BCR-ABL1-induced leukemogenesis and autophagic targeting by arsenic trioxide

We have recently shown that arsenic trioxide (As₂O₃) is a potent inducer of autophagic degradation of the BCR-ABL1 oncoprotein, which is the cause of chronic myeloid leukemia (CML) and Ph+ acute lymphoid leukemia (Ph+ ALL). Our recently published work has shown that pharmacological inhibition of autophagy or molecularly targeting of elements of the autophagic machinery partially reverses the suppressive effects of As₂O₃ on primitive leukemic precursors from CML patients. Altogether, our studies have provided direct evidence that arsenic-induced, autophagy-mediated, degradation of BCR-ABL1 is an important mechanism for the generation of the effects of As₂O₃ on BCR-ABL1 transformed leukemic progenitors. These studies raise the potential of future clinical-translational efforts employing combinations of arsenic trioxide with autophagy-modulating agents to promote elimination of early leukemic progenitors and, possibly, leukemia-initiating stem cells.     

Combination of arsenic trioxide and cisplatin synergistically inhibits both hexokinase activity and viability of Ehrlich ascites carcinoma cells

Hexokinase‐2 is overexpressed in several carcinomas including breast cancer to sustain energy for rapidly dividing cells and associates with chemoresistance. However, the impact of chemo drugs (alone or in combination) on hexokinase activity and autophagic cell death is unclear. In this report, we used an in vivo murine adenocarcinoma model to validate the effects of As₂O₃ and cisplatin on hexokinase activity and autophagic cancer cell death. We found that the two drugs inhibit hexokinase activity and induce autophagic marker, beclin 1 expression. Interestingly, combining As₂O₃ with cisplatin synergistically enhanced these effects and alleviated oxidative stress often encountered in As₂O₃ treatment. Altogether, our data provide direct evidence that inhibition of hexokinase activity and induction of autophagic cell death are mediating the antineoplastic effects of As₂O₃ and cisplatin. Our findings raise the potential of combining As₂O₃ with cisplatin as an approach to augment cisplatin‐induced cell death and combat cisplatin chemoresistance in cancer.     

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.     

Potent antitumor activities of recombinant human PDCD5 protein in combination with chemotherapy drugs in K562 cells

Conventional chemotherapy is still frequently used. Programmed cell death 5 (PDCD5) enhances apoptosis of various tumor cells triggered by certain stimuli and is lowly expressed in leukemic cells from chronic myelogenous leukemia patients. Here, we describe for the first time that recombinant human PDCD5 protein (rhPDCD5) in combination with chemotherapy drugs has potent antitumor effects on chronic myelogenous leukemia K562 cells in vitro and in vivo. The antitumor efficacy of rhPDCD5 protein with chemotherapy drugs, idarubicin (IDR) or cytarabine (Ara-C), was examined in K562 cells in vitro and K562 xenograft tumor models in vivo. rhPDCD5 protein markedly increased the apoptosis rates and decreased the colony-forming capability of K562 cells after the combined treatment with IDR or Ara-C. rhPDCD5 protein by intraperitoneal administration dramatically improved the antitumor effects of IDR treatment in the K562 xenograft model. The tumor sizes and cell proliferation were significantly decreased; and TUNEL positive cells were significantly increased in the combined group with rhPDCD5 protein and IDR treatment compared with single IDR treatment groups. rhPDCD5 protein, in combination with IDR, has potent antitumor effects on chronic myelogenous leukemia K562 cells and may be a novel and promising agent for the treatment of chronic myelogenous leukemia.     

Arsenic Trioxide and Resveratrol Show Synergistic Anti-Leukemia Activity and Neutralized Cardiotoxicity

Cardiotoxicity is an aggravating side effect of many clinical antineoplastic agents such as arsenic trioxide (As₂O₃), which is the first-line treatment for acute promyelocytic leukemia (APL). Clinically, drug combination strategies are widely applied for complex disease management. Here, an optimized, cardiac-friendly therapeutic strategy for APL was investigated using a combination of As₂O₃ and genistein or resveratrol. Potential combinations were explored with respect to their effects on mitochondrial membrane potential, reactive oxygen species, superoxide dismutase activity, autophagy, and apoptosis in both NB4 cells and neonatal rat left ventricular myocytes. All experiments consistently suggested that 5 µM resveratrol remarkably alleviates As₂O₃-induced cardiotoxicity. To achieve an equivalent effect, a 10-fold dosage of genistein was required, thus highlighting the dose advantage of resveratrol, as poor bioavailability is a common concern for its clinical application. Co-administration of resveratrol substantially amplified the anticancer effect of As₂O₃ in NB4 cells. Furthermore, resveratrol exacerbated oxidative stress, mitochondrial damage, and apoptosis, thereby reflecting its full range of synergism with As₂O₃. Addition of 5 µM resveratrol to the single drug formula of As₂O₃ also further increased the expression of LC3, a marker of cellular autophagy activity, indicating an involvement of autophagy-mediated tumor cell death in the synergistic action. Our results suggest a possible application of an As₂O₃ and resveratrol combination to treat APL in order to achieve superior therapeutics effects and prevent cardiotoxicity.     

