ATR, PML, and CHK2 play a role in arsenic trioxide-induced apoptosis

Arsenic trioxide (ATO) is a potent anti-leukemic chemotherapeutic agent for acute promyelocytic leukemia (APL) that results from a t (15, 17) chromosomal translocation that produces PML-RARalpha, a fusion protein between a tumor suppressor PML and the retinoic acid receptor RARalpha. APL patients are initially treated with retinoic acid, but most develop resistance and relapse. In contrast, ATO induces prolonged remissions even in the relapsed cases. However, the molecular mechanisms by which ATO kills the leukemic cells are not fully understood. We find that ATO induces apoptosis, at least in part, by activating proapoptotic kinase Chk2. ATO does this by stimulating ATR (ataxia telangiectasia mutated and Rad3-related) kinase, a Chk2-activating kinase. In conjunction, ATO degrades PML-RARalpha, resulting in the restoration of PML, which is required for autophosphorylation and full activation of Chk2. As a result, the p53-dependent apoptosis pathway is activated. Based on this, we propose that a pathway composed of ATR, PML, Chk2, and p53 plays a role in ATO-mediated apoptosis, a notion that is consistent with the observation that Chk2 is genetically intact and mutations in the p53 gene are extremely rare in APL.     

Arsenic trioxide stimulates SUMO-2/3 modification leading to RNF4-dependent proteolytic targeting of PML

We have recently reported that poly-SUMO-2/3 conjugates are subject to a ubiquitin-dependent proteolytic control in human cells. Here we show that arsenic trioxide (ATO) increases SUMO-2/3 modification of promyelocytic leukemia (PML) leading to its subsequent ubiquitylation in vivo. The SUMO-binding ubiquitin ligase RNF4 mediates this modification and causes disruption of PML nuclear bodies upon treatment with ATO. Reconstitution of SUMO-dependent ubiquitylation of PML by RNF4 in vitro and in a yeast trans vivo system revealed a preference of RNF4 for chain forming SUMOs. Polysumoylation of PML in response to ATO thus leads to its recognition and ubiquitylation by RNF4.     

PML/RARalpha fusion protein mediates the unique sensitivity to arsenic cytotoxicity in acute promyelocytic leukemia cells: Mechanisms involve the impairment of cAMP signaling and the aberrant regulation of NADPH oxidase

Acute promyelocytic leukemia (APL) cells are characterized by PML/RARalpha fusion protein, high responsiveness to arsenic trioxide (ATO)-induced cytotoxicity and an abundant generation of reactive oxygen species (ROS). In this study we investigated the association among these three features in APL-derived NB4 cells. We found that NADPH oxidase-derived ROS generation was more abundant in NB4 cells compared with monocytic leukemia U937 cells. By using PR9, a sub-line of U937 stably transduced with the inducible PML/RARalpha expression vectors, we attributed disparities on ROS generation and ATO sensitivity to the occurrence of PML/RARalpha fusion protein, since PML/RARalpha-expressing cells appeared higher NADPH oxidase activity, higher ROS level and higher sensitivity to ATO. On the other hand, the basal intensity of cAMP signaling pathway was compared between NB4 and U937 as well as between PR9 cells with or without PML/RARalpha, demonstrating that PML/RARalpha-expressing cells had an impaired cAMP signaling pathway which relieved its inhibitory effect on NADPH oxidase derived ROS generation. In summary, the present study demonstrated the correlation of PML/RARalpha with cAMP signaling pathway, NADPH oxidase and ROS generation in APL cells. PML/RARalpha that bestows NB4 cells various pathological features, paradoxically also endows these cells with the basis for susceptibility to ATO-induced cytotoxcity.     

Alterations in Glutathione Levels and Apoptotic Regulators Are Associated with Acquisition of Arsenic Trioxide Resistance in Multiple Myeloma

Arsenic trioxide (ATO) has been tested in relapsed/refractory multiple myeloma with limited success. In order to better understand drug mechanism and resistance pathways in myeloma we generated an ATO-resistant cell line, 8226/S-ATOR05, with an IC50 that is 2–3-fold higher than control cell lines and significantly higher than clinically achievable concentrations. Interestingly we found two parallel pathways governing resistance to ATO in 8226/S-ATOR05, and the relevance of these pathways appears to be linked to the concentration of ATO used. We found changes in the expression of Bcl-2 family proteins Bfl-1 and Noxa as well as an increase in cellular glutathione (GSH) levels. At low, clinically achievable concentrations, resistance was primarily associated with an increase in expression of the anti-apoptotic protein Bfl-1 and a decrease in expression of the pro-apoptotic protein Noxa. However, as the concentration of ATO increased, elevated levels of intracellular GSH in 8226/S-ATOR05 became the primary mechanism of ATO resistance. Removal of arsenic selection resulted in a loss of the resistance phenotype, with cells becoming sensitive to high concentrations of ATO within 7 days following drug removal, indicating changes associated with high level resistance (elevated GSH) are dependent upon the presence of arsenic. Conversely, not until 50 days without arsenic did cells once again become sensitive to clinically relevant doses of ATO, coinciding with a decrease in the expression of Bfl-1. In addition we found cross-resistance to melphalan and doxorubicin in 8226/S-ATOR05, suggesting ATO-resistance pathways may also be involved in resistance to other chemotherapeutic agents used in the treatment of multiple myeloma.     

