Cell Biology International 32 (2008) 1497e1505www.elsevier.com/locate/cellbi

Realgar-induced differentiation is associated with MAPK pathwaysin HL-60 cells

Nan Wang a, Li-Wen Wang b, Bao-Di Gou a,*, Tian-Lan Zhang a,b,**, Kui Wang a

a Department of Chemical Biology, Peking University School of Pharmaceutical Sciences, 38 Xueyuan Road, Beijing 100083, PR Chinab Department of Chemical Biology, Capital Medical University School of Pharmaceutical Sciences, 10 Xitoutiao, You An Men, Beijing 100069, PR China

Received 15 February 2008; revised 14 May 2008; accepted 12 August 2008

Abstract

The clinical efficacy and safety of realgar (arsenic sulfide, As4S4) in the treatment of acute promyelocytic leukemia in China have given riseto an upsurge in research on the underlying mechanism. We prepared realgar nanoparticles (RNPs) to examine their effect on the differentiationof HL-60 cells. Treatment with RNPs at 6 mM for 72 h induced cell differentiation that was assessed by morphological change, NBT reductiveability, and elevation of CD11b expression at both mRNA and protein levels. The RNP-induced differentiation was synergized, enhanced andsuppressed by the inhibition of p38 MAPK, JNK and ERK pathways, respectively. Our findings demonstrate that MAPK signaling pathways areclosely related to the RNP-induced differentiation in HL-60 cells.� 2008 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved.

Keywords: Realgar; Arsenic sulfide; Differentiation; MAPK pathway; HL-60

1. Introduction

Medicinal use of arsenicals has a long history (Waxmanand Anderson, 2001; Evens et al., 2004), but contemporarystudies show that arsenic trioxide (ATO, As2O3) remainseffective in the treatment of acute promyelocytic leukemia(APL; Shen et al., 1997; Soignet et al., 2001; Zhang et al.,2001; Miller et al., 2002). Unfortunately, the clinical activityof ATO has been somewhat tempered by its acute toxicity aswell as association with carcinogenesis (Huang et al., 1998;Lu et al., 2002). Realgar (arsenic sulfide, As4S4) is much lessacutely toxic than ATO, and has potent anti-leukemic activityin combination with imatinib (Yin et al., 2004). Realgartreatment alone is also effective in inducing complete

Abbreviations: RNP, realgar nanoparticle; ATO, arsenic trioxide; APL,

acute promyelocytic leukemia; MAPK, mitogen-activated protein kinase;

NBT, nitroblue tetrazolium; PMA, phorbol 12-myristate 13-acetate.

* Corresponding author. Tel.: þ86 10 82801539; fax: þ86 10 62015584.

** Corresponding author. Fax: þ86 10 83911533.

E-mail addresses: b.gou@hsc.pku.edu.cn (B.-D. Gou), tlzhang@hsc.pku.

edu.cn (T.-L. Zhang).

1065-6995/$ - see front matter � 2008 International Federation for Cell Biology.

doi:10.1016/j.cellbi.2008.08.017

remission both in patients with newly diagnosed APL and inthose with APL relapse (Lu et al., 2002). These findings havegiven rise to an upsurge in research on the underlying mech-anism of action.

The mechanistic studies at cellular and molecular levelshampered by the poor water-solubility of realgar have recentlychanged due to flourishing nanotechnology. Reduced size ofrealgar nanoparticles (RNPs) enhances their bioavailability tohuman gynecological cell lines (CI80-13S, OVCAR, OVCAR-3 and HeLa) (Wu and Ho, 2006). The apoptosis-inducingeffect of RNPs has been reported in human endothelial cellline ECV-30 (Deng et al., 2001), promyelocytic leukemia cellline HL-60 (Ye et al., 2004) and leukemic monocytelymphoma cell line U937 (Wang et al., 2007).

Compared with the induction of apoptosis, differentiationtherapy is less toxic due to lower doses being required and thusmay provide an alternative strategy (Wang and Chen, 2000).HL-60 cell line retains the ability to differentiate in responseto a variety of inducers and is a helpful model system forelucidating hemopoietic differentiation (Birnie, 1998). In celldifferentiation, the importance of mitogen-activated proteinkinase (MAPK) superfamily (ERK1/2, JNK and p38) has been

Published by Elsevier Ltd. All rights reserved.

1498 N. Wang et al. / Cell Biology International 32 (2008) 1497e1505

recognized. The classical MAPK pathway (ERK1/2) is pref-erentially activated in response to growth factors, cytokinesand phorbol esters. The JNK and p38 MAPK pathways areresponsive to stress stimuli ranging from osmotic shock andionizing radiation to inflammatory cytokines (Chang andKarin, 2001). Different members of the MAPK family havebeen modulated during myeloid differentiation by a variety ofstimuli (Miranda et al., 2002; Kim et al., 2005; Wang et al.,2005).

The aim of the present study was to investigate the effectsof RNPs on the differentiation of HL-60 cells, with emphasison the involvement of MAPK signaling pathways in the RNP-induced differentiation.

Fig. 1. Effects of RNPs on proliferation, viability and cycle distribution of HL-60 c

Cell proliferation; (B) the viable cells were determined using trypan blue exclusion

one experiment. *The difference is significant (P< 0.05) from the control. (C) DN

staining with propidium iodide. These results are representative of 3 independent

2. Materials and methods

2.1. Reagents and antibodies

Realgar, nitroblue tetrazolium (NBT), phorbol 12-myristate13-acetate (PMA), DMSO, Hoechst 33258, MEK inhibitorPD98059, p38 MAPK inhibitor SB202190 and JNK inhibitorSP600125 were purchased from Sigma Chemical Co (St.Louis, MO, USA). These inhibitors were stored at �20 �Cunder light-free conditions. