dose‐dependent cardiotoxicity. The mechanism of doxorubicin‐induced cardiotoxicity may involve various signalling pathways including free radical generation, peroxynitrite formation, calcium overloading, mitochondrial dysfunction and alteration in apoptosis and autophagy. Interestingly, the use of resveratrol in combination with doxorubicin has been reported to prevent cardiac toxicity as well as to exert a synergistic effect against tumour cells both in vivo and in vitro. Thus, the aim of this review is to summarize current knowledge and to elucidate the protective effect of resveratrol in doxorubicin‐induced cardiotoxicity.     

Autophagy Is a Critical Mechanism for the Induction of the Antileukemic Effects of Arsenic Trioxide

Arsenic trioxide (As₂O₃) exhibits potent antitumor effects in vitro and in vivo, but the precise mechanisms by which it generates such responses are not well understood. We provide evidence that As₂O₃ is a potent inducer of autophagy in leukemia cells. Such induction of autophagy by As₂O₃ appears to require activation of the MEK/ERK pathway but not the AKT/mammalian target of rapamycin or JNK pathways. In efforts to understand the functional relevance of arsenic-induced autophagy, we found that pharmacological inhibitors of autophagy or molecular targeting of beclin 1 or Atg7 results in reversal of the suppressive effects of As₂O₃ on leukemic cell lines and primary leukemic progenitors from acute myelogenous leukemia patients. Altogether, our data provide direct evidence that autophagic cell death is critical for the generation of the effects of As₂O₃ on acute myelogenous leukemia cells and raise the potential of modulation of elements of the autophagic machinery as an approach to enhance the antitumor properties of As₂O₃ and possibly other heavy metal derivatives.     

Attenuation of arsenic trioxide induced cardiotoxicity through flaxseed oil in experimental rats

Objectives: Arsenic trioxide (As₂O₃) is a potent drug for acute promyelocytic leukaemia, but its clinical trials are allied with some serious adverse events mainly cardiac functional abnormalities. So the objective of our investigation is to identify the cardioprotective action of flaxseed oil (FSO), a natural compound against As₂O₃ induced cardiotoxicity.

Methods: Male wistar rats were treated with As₂O₃ (4?mg/kg) to induce cardiotoxicity. FSO (250 and 500?mg/kg) was given in combination with As₂O₃ for evaluating its cardioprotective efficacy.

Results: Treatment with As₂O₃ resulted in deposition of arsenic in heart tissue, increased cardiac marker enzymes release, lipid peroxidation (LPO), oxidative insults and pathological damages in the heart. Co-treatment with FSO (500?mg/kg) significantly reduced the arsenic accumulation, cardiac marker enzymes, LPO and cardiac structural alterations. FSO treatment significantly improved cardiac glutathione content, antioxidant enzymes and reduced the pathological damages in cardiac tissue. Gas chromatographic–mass spectrometry analysis revealed that the major fatty acid content in the FSO is alpha-linolenic acid, which has a strong milieu in cardiac health.

Conclusion: The results of the current investigation suggested that FSO is an effective agent in reducing arsenic-induced cardiac toxicity and can be used as an adjunct/dietary supplement for the cancer patients on As₂O₃ therapy.     

Different effects of genistein and resveratrol on oxidative DNA damage in vitro

Previous studies have demonstrated that phenolic compounds, including genistein (4′,5,7-trihydroxyisoflavone) and resveratrol (3,4′,5-trihydroxystilbene), are able to protect against carcinogenesis in animal models. This study was undertaken to examine the ability of genistein and resveratrol to inhibit reactive oxygen species (ROS)-mediated strand breaks in φX-174 plasmid DNA. H₂O₂/Cu(II) and hydroquinone/Cu(II) were used to cause oxidative DNA strand breaks in the plasmid DNA. We demonstrated that the presence of genistein at micromolar concentrations resulted in a marked inhibition of DNA strand breaks induced by either H₂O₂/Cu(II) or hydroquinone/Cu(II). Genistein neither affected the Cu(II)/Cu(I) redox cycle nor reacted with H₂O₂ suggest that genistein may directly scavenge the ROS that participate in the induction of DNA strand breaks. In contrast to the inhibitory effects of genistein, the presence of resveratrol at similar concentrations led to increased DNA strand breaks induced by H₂O₂/Cu(II). Further studies showed that in the presence of Cu(II), resveratrol, but not genistein was able to cause DNA strand breaks. Moreover, both Cu(II)/Cu(I) redox cycle and H₂O₂ were shown to be critically involved in resveratrol/copper-mediated DNA strand breaks. The above results indicate that despite their similar in vivo anticarcinogenic effects, genistein and resveratrol appear to exert different effects on oxidative DNA damage in vitro.     