Triterpenoid CDDO-Im downregulates PML/RARalpha expression in acute promyelocytic leukemia cells

The triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) induces differentiation and apoptosis of diverse human tumor cells. In the present study, we examined the effects of the CDDO imidazolide imide (CDDO-Im) on the NB4 acute promyelocytic leukemia (APL) cell line and primary APL cells. The results show that CDDO-Im selectively downregulates expression of the PML/retinoic receptor alpha fusion protein by a caspase-dependent mechanism and sensitizes APL cells to the differentiating effects of all-trans retinoic acid (ATRA). CDDO-Im treatment of APL cells was also associated with disruption of redox balance and activation of the extrinsic apoptotic pathway. In concert with these results, CDDO-Im sensitizes APL cells to arsenic trioxide (ATO)-induced apoptosis. Our findings indicate that CDDO-Im may be effective in the treatment of APL by: (i) downregulation of PML/RARalpha; (ii) enhancement of ATRA-induced differentiation; and (iii) sensitization of ATO-induced APL cell death.     

VEGFA Expression Is Inhibited by Arsenic Trioxide in HUVECs through the Upregulation of Ets-2 and miRNA-126

Arsenic trioxide (ATO) has been used to treat patients with acute promyelocytic leukemia. Recently, studies have shown that ATO can induce apoptosis in leukemic cells and blood vessel endothelial cells in a time- and dose-dependent manner through the inhibition of vascular endothelial growth factor A (VEGFA) production. VEGFA is a key factor in angiogenesis initiation. Targeted inhibition of VEGF or VEGFA expression can suppress angiogenesis; however, little is known about the mechanism by which ATO inhibits VEGFA expression. In this study, we investigated the role of miRNA-126 in the mechanism of action of ATO in human umbilical vein endothelial cells (HUVECs). ATO significantly decreased the viability and proliferation of HUVECs and decreased their migration at 48 h. Cell proliferation was inhibited by 50% (IC50) when 5.0 μmol/L ATO was used. ATO treatment induced miR-126 upregulation and HUVEC apoptosis. Transfection with a miR-126 mimic significantly downregulated VEGFA mRNA levels, and transfection with a miR-126 inhibitor significantly upregulated VEGFA mRNA levels. Finally, we showed that ATO treatment upregulated Ets-2 and miR-126 expression in HUVECs. These results demonstrate that ATO inhibits the growth of HUVECs and induces apoptosis by downregulating VEGFA. One mechanism by which this occurs is Ets-2 upregulation, which results in an increase in miR-126 levels and downregulation of VEGFA expression.     

Arsenic Trioxide Inhibits Hepatitis C Virus RNA Replication through Modulation of the Glutathione Redox System and Oxidative Stress

