Arsenic trioxide induces depolymerization of microtubules in an acute promyelocytic leukemia cell line

doi:10.5045/kjh.2012.47.2.105

Korean J Hematol. 2012 June; 47(2): 105–112.

Published online 2012 June 26. doi: 10.5045/kjh.2012.47.2.105

PMCID: PMC3389058

Arsenic trioxide induces depolymerization of microtubules in an acute promyelocytic leukemia cell line

Jin Ho Baek,¹^# Chang Hoon Moon,²^# Seung Joo Cha,² Hee Soon Lee,² Eui-Kyu Noh,^1,² Hawk Kim,^1,² Jong-Ho Won,³ and Young Joo Min^1,²

¹Division of Hematology and Oncology, Department of Internal Medicine, Ulsan University Hospital, University of Ulsan College of Medicine, Ulsan, Korea.

²Biomedical Research Center, Ulsan University Hospital, University of Ulsan College of Medicine, Ulsan, Korea.

³Division of Hematology-Oncology, Soonchunhyang University College of Medicine, Seoul, Korea.

Corresponding author.

Correspondence to Young Joo Min, M.D., Ph.D. Biomedical Research Center, Ulsan University Hospital, University of Ulsan College of Medicine, 290-3, Jeonha-dong, Dong-gu, Ulsan 682-714, Korea. Tel: +82-52-250-8832, Fax: +82-52-250-8520, Email: yjmin/at/uuh.ulsan.kr

^#These authors equally contributed to this work.

Received March 9, 2012; Revised May 7, 2012; Accepted May 16, 2012.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

Arsenic trioxide (As₂O₃) is a well-known and effective treatment that can result in clinical remission for patients diagnosed with acute promyelocytic leukemia (APL). The biologic efficacy of As₂O₃ in APL and solid tumor cells has been explained through its actions on anti-proliferation, anti-angiogenesis, and apoptotic signaling pathways. We theorize that As₂O₃ activates a pathway that disrupts microtubule dynamics forming abnormal, nonfunctioning mitotic spindles, thus preventing cellular division. In this study, we investigated how As₂O₃ induces apoptosis by causing microtubule dysfunction.

Methods

Cultured NB4 cells were treated with As₂O₃, paclitaxel, and vincristine. Flow cytometric analysis was then performed. An MTT assay was used to determine drug-mediated cytotoxicity. For tubulin polymerization assay, each polymerized or soluble tubulin was measured. Microtubule assembly-disassembly was measured using a tubulin polymerization kit. Cellular microtubules were also observed with fluorescence microscopy.

Results

As₂O₃ treatment disrupted tubulin assembly resulting in dysfunctional microtubules that cause death in APL cells. As₂O₃ markedly enhanced the amount of depolymerized microtubules. The number of microtubule posttranslational modifications on an individual tubulin decreased with As₂O₃ concentration. Immunocytochemistry revealed changes in the cellular microtubule network and formation of polymerized microtubules in As₂O₃-treated cells.

Conclusion

The microtubules alterations found with As₂O₃ treatment suggest that As₂O₃ increases the depolymerized forms of tubulin in cells and that this is potentially due to arsenite's negative effects on spindle dynamics.

Keywords: Acute promyelocytic leukemia, Arsenic trioxide, Tubulin, Apoptosis, Antimitotic agents

INTRODUCTION

Arsenic compounds were used therapeutically by the ancients and are also widely utilized in traditional Chinese medicine. Arsenic trioxide (As₂O₃) is a cancer therapeutic that can be used alone or in combination with other agents to treat patients with acute promyelocytic leukemia (APL). Notably, As₂O₃ treatment has been known to achieve high rates of remission. In newly diagnosed patients, complete remission rates have ranged from 86% to 95%, and, in relapsed patients, rates of 80% to 93% have been reported [1]. Moreover, As₂O₃ has shown to exhibit anticancer properties in diseases such as multiple myeloma and myelodysplastic syndromes. As such, there has been great interest in As₂O₃ as a chemotherapeutic drug. Arsenic affects numerous intracellular signal transduction pathways and causes many alterations in cellular function. These actions of arsenic may result in the induction of apoptosis, the inhibition of growth and angiogenesis, and the promotion of differentiation [2]. However, the mechanism whereby arsenic specifically targets tumor cells is not clearly understood.

Of the various target elements affected by arsenic, microtubules are critical elements in a wide variety of fundamental cell functions, including mitosis, motility, and intracellular vesicular transport [3]. As such, microtubules are a good target for many natural toxins as well as chemotherapeutic drugs [4, 5]. Disruption of microtubule dynamics induces abnormal formation or loss of a functional mitotic spindle, both of which interrupt cell division and cause cell death. Several lines of evidence have previously been reported to suggest that As₂O₃ affects spindle formation/function by modulating assembly and/or disassembly of tubulins in several cancer cell lines, including leukemic cells [6, 7]. Together, these data suggest that As₂O₃ may mimic microtubule poisons and interrupt mitosis [7].

To date, a few reports have indicated that, depending on the cell line, As₂O₃ treatment results in cell-cycle arrest at the G₁ or G₂/M phase via microtubule dysfunction [8]. Other contrastive opinions for the As₂O₃ mechanism of action on the assembly/disassembly of tubulin in tumor cell lines have also been provided [7-9]. Li and Broome reported that As₂O₃ treatment resulted in cell-cycle arrest at metaphase in myeloid leukemia cells through noncompetitive disruption of GTP binding to β-tubulin and inhibition of GTP-induced tubulin polymerization [9]. Ling et al. reported that As₂O₃ caused a paclitaxel-like, concentration-dependent, tubulin polymerization in vitro and altered the microtubule network as determined by cell morphology and electron microscopy (EM) analysis [8].

In this study, we wanted to show that As₂O₃ might induce apoptosis by causing microtubule dysfunction. We investigated whether As₂O₃ promotes or inhibits the polymerization of microtubules in an APL cell line. Thus, we hoped to better understand the unique mechanism underlying the action of As₂O₃.

MATERIALS AND METHODS

1. Cell culture and compounds

Acute promyelocytic leukemia (APL) NB4 cells (provided by Dr. J. H. Won of Soon Chun Hyang University College of Medicine, Korea) were maintained as suspension cultures in PRMI-1640 (Gibco, Invitrogen Ltd, UK) containing 10% fetal bovine serum (Gibco, Invitrogen Ltd, UK) and 1× penicillin-streptomycin (Gibco, Invitrogen Ltd, UK). Cells were maintained at 37 [degree celsius]

in water-saturated atmosphere containing 5% CO₂. As₂O₃ was purchased from Sigma Chemical Inc. and dissolved in 1 M NaOH as stock solution. Paclitaxel was purchased from BMS Pharmaceutical Korea Limited and a stock solution of paclitaxel (Taxol®, Korea) was prepared as per the manufacturer's suggestion. Vincristine was purchased from Reyon Pharmaceutical Co., LTD and prepared at a concentration of 0.1 M in PBS. Monoclonal anti-α-tubulin, anti-acetylated α-tubulin, anti-glycealdehyde-3-phosphate dehydrogenase (GAPDH), and HRP-conjugated secondary antibody were purchased from Santa Cruz Biotechnology, Inc., CA, USA.

2. Flow cytometric analysis

NB4 cells (2×10⁶ cells) were treated with each drug at the indicated concentration for 24 hours. Cells were washed with PBS by spinning at 1,000 rpm for 5 min at 4 [degree celsius]

and fixed with 1 mL of ice-cold 95% ethanol drop-wise while vortexing. The fixed cells were incubated in ice-cold 95% ethanol for at least 30 min and then centrifuged as described above. The pellets were resuspended in 1 mL PBS and 100 µL of 1 mg/mL propidium iodide (PI) for 5-10 min at room temperature. Flow cytometric analysis was performed using a FACSCalibur Flow Cytometer and Cell Quest software (BD biosciences, San Jose, CA, USA).

3. Cytotoxicity assay

To determine drug-mediated cytotoxicity, an MTT assay was performed. This was carried out using the MTS reagent of the Cell Titer 96 Aqueous One Solution (Promega Corp., USA) with absorbance measured at 490 nm, as previously described [10]. Briefly, NB4 cells were seeded in 0.5×10⁵ cells per well for 24 hrs at 37 [degree celsius]

and 5% CO₂. After treatment with the indicated drug concentrations, cells were reincubated for 24 hrs and 48 hrs. Twenty microliters of the MTS solution was added to each well and incubated at 37 [degree celsius]

. Thereafter, the absorbance (A) values of each well was determined using a microplate spectrophotometer (Bio-Tek Instruments Inc., USA). The percentage of viable cells was calculated using the background-corrected absorbance as follows:

% cytotoxicity=(1-A of experimental well/A of positive control well)×100

4. Tubulin polymerization assay

Cells were plated in 24-well plates, grown for 1 day (60-80% confluency), and treated with either no drug or varying concentrations of drugs at 37 [degree celsius]

for 6 hours. After the medium was removed, cells were rinsed in 1× PBS at 22 [degree celsius]

, harvested at the same temperature in lysis buffer containing 0.1 M PIPES, 1 mM EGTA, 1 mM MgSO₄, 30% glycerol, 5% DMSO, 5 mM GTP, 0.125% NP-40, and protease inhibitor cocktail (Calbiochem, Merck KGaA, Darmstadt, Germany), and then centrifuged at 13,200 rpm at 22 [degree celsius]

for 30 min followed by vortexing, to separate polymerized (P) from soluble (S) tubulin [11]. Pellets of polymerized "P" tubulin were resuspended in a volume of lysis buffer equal to the soluble "S" fraction. Each had gel sample buffer added and equal aliquots separated on 10% SDS-PAGE gels were transferred to PVDF membranes (Whatman). The membranes were blocked with 5% skim milk for 2 hrs in TBS buffer containing 0.1% Tween 20 and either the α-tubulin antibody (SantaCruze Biotechnology, CA, USA) or acetylated α-tubulin (SantaCruze Biotechnology, CA, USA). Antibodies were incubated overnight at 4 [degree celsius]

with constant shaking. After washing, an HRP-conjugated anti-mouse antibody was applied at a dilution of 1:10,000. Secondary antibody binding was carried out at room temperature for 2 hrs with shaking. To visualize the protein bands, chemiluminescence detection was performed with the Chemiluminescent kit (Bio-Rad Laboratories, CA, USA). The blots were reprobed with a monoclonal antibody against GAPDH, and the results were used as loading controls. Band intensities were quantified by densitometry using Bio-Rad software.

5. Tubulin assembly in vitro

The effects of As₂O₃ on the microtubule assembly-disassembly process were determined using a tubulin polymerization kit (Cytoskeleton, Denver, CO, USA). Purified bovine brain tubulin was resuspended on ice in ice-cold buffer (80 mM PIPES at pH 6.9, 2 mM MgCl₂, 0.5 mM EGTA, 15% glycerol, 1 mM GTP) and 100 µL of the suspension (3 mg/mL) was aliquoted into half of a 96-well plate pre-warmed to 37 [degree celsius]

as per the manufacturer's instructions. The assay was determined by measuring the change of absorbance (340 nm) at room temperature using a spectrophotometer every 5 min for 70 min (µQuant, Bio-Tek Instruments Inc., USA).

6. Immunocytochemitry

After treatment with As₂O₃, paclitaxel, and vincristine for 6 hours, cells were washed with PBS solution and the cell suspension was then incubated on a glass slides coated with poly-L-lysine for 30 min at room temperature. After aspirating the excess cell suspension, the cells were fixed with 3.7% formaldehyde in PBS for 10 minutes and permeabilized with 50 µL of BD Cytofix/Cytoperm solution (BD Biosciences, San Jose, CA, USA) for 25 min at 4 [degree celsius]

. Permeabilize, fixed cells were washed twice with 100 µL of 1× BD Perm/Wash buffer (BD Biosciences, San Jose, CA, USA) for 5 min. Fixed cells were incubated with a monoclonal anti-α tubulin antibody (1:50; Santa Cruz Biotechnology, CA, USA) at room temperature for 1 hr. After washing 3 times in 100 µL with 1× BD Perm/Wash buffer (BD Biosciences, San Jose, CA, USA) for 5 min, cells were reincubated with FITC-conjugated second antibody (1:100, Santa Cruz Biotechnology, CA, USA) in the dark for 1 hr. Cellular microtubules were imaged using an Olympus IX-81 fluorescence microscope.

RESULTS

1. Effect of As₂O₃ on NB4 cell growth and apoptosis

In a previously reported study, As₂O₃-induced anti-proliferation in various tumor cell lines was evaluated and a clinical study revealed that the effective dose of As₂O₃ in vivo/vitro was 0.5-3 µM [9, 12]. However, to confirm As₂O₃ cytotoxicity in NB4 cells, we examined different concentration of As₂O₃ and measured cytotoxicity via an MTT assay after 24 and 48 hrs of treatment. As shown in Fig. 1A, the cytotoxic effect of As₂O₃ was dose and time dependent. At 5 µM As₂O₃, NB4 cell growth was inhibited by more than 50%. We next examined whether the inhibition of cell growth by As₂O₃ could cause apoptosis. After NB4 cells were treated with the indicated concentration of As₂O₃ for 24 hrs, As₂O₃-induced apoptosis was determined by propidium iodide (PI) staining and compared with controls for 5 nM and 7 nM paclitaxel treatments. As shown in Fig. 1B and Table 1, paclitaxel, a microtubule-targeting drug, induced G₂/M phase arrest and caused apoptosis. At a low concentration (<1 µM As₂O₃), <13% of cells were in the G₂/M phase but at a concentration of >3 µM, >40% of cells were in the G₂/M phase. Similar to paclitaxel treatment, As₂O₃ treatment resulted in a concentration-dependent accumulation of NB4 cells in the G₂/M phase, although As₂O₃-treated cells resulted in a higher concentration in this phase than when cells were treated with paclitaxel. Cell cycle arrest induced by As₂O₃ treatment is initiated by the dissipation of the mitochondrial membrane potential (MMP), which leads to the release of cytochrome c (data not shown). Together, these data indicate that As₂O₃-induced apoptosis was preceded by arrest in the G₂/M phase of the cell cycle similar to paclitaxel-induced apoptosis.

Fig. 1

Induction of cytotoxicity and cell cycle arrest by As₂O₃. (A) To show the inhibitory effects of As₂O₃ on the cell proliferation, exponentially growing cells were treated with the indicated concentrations of As₂O₃ for 24 hour and 48 hours, and cell proliferation (more ...)

Table 1

Cell cycle distribution of cells treated with arsenic trioxide and paclitaxel for 24 hours.

2. Effect of As₂O₃ on tubulin polymerization

Because As₂O₃ treatment markedly blocked the cell cycle in the G₂/M phase, we became interested in testing whether As₂O₃ would disrupt or enhance tubulin polymerization in NB4 cells. To test this hypothesis, we examined the affects of tubulin polymerization and depolymerization on the treatment at various concentrations of As₂O₃ for a 6 hours treatment. Vincristine and paclitaxel were used as positive controls and all experiments were performed by separating polymerized "P" from soluble "S" tubulin at 22 [degree celsius]

. As shown in Fig. 2A, vincristine or paclitaxel enhanced the cellular levels of the "S" fraction and "P" fraction in the cell lysates, similar to previously published reports. When cells were treated with increasing concentrations of As₂O₃, the amount of tubulin in the depolymerized fraction, "S", was increased compared with untreated cells. The baseline proportion of α-tubulin in the soluble fraction was 59.6%, while the depolymerized proportion observed after treatment was approximately 52-72%. The tubulin shift to the "S" fraction was exactly noted over the 5-20 µM As₂O₃ range. As such, the increase in the number of cells in arrest with As₂O₃ found in the apoptosis study correlated with the increased "S" fraction of tubulin and was greater than that in the control cells. These data clearly indicate that As₂O₃ induces tubulin depolymerization, although the effect is not as strong as vincristine-induced depolymerization.

Fig. 2

Alterations in tubulin polymerization induced by As₂O₃. (A) To demonstrate that the fraction of depolymerized tubulin is increased after treatment with As₂O₃, NB4 cells were treated with drugs or without drugs as a control for 6 hours at the indicated (more ...)