Arsenic trioxide induces apoptosis through JNK and ERK in human mesothelioma cells

Malignant mesothelioma is an aggressive tumor of serosal surfaces, which is refractory to current treatment options. Arsenic trioxide (As₂O₃) is used clinically to treat acute promyelocytic leukemia, and also to inhibit proliferation of several solid tumors including hepatoma, esophageal, and gastric cancer in vitro. Here we found that As₂O₃ inhibited cell viability of a mesothelioma cell line, NCI‐H2052. As₂O₃ induced apoptosis of NCI‐H2052 cells, which was accompanied by activation of c‐Jun NH₂‐terminal kinase (JNK)1/2, extracellular signal‐regulated kinase (ERK)1/2, and caspase‐3. zVAD‐fmk, a broad‐spectrum caspase inhibitor, inhibited As₂O₃‐induced apoptosis and activation of caspase‐3, but not that of JNK1/2 and ERK1/2. Small interfering RNAs (siRNAs) targeting JNK1/2 suppressed As₂O₃‐induced caspase‐3 activation and apoptosis, indicating that JNK1/2 regulate As₂O₃‐induced apoptosis though caspase cascade. Furthermore, JNK1 siRNA abrogated As₂O₃‐induced JNK2 phosphorylation and JNK2 siRNA abrogated As₂O₃‐induced JNK1 phosphorylation, suggesting that JNK1 and JNK2 interact with each other. Moreover, JNK1 siRNA, but not JNK2 siRNA, abrogated As₂O₃‐induced ERK1/2 phosphorylation. JNK2 siRNA together with PD98059, a specific MAPK/ERK kinase inhibitor, suppressed As₂O₃‐induced apoptosis more significantly than JNK2 siRNA alone. These results indicated that As₂O₃ induces apoptosis of NCI‐H2052 cells mainly through JNK1/2 activation, and that ERK1/2 is involved in As₂O₃‐induced apoptosis when JNK1/2 are inactivated. J. Cell. Physiol. 226: 762–768, 2011. © 2010 Wiley‐Liss, Inc.     

ATO/ATRA/Anthracycline-Chemotherapy Sequential Consolidation Achieves Long-Term Efficacy in Primary Acute Promyelocytic Leukemia

The combination of all-trans retinoic acid (ATRA) and arsenic trioxide (As₂O₃, ATO) has been effective in obtaining high clinical complete remission (CR) rates in acute promyelocytic leukemia (APL), but the long-term efficacy and safety among newly diagnosed APL patients are unclear. In this retrospective study, total 45 newly diagnosed APL patients received ATRA/chemotherapy combination regimen to induce remission. Among them, 43 patients (95.6%) achieved complete remission (CR) after induction therapy, followed by ATO/ATRA/anthracycline-based chemotherapy sequential consolidation treatment with a median follow-up of 55 months. In these patients, the estimated overall survival (OS) and the relapse-free survival (RFS) were 94.4%±3.9% and 94.6±3.7%, respectively. The toxicity profile was mild and reversible. No secondary carcinoma was observed. These results demonstrated the high efficacy and minimal toxicity of ATO/ATRA/anthracycline-based chemotherapy sequential consolidation treatment for newly diagnosed APL in long-term follow-up, suggesting a potential frontline therapy for APL.     

Ellagic acid mitigates arsenic‐trioxide‐induced mitochondrial dysfunction and cytotoxicity in SH‐SY5Y cells

In the current study, neuroprotective significance of ellagic acid (EA, a polyohenol) was explored by primarily studying its antioxidant and antiapoptotic potential against arsenic trioxide (As₂O₃)‐induced toxicity in SH‐SY5Y human neuroblastoma cell lines. The mitigatory effects of EA with particular reference to cell viability and cytotoxicity, the generation of reactive oxygen species, DNA damage, and mitochondrial dynamics were studied. Pretreatment of SH‐SY5Y cells with EA (10 and 20 μM) for 60 min followed by exposure to 2 μM As₂O₃ protected the SH‐SY5Y cells against the harmful effects of the second. Also, EA pre‐treated groups expressed improved viability, repaired DNA, reduced free radical generation, and maintained altered mitochondrial membrane potential than those exposed to As₂O₃ alone. EA supplementation also inhibited As₂O₃‐induced cytochrome c expression that is an important hallmark for determining mitochondrial dynamics. Thus, the current investigations are more convinced for EA as a promising candidate in modulating As₂O₃‐induced mitochondria‐mediated neuronal toxicity under in vitro system.