Arsenic trioxide (ATO), a therapeutic reagent used for the treatment of acute promyelocytic leukemia, has recently been reported to increase human immunodeficiency virus type 1 infectivity. However, in this study, we have demonstrated that replication of genome-length hepatitis C virus (HCV) RNA (O strain of genotype 1b) was notably inhibited by ATO at submicromolar concentrations without cell toxicity. RNA replication of HCV-JFH1 (genotype 2a) and the release of core protein into the culture supernatants were also inhibited by ATO after the HCV infection. To clarify the mechanism of the anti-HCV activity of ATO, we examined whether or not PML is associated with this anti-HCV activity, since PML is known to be a target of ATO. Interestingly, we observed the cytoplasmic translocation of PML after treatment with ATO. However, ATO still inhibited the HCV RNA replication even in the PML knockdown cells, suggesting that PML is dispensable for the anti-HCV activity of ATO. In contrast, we found that N-acetyl-cysteine, an antioxidant and glutathione precursor, completely and partially eliminated the anti-HCV activity of ATO after 24 h and 72 h of treatment, respectively. In this context, it is worth noting that we found an elevation of intracellular superoxide anion radical, but not hydrogen peroxide, and the depletion of intracellular glutathione in the ATO-treated cells. Taken together, these findings suggest that ATO inhibits the HCV RNA replication through modulation of the glutathione redox system and oxidative stress.Hepatitis C virus (HCV) is the causative agent of chronic hepatitis, which progresses to liver cirrhosis and hepatocellular carcinoma. HCV is an enveloped virus with a positive single-stranded 9.6-kb RNA genome, which encodes a large polyprotein precursor of approximately 3,000 amino acid residues. This polyprotein is cleaved by a combination of the host and viral proteases into at least 10 proteins in the following order: core, envelope 1 (E1), E2, p7, nonstructural 2 (NS2), NS3, NS4A, NS4B, NS5A, and NS5B (30).Alpha interferon has been used as an effective anti-HCV reagent in clinical therapy for patients with chronic hepatitis C. The current combination treatment with pegylated alpha interferon and ribavirin, a nucleoside analogue, has been shown to improve the sustained virological response rate to more than 50% (15). However, the adverse effects of the combination therapy and the limited efficacy against genotype 1b warrant the development of new anti-HCV reagents.Arsenic trioxide (ATO) (As₂O₃, arsenite) has been used as a therapeutic reagent in acute promyelocytic leukemia, which bears an oncogenic PML-retinoic acid receptor alpha fusion protein resulting from chromosomal translocation (51, 52, 68, 70). The ATO treatment induces complete remission through degradation of the aberrant PML-retinoic acid receptor α (70). The PML tumor suppressor protein is required for formation of the PML nuclear body (PML-NB), also known as nuclear dot 10 or the PML oncogenic domain, which is often disrupted by infection with DNA viruses, such as herpes simplex virus type 1, human cytomegalovirus, and Epstein-Barr virus (17). The treatment with ATO results in degradation of the PML protein and disruption of the PML-NB (70). Therefore, ATO has been become a useful probe for investigating the functions of the PML-NB, including cell growth, apoptosis, stress response, and viral infection. Indeed, ATO has been shown to increase retroviral infectivity, such as human immunodeficiency virus type 1 (HIV-1) and murine leukemia virus infectivity, but the mechanisms of this change are not well understood (5, 6, 32, 44, 47, 50, 57). In contrast, ATO was recently reported to inhibit the replication of HCV subgenomic replicon RNA (24). However, it also remains unclear how ATO inhibits the HCV RNA replication. In this study, using genome-length HCV RNA replication systems, we investigated the molecular mechanism(s) of the anti-HCV activity of ATO, and we provide evidence that ATO inhibits HCV RNA replication through modulation of the glutathione redox system and oxidative stress.     

Microtubule-Associated Protein 1 Light Chain 3 Interacts with and Contributes to Growth Inhibiting Effect of PML

Previously we reported that the expression of promyelocytic leukemia (PML)-retinoic acid receptor alpha (RARα) fusion gene, which is caused by specific translocation (15;17) in acute promyelocytic leukemia, can enhance constitutive autophagic activity in leukemic and nonleukemic cells, and PML overexpression can sequestrate part of microtubule-associated protein light chain 3 (LC3) protein in PML nuclear bodies, suggesting that LC3 protein also distributes into nuclei although it is currently thought to function primarily in the cytoplasm, the site of autophagosomal formation. However, its potential significance of nucleoplasmic localizations remains greatly elusive. Here we demonstrate that PML interacts with LC3 in a cell type-independent manner as assessed by Co-IP assay and co-localization observation. Overexpressed PML significantly coprecipitates with endogenous and nuclear LC3 protein. Furthermore, a fraction of endogenous PML protein is found to be co-localized with LC3 protein under steady state condition, which is further enhanced by IFNα induction, indicating that PML up-regulation potentiates this interaction. Additionally, DsRed-PML associates with EGFP-LC3 during telophase and G1 phase but not in metaphase and anaphase. Two potential LC3-interacting region (LIR) motifs in PML are required for interaction of PML with LC3 while this association is independent of autophagic activity. Finally, we show that interaction between PML and LC3 contributes to cell growth inhibition function of PML. Considering that PML is an important tumor suppressor, we propose that nuclear portion of LC3 protein may associate with PML to control cell growth for prevention and inhibition of cancer occurrence and development.     

Differential effect of ascorbic acid and n-acetyl-L-cysteine on arsenic trioxide-mediated oxidative stress in human leukemia (HL-60) cells

Arsenic trioxide (ATO) has been recommended for the treatment of refractory cases of acute promyelocytic leukemia (APL). Recent studies in our laboratory indicated that oxidative stress plays a key role in ATO-induced cytotoxicity in human leukemia (HL-60) cells. In the present investigation, we performed the MTT assay and trypan blue exclusion test for cell viability. We also performed the thiobarbituric acid test to determine the levels of malondialdehyde (MDA) production in HL-60 cells coexposed to either ascorbic acid (AA) and ATO or to n-acetyl-L-cysteine (NAC) and ATO. The results of MTT assay indicated that AA exposure potentiates the cytotoxicity of ATO in HL-60 cells, as evidenced by a gradual increase in MDA levels with increasing doses of AA. In contrary, the addition of NAC to ATO-treated HL-60 cells resulted in a dose-dependent decrease of MDA production. From these results, we conclude that the addition of the AA to ATO-treated HL-60 cells enhances the formation of reactive oxygen species (ROS), whereas the addition of NAC under the same experimental condition significantly (p < .05) decreases the level of ROS formation. On the basis of these direct in vitro findings, our studies provide evidence that AA may extend the therapeutic spectrum of ATO. The coadministration of NAC with ATO shows a potential specificity for tumor cells, indicating that it may not enhance the clinical outcome associated with ATO monotherapy in vivo.     

Synergistic effects of arsenic trioxide and radiation in osteosarcoma cells through the induction of both autophagy and apoptosis

Osteosarcoma is the most common primary malignant bone tumor, occurring mainly in children and adolescents, and survival largely depends on their response to chemotherapy. However, the risk of relapse and adverse outcomes is still high. We investigated the synergistic anti-cancer effects of ionizing radiation combined with arsenic trioxide (ATO) and the mechanisms underlying apoptosis or autophagy induced by combined radiation and ATO treatment in human osteosarcoma cells. We found that exposure to radiation increased the population of HOS cells in the G(2)/M phase within 12 h in a time-dependent manner. Radiation combined with ATO induced a significantly prolonged G(2)/M arrest, consequently enhancing cell death. Furthermore, combined treatment resulted in enhanced ROS generation compared to treatment with ATO or radiation alone. The enhanced cytotoxic effect of combined treatment occurred from the increased induction of autophagy and apoptosis through inhibition of the PI3K/Akt signaling pathway in HOS cells. The combined treatment of HOS cells pretreated with Z-VAD, 3-MA or PEG-catalase resulted in a significant reduction of cytotoxicity. In addition, G(2)/M arrest and ROS generation could be involved in the underlying mechanisms. The data suggest that a combination of radiation and ATO could be a new potential therapeutic strategy for the treatment of osteosarcoma.     

Elevated cerebrospinal fluid leukocyte count and protein concentration at diagnosis: independent risk factors in children with acute lymphoblastic leukemia

The aim of this study was to investigate whether determination of the initial cerebrospinal fluid (CSF) protein concentration and leukocyte count in children with acute lymphoblastic leukemia (ALL) could yield useful information about the patient's central nervous system status and prognosis. The population-based unselected series comprised 160 children. The mean follow-up time was 72 months (range 25-143 months). Both the CSF protein concentration and the leukocyte count, if elevated, were significantly, although not independently, associated with diminished probability of event-free survival. The patients were divided into three groups for the final analyses: those without any abnormalities in the CSF (n = 133), those with elevated protein concentration and/or elevated leukocyte count, but with no malignant lymphoblasts in the CSF (n = 21), and those with malignant lymphoblasts in the CSF (n = 6). The probabilities of 5-year event-free survival for the first and second group were 65% and 15%; the probability of 2-year event-free survival for the third group was 17%. These differences were statistically significant (p less than 0.001). In multivariate analysis the relative risks of death or relapse for these groups were 1, 2.8 (95% confidence limits 1.5-4.9), and 7.6 (2.4-24.3), respectively (p less than 0.001). The inclusion of an elevated CSF protein concentration or leukocyte count in the risk group criteria of further trials should be considered.     

EVI-1 modulates arsenic trioxide induced apoptosis through JNK signalling pathway in leukemia cells

High expression of the oncogene ecotropic viral integration site-1 (EVI-1) is an independent negative prognostic indicator of survival in leukemia patients. This study aimed to examine the effects of arsenic trioxide (ATO) on EVI-1 in acute myeloid leukemia (AML). Mononuclear cells were isolated from the bone marrow and peripheral blood of AML patients and healthy donors. EVI-1 expression in hematopoietic cells was evaluated by RT-qPCR and Western blot analysis. EVI-1 was highly expressed in both primary AML and leukemia cell lines (THP-1 and K562). ATO down-regulated EVI-1 mRNA in zebrafish in vivo as well as in primary leukemia cells and THP-1 and K562 cells in vitro. Additionally, ATO treatment induced apoptosis, down-regulated both EVI-1 mRNA and oncoprotein expression, increased the expression of pro-apoptosis proteins, and decreased the expression of anti-apoptotic proteins in leukemia cells in vitro. EVI-1 expression in leukemia cells (THP-1 and K562) transduced with EVI-1 siRNA was significantly reduced. Silencing EVI-1 had a significant effect on the activation of the JNK pathway and the induction of leukemia cell apoptosis. ATO may downregulate EVI-1 mRNA and oncoprotein levels and block the inhibitory effects of EVI-1 on the JNK pathway, which activates the JNK apoptotic pathway, thereby leading to the apoptosis of EVI-1 in AML patients.