Since As₂O₃ reduced the cytotoxicity of antimicrotubule drugs in SK-N-SH cells [13], we investigated whether the combination of paclitaxel and As₂O₃ synergistically affected tubulin assembly in NB4 cells. To examine to the synergistic mechanism of As₂O₃ and paclitaxel treatment on microtubule formation, we treated cells with 5 µM As₂O₃ alone, 10 µM As₂O₃ alone, and 40 nM paclitaxel alone, or in combination at the same concentrations for 7 hours. The results shown in Fig. 2B reveal that the combined effects of As₂O₃ and paclitaxel did not influence the tubulin polymerization and microtubule assembly.

3. Effect of As₂O₃ on microtubule stability

It is well established that tubulin can be altered by post-translational modifications, and several of these are exclusive to α-tubulin. Although the precise relationship between these tubulin modifications and microtubule stability has not been elucidated, several changes are definitive markers of microtubule stability [14, 15]. Therefore, we examined the extent of acetylation of α-tubulin, a posttranslational modification indicative of stabilized microtubules, often observed following exposure of cells to microtubule stabilizing agents (i.e., paclitaxel and epothilone) [16]. As shown in Fig. 3, we assessed the expression levels of modified α-tubulin forms in separated fractions of NB4 cell lysates. The proportions below the bands represent the expression quantity in each fraction. We observed a marked increase in the amount of acetylated α-tubulin (<90%) in the polymerized fraction (P) of paclitaxel treated cells, compared to the controls. In addition, the proportion of polymerized fraction (P) with As₂O₃ treatment was decreased (<62%) compared to untreated cells. Therefore, these data indicate that As₂O₃ increases microtubule dynamics by interfering with post-translational modifications involved in microtubule stability.

Fig. 3

Effects of As₂O₃ on microtubule stabilization. NB4 cells were treated with drugs or no drugs as a control, for 6 hours and 24 hours at the indicated concentration. Lysates were separated into polymerized (P) or soluble (S) fractions. The blots probed (more ...)

4. Effect of As₂O₃ on tubulin polymerization in vitro

Because As₂O₃ treatment markedly increased the disassembled portion of tubulin and influenced the stability of microtubules, we became interested in testing whether As₂O₃ could directly affect tubulin. To test this hypothesis, tubulin polymerization and depolymerization in vitro were examined at 37 [degree celsius]

in a reaction mixture containing purified tubulin and GTP, in the presence or absence of As₂O₃. Fig. 4 shows that As₂O₃ significantly promotes tubulin depolymerization, an effect similar to that induced by vincristine. Thus, these data suggest that As₂O₃ has directly bounds tubulin and induces tubulin depolymerization.

Fig. 4

Effects of As₂O₃ on microtubule polymerization in vitro. Purified tubulin from bovine brain tissue (Cytoskeleton) was incubated at 37 [degree celsius]

in reaction mixtures containing 1 mM GTP, 10 nM paclitaxel (Taxol), 10 nM vincristine, 5 µM As₂O₃ and (more ...)

5. Effect of As₂O₃ on cellular microtubules

Because vincristine-like, tubulin-destabilizing activity was found in vitro, we tested whether As₂O₃ treatment affected the cellular microtubule network. NB4 cells were treated with the indicated concentration of paclitaxel, vincristine, and As₂O₃ or with no drug-treatment as a control. After 6 hours of incubation, the microtubule network was visualized by immunocytochemistry. As shown in Fig. 5, the microtubule network in control cells exhibited normal cytoskeletal arrangement and organization. Treatment with vincristine resulted in microtubule depolymerization with a decrease in the density of cellular microtubule and disappearance of microtubule bundles surrounding in the cytoplasm. As₂O₃ treatment resulted in findings similar to those seen with vincristine-induced microtubule changes (e.g., decreased microtubule density). In contrast, paclitaxel treatment induced microtubule polymerization with long, thick cytoplasmic microtubules.

Fig. 5

Effects of As₂O₃ on the organization of cellular microtubule network. NB4 cells were treated with 5 µM As₂O₃, 40 nM paclitaxel, and 20 nM vincristine. Mock-treated cells were used as a control. After a 6-hour incubation, cells were harvested and (more ...)

DISCUSSION

The microtubules are well recognized as a chemotherapeutic target, and there is an ongoing search for high efficacy microtubule targeting agents. In cultured, malignant cells, microtubules contribute to various mechanisms, including drug efflux transporter [17], altered tubulin isotype expression [5, 18], and tubulin mutation [19]. Therefore, it is suggested that arsenic compounds might be useful for the treatment of some instances of human cancer [20].

Our data demonstrates that As₂O₃ efficiently inhibits proliferation of NB4 cells and, in a previous study, the IC₅₀ value for As₂O₃ in MCF-7 breast carcinoma and A2780 ovarian carcinoma cell lines was approximately 3 µM, a concentration very close to the effective serum concentration for successfully treating patients with acute promyelocytic leukemia in the clinic [8]. Although it was a different type of cancer cells line, the cytotoxicity of As₂O₃ at about 50% was in the range of the 5 µM for NB4 cells (Fig. 1A). This evidence indicates that the efficiency of As₂O₃ was similar to previously described reports. Some investigators also report that As₂O₃ causes DNA damage, oxidative stress, and mitochondrial dysfunction [21-23]. In addition, As₂O₃ treatment blocked the cell cycle in these cell line. In the present study, we found that As₂O₃ treatment resulted in cell-cycle arrest at the G₂/M phase in NB4 cells (Fig. 1B).

We also considered whether As₂O₃-induced G₂/M phase arrest is due to its interaction with tubulin, thus resulting in abnormal tubulin polymerization. To test this, we have taken advantage of an assay designed by Minotti et al. [24] to separate polymerized "P" from "S" soluble tubulin fractions by centrifugation. This procedure, and subsequent modifications of it, has been widely utilized to quickly determine the percentage of tubulin polymers in cells under a variety of experimental conditions. An increase in the "S" fraction serves as an indicator of tubulin destabilization. In the present study, we were able to show that the presence of unstable microtubules in the treated cell populations by several criteria. First, compared to the controls, an increased proportion of tubulin was found in the soluble tubulin fractions. Soluble tubulin amounts were present in an As₂O₃ concentration-dependent manner. The effect of As₂O₃ on tubulin depolymerization was weaker than that of vincristine (Fig. 2). These data suggest that As₂O₃ directly or indirectly induces the disruption of microtubule formation via tubulin polymerization in contrast to data published by Ling et al., who reported that As₂O₃ increases tubulin polymerization [8]. In addition, the disassembly of the microtubule network after treatment with As₂O₃ suggests that it acts as a microtubule depolymerizing agent. We were able to demonstrate that α-tubulin posttranslational modifications correspond to increased microtubule stability. Tubulin can undergo numerous posttranslational modifications, including phosphorylation, polyglutamylation, polyglyclation, deglutamylation, acetylation, and tyrosination/detyrosination. Although the functions of these α-tubulin modifications remain unclear, deglutamylation, acetylation, and detyrosination are indicative of stable microtubules [14, 15, 25]. Acetylation occurs on lysine 40 near the amino terminus of α-tubulin and does not appear to increase the levels of stable microtubules but instead accumulates in existing stable microtubules [14, 15]. Glu-tubulin is formed when the last residue on α-tubulin, a tyrosine, is removed by tubulin-carboxypeptidase and the glutamic acid is exposed [25]. As such, the phenomenon observed here, depolymerization of microtubule by As₂O₃, could be explained, in part, by alteration of microtubule stability through microtubule modification. As compared to paclitaxel treatment, As₂O₃ significantly decreased acetylated α-tubulin (Fig. 3), this suggests that microtubule stabilization may play an important role in As₂O₃-mediated microtubule depolymerization.

Next, we tested the effect of As₂O₃, paclitaxel, and vincristine on tubulin assembly in a cell-free in vitro system (Fig. 4). We found that the effect of As₂O₃ was similar to that of vincristine but different from paclitaxel. This does not rule out the possibility that As₂O₃ may bind to polymerized tubulins and stabilize microtubules. The reason for the appearance of tubulin depolymerization may be explained if As₂O₃ acts as a noncompetitive inhibitor, thus interfering with the GTP binding domain on β-tubulin [9]. In this study, it is notable that the same concentration of As₂O₃ was capable of inhibiting the assembly of tubulin, but these concentrations were also found to elicit some differences in the cell-based assays or the cell-free systems. The intracellular conditions may provide an ideal environment for the reaction of As₂O₃ with microtubules whereas the reaction conditions of these widely used cell-free microtubule assembly-disassembly assays may not facilitate the As₂O₃-microtubule interaction. Indeed, this phenomenon has been observed before by using other tubulin ligands, although the discrepancies were not as pronounced as in this study [26].

Finally, indirect immunofluorescence technique allowed us to detect morphological changes in the microtubule network, such as alterations in microtubule organization and arrangement. The changes in microtubule length and density constitute an appropriate method to qualitatively assess the intracellular microtubule polymerization or depolymerization caused by anti-tubulin agents. The results from our tubulin depolymerization experiments indicated that, similar to the effect of vincristine treatment, As₂O₃ treatment prevented tubulin polymerization. To validate these results, changes in the cellular microtubule network were observing immunocytochemically. We found that the effect of As₂O₃ was similar to that of vincristine (Fig. 5). Thick bundles of microtubule network surrounding cytoplasm were observed in paclitaxel-treated cells, whereas shortened depolymerized microtubules were observed in As₂O₃- and vincristine-treated cells.

In summary, our data demonstrate that As₂O₃ is efficacious in suppressing cell growth in NB4 cells. As₂O₃ directly or indirectly interferes with microtubules and blocks the cell cycle at the G₂/M phase. The effects of As₂O₃ on microtubule assembly are vincristine-like, but also distinct from vincristine. As₂O₃ is less active than vincristine in NB4 cells and is diametrically opposite of the observations published by Ling et al. who reported that As₂O₃ induced tubulin polymerization [8]. The unique mechanism that allows for As₂O₃ to induce both cell death and cell cycle arrest, and to cause microtubule depolymerization, makes it an ideal candidate for antineoplastic therapy. This also suggests that As₂O₃ can be used at a low concentration and still selectively target the microtubules of rapidly dividing tumor cells, thus minimizing its general toxicity. Evidence that tubulin plays an important role in the treatment of leukemia raises the possibility for the development of a rationally designed arsenic-based antimitotic agent.

Footnotes

This work was supported by Priority Research Center Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2010-0029621).

References

1. Kamimura T, Miyamoto T, Harada M, Akashi K. Advances in therapies for acute promyelocytic leukemia. Cancer Sci. 2011;102:1929–1937. [PubMed]

2. Miller WH, Jr, Schipper HM, Lee JS, Singer J, Waxman S. Mechanisms of action of arsenic trioxide. Cancer Res. 2002;62:3893–3903. [PubMed]

3. Fojo T, Menefee M. Mechanisms of multidrug resistance: the potential role of microtubule-stabilizing agents. Ann Oncol. 2007;18(Suppl 5):v3–v8. [PubMed]

4. Barlow SB, Gonzalez-Garay ML, Cabral F. Paclitaxel-dependent mutants have severely reduced microtubule assembly and reduced tubulin synthesis. J Cell Sci. 2002;115:3469–3478. [PubMed]

5. Kavallaris M, Tait AS, Walsh BJ, et al. Multiple microtubule alterations are associated with Vinca alkaloid resistance in human leukemia cells. Cancer Res. 2001;61:5803–5809. [PubMed]

6. Jordan MA, Wilson L. Microtubules as a target for anticancer drugs. Nat Rev Cancer. 2004;4:253–265. [PubMed]

7. Huang SC, Lee TC. Arsenite inhibits mitotic division and perturbs spindle dynamics in HeLa S3 cells. Carcinogenesis. 1998;19:889–896. [PubMed]

8. Ling YH, Jiang JD, Holland JF, Perez-Soler R. Arsenic trioxide produces polymerization of microtubules and mitotic arrest before apoptosis in human tumor cell lines. Mol Pharmacol. 2002;62:529–538. [PubMed]

9. Li YM, Broome JD. Arsenic targets tubulins to induce apoptosis in myeloid leukemia cells. Cancer Res. 1999;59:776–780. [PubMed]

10. Yan J, Xu YH. Tributyrin inhibits human gastric cancer SGC-7901 cell growth by inducing apoptosis and DNA synthesis arrest. World J Gastroenterol. 2003;9:660–664. [PubMed]

11. Poruchynsky MS, Kim JH, Nogales E, et al. Tumor cells resistant to a microtubule-depolymerizing hemiasterlin analogue, HTI-286, have mutations in alpha- or beta-tubulin and increased microtubule stability. Biochemistry. 2004;43:13944–13954. [PubMed]

12. Chen GQ, Zhu J, Shi XG, et al. In vitro studies on cellular and molecular mechanisms of arsenic trioxide (As2O3) in the treatment of acute promyelocytic leukemia: As2O3 induces NB4 cell apoptosis with downregulation of Bcl-2 expression and modulation of PML-RAR alpha/PML proteins. Blood. 1996;88:1052–1061. [PubMed]

13. Carré M, Carles G, André N, et al. Involvement of microtubules and mitochondria in the antagonism of arsenic trioxide on paclitaxel-induced apoptosis. Biochem Pharmacol. 2002;63:1831–1842. [PubMed]

14. Palazzo A, Ackerman B, Gundersen GG. Cell biology: Tubulin acetylation and cell motility. Nature. 2003;421:230. [PubMed]

15. Infante AS, Stein MS, Zhai Y, Borisy GG, Gundersen GG. Detyrosinated (Glu) microtubules are stabilized by an ATP-sensitive plus-end cap. J Cell Sci. 2000;113:3907–3919. [PubMed]

16. Low JA, Wedam SB, Lee JJ, et al. Phase II clinical trial of ixabepilone (BMS-247550), an epothilone B analog, in metastatic and locally advanced breast cancer. J Clin Oncol. 2005;23:2726–2734. [PubMed]

17. Gottesman MM, Fojo T, Bates SE. Multidrug resistance in cancer: role of ATP-dependent transporters. Nat Rev Cancer. 2002;2:48–58. [PubMed]

18. Orr GA, Verdier-Pinard P, McDaid H, Horwitz SB. Mechanisms of Taxol resistance related to microtubules. Oncogene. 2003;22:7280–7295. [PubMed]

19. Hari M, Wang Y, Veeraraghavan S, Cabral F. Mutations in alpha- and beta-tubulin that stabilize microtubules and confer resistance to colcemid and vinblastine. Mol Cancer Ther. 2003;2:597–605. [PubMed]

20. Freitas RA, Silva dos, Gimenes Teixeira HL, et al. Apoptosis induction by (+)alpha-tocopheryl succinate in the absence or presence of all-trans retinoic acid and arsenic trioxide in NB4, NB4-R2 and primary APL cells. Leuk Res. 2009;33:958–963. [PubMed]

21. Dong JT, Luo XM. Arsenic-induced DNA-strand breaks associated with DNA-protein crosslinks in human fetal lung fibroblasts. Mutat Res. 1993;302:97–102. [PubMed]

22. Van Wijk R, Welters M, Souren JE, Ovelgonne H, Wiegant FA. Serum-stimulated cell cycle progression and stress protein synthesis in C3H10T1/2 fibroblasts treated with sodium arsenite. J Cell Physiol. 1993;155:265–272. [PubMed]

23. Yih LH, Lee TC. Arsenite induces p53 accumulation through an ATM-dependent pathway in human fibroblasts. Cancer Res. 2000;60:6346–6352. [PubMed]

24. Minotti AM, Barlow SB, Cabral F. Resistance to antimitotic drugs in Chinese hamster ovary cells correlates with changes in the level of polymerized tubulin. J Biol Chem. 1991;266:3987–3994. [PubMed]

25. MacRae TH. Tubulin post-translational modifications-enzymes and their mechanisms of action. Eur J Biochem. 1997;244:265–278. [PubMed]

26. Jiang JD, Wang Y, Roboz J, Strauchen J, Holland JF, Bekesi JG. Inhibition of microtubule assembly in tumor cells by 3-bromoacetylamino benzoylurea, a new cancericidal compound. Cancer Res. 1998;58:2126–2133. [PubMed]

Articles from The Korean Journal of Hematology are provided here courtesy of
Korean Society of Hematology