SnoN/SkiL,a TGFβ signaling mediator

Effective treatment of cancer cells with chemotherapeutic drugs relies on their ability to induce cell death, making the discovery of their mechanisms of action crucial. Arsenic trioxide (As₂O₃), used in the treatment of promyelocytic leukemia (PML), triggers cell death in several solid tumor cell lines including ovarian carcinomas. While As₂O₃ is remarkably cytotoxic in human ovarian cancer cells, its mechanism of action is poorly understood. We recently investigated the effects of As₂O₃ on several transforming growth factor-β (TGFβ) signaling mediators to better understand its cell death mechanism. Indeed, dysregulated (TGFβ) signaling is typical of ovarian cancers. Based on our findings, we propose that As₂O₃ induces a Beclin 1-independent autophagic pathway in ovarian carcinoma cells by modulating SnoN/SkiL expression, implicating SnoN as a novel therapeutic target for ovarian cancers.     

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.     

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.     

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.     

Arsenic trioxide induces G2/M arrest in hepatocellular carcinoma cells by increasing the tumor suppressor PTEN expression

Arsenic trioxide (As₂O₃), an effective agent against acute promyelocytic leukemia, has been reported to inhibit the viability of solid tumors cell lines recently. The detailed molecular mechanism underlying the As₂O₃‐induced inactivation of the cdc2 and possible functional role of PTEN in the observed G2/M arrest has yet to be elucidated. Here, we assessed the role of PTEN in regulation of As₂O₃‐mediated G2/M cell cycle arrest in Hepatocellular carcinoma cell lines (HepG2 and SMMC7721). After 24 h following treatment, As₂O₃ induced a concentration‐dependent accumulation of cells in the G2/M phase of the cell cycle. The sustained G2/M arrest by As₂O₃ is associated with decreased cdc2 protein and increased phospho‐cdc2(Tyr15). As₂O₃ treatment increased Wee1 levels and decreased phospho‐Wee1(642). Moreover, As₂O₃ substantially decreased the Ser473 and Thr308 phosphorylation of Akt and upregulated PTEN expression. Downregulation of PTEN by siRNA in As₂O₃‐treated cells increased phospho‐Wee1(Ser642) while decreased phospho‐cdc2(Tyr15), resulting in decreased the G2/M cell cycle arrest. Therefore, induction of G2/M cell cycle arrest by As₂O₃ involved upregulation of PTEN. J. Cell. Biochem. 113: 3528–3535, 2012. © 2012 Wiley Periodicals, Inc.     

(−)‐Epicatechin rescues the As2O3‐induced HERG K+ channel deficiency possibly through upregulating transcription factor SP1 expression

(?)‐Epicatechin (EPI) has beneficial effects on the cardiovascular disease. The human ether‐a‐go‐go‐related gene (HERG) potassium channel is crucial for repolarization of cardiac action potential. Dysfunction of the HERG channel can cause long QT syndrome type 2 (LQT2). Arsenic trioxide (As₂O₃) has shown efficacy in the treatment of acute promyelocytic leukemia. However, As₂O₃ can induce the deficiency of HERG channel and cause LQT2. In this study, we examined whether EPI could rescue the As₂O₃‐induced HERG channel deficiency. We found that 3 μM EPI obviously increased protein expression and current of HERG channel. EPI was able to recover the protein expression and current of HERG channel disrupted by As₂O₃. EPI was able to increase the expression of SP1 protein and recover the expression of SP1 protein disrupted by As₂O₃. In addition, EPI significantly shortened action potential duration prolonged by As₂O₃. Our data suggest that EPI rescues As₂O₃‐induced HERG channel deficiency through upregulating SP1 expression.     

Inhibitory role of TGIF in the As<Subscript>2</Subscript>O<Subscript>3</Subscript>-regulated p21<Superscript><Emphasis Type="Italic">WAF1/CIP1</Emphasis></Superscript> expression

Although arsenic is an infamous carcinogen, it has been effectively used to treat acute promyelocytic leukemia, and can induce cell cycle arrest or apoptosis in human solid tumors. Previously, we had demonstrated that opposing effects of ERK1/2 and JNK on p21 expression in response to arsenic trioxide (As₂O₃) are mediated through the Sp1 responsive elements of the p21 promoter in A431 cells. Presently, we demonstrate that Sp1, and c-Jun functionally cooperate to activate p21 promoter expression through Sp1 binding sites (−84/−64) by using DNA affinity binding, chromatin immunoprecipitation, and promoter assays. Surprisingly, As₂O₃-induced c-Jun(Ser63/73) phosphorylation can recruit TGIF/HDAC1 to the Sp1 binding sites and then suppress p21 promoter activation. We suggest that, after As₂O₃ treatment, the N-terminal domain of c-Jun phosphorylation by JNK recruits TGIF/HDAC1 to the Sp1 sites and then represses p21 expression. That is, TGIF is involved in As₂O₃-inhibited p21 expression, and then blocks the cell cycle arrest.     

Participation of autophagy in renal ischemia/reperfusion injury

Renal ischemia-reperfusion (I/R) injury is inevitable in transplantation, and it results in renal tubular epithelial cells undergoing cell death. We observed an increase in autophagosomes in the tubular epithelial cells of I/R-injured mouse models, and in biopsy specimens from human transplanted kidney. However, it remains unclear whether autophagy functions as a protective pathway, or contributes to I/R-induced cell death. Here, we employed the human renal proximal tubular epithelial cell line HK-2 in order to explore the role of autophagy under hypoxia (1% O₂) or activation of reactive oxygen species (500 μM H₂O₂). When compared to normoxic conditions, 48 h of hypoxia slightly increased LC3-labeled autophagic vacuoles and markedly increased LAMP2-labeled lysosomes. We observed similar changes in the mouse IR-injury model. We then assessed autophagic generation and degradation by inhibiting the downstream lysosomal degradation of autophagic vacuoles using lysosomal protease inhibitor. We found that autophagosomes increased markedly under hypoxia in the presence of lysosomal protease inhibitors, thus suggesting that hypoxia induces high turnover of autophagic generation and degradation. Furthermore, inhibition of autophagy significantly inhibited H₂O₂-induced cell death. In conclusion, high turnover of autophagy may lead to autophagic cell death during I/R injury.     

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.     

A rapid and high content assay that measures cyto-ID-stained autophagic compartments and estimates autophagy flux with potential clinical applications

The lack of a rapid and quantitative autophagy assay has substantially hindered the development and implementation of autophagy-targeting therapies for a variety of human diseases. To address this critical issue, we developed a novel autophagy assay using the newly developed Cyto-ID fluorescence dye. We first verified that the Cyto-ID dye specifically labels autophagic compartments with minimal staining of lysosomes and endosomes. We then developed a new Cyto-ID fluorescence spectrophotometric assay that makes it possible to estimate autophagy flux based on measurements of the Cyto-ID-stained autophagic compartments. By comparing to traditional autophagy approaches, we found that this assay yielded a more sensitive, yet less variable, quantification of the stained autophagic compartments and the estimate of autophagy flux. Furthermore, we tested the potential application of this autophagy assay in high throughput research by integrating it into an RNA interference (RNAi) screen and a small molecule screen. The RNAi screen revealed WNK2 and MAP3K6 as autophagy-modulating genes, both of which inhibited the MTOR pathway. Similarly, the small molecule screen identified sanguinarine and actinomycin D as potent autophagy inducers in leukemic cells. Moreover, we successfully detected autophagy responses to kinase inhibitors and chloroquine in normal or leukemic mice using this assay. Collectively, this new Cyto-ID fluorescence spectrophotometric assay provides a rapid, reliable quantification of autophagic compartments and estimation of autophagy flux with potential applications in developing autophagy-related therapies and as a test to monitor autophagy responses in patients being treated with autophagy-modulating drugs.     

Opposite effect of ERK1/2 and JNK on p53-independent p21 WAF1/CIP1 activation involved in the arsenic trioxide-induced human epidermoid carcinoma A431 cellular cytotoxicity

Summary 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 p21 ^WAF1/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.     

PML-RARα enhances constitutive autophagic activity through inhibiting the Akt/mTOR pathway

Autophagy is a highly conserved, closely regulated homeostatic cellular activity that allows for the bulk degradation of long-lived proteins and cytoplasmic organelles. Its roles in cancer initiation and progression and in determining the response of tumor cells to anticancer therapy are complicated, and only limited investigation has been conducted on the potential significance of autophagy in the pathogenesis and therapeutic response of acute myeloid leukemia. Here we demonstrate that the inducible or transfected expression of the acute promyelocytic leukemia (APL)-specific PML-RARα, but not PLZF-RARα or NPM-RARα, fusion protein upregulates constitutive autophagy activation in leukemic and nonleukemic cells, as evaluated by hallmarks for autophagy including transmission electron microscopy. The significant increase in autophagic activity is also found in the leukemic cells-infiltrated bone marrow and spleen from PML-RARα-transplanted leukemic mice. The autophagy inhibitor 3-methyladenine significantly abrogates the autophagic events upregulated by PML-RARα, while the autophagic flux assay reveals that the fusion protein induces autophagy by increasing the on-rate of autophagic sequestration. Furthermore, this modulation of autophagy by PML-RARα is possibly mediated by a decreased activation of the Akt/mTOR pathway. Finally, we also show that autophagy contributes to the anti-apoptotic function of the PML-RARα protein. Given the critical role of the PML-RARα oncoprotein in APL pathogenesis, this study suggests an important role of autophagy in the development and treatment of this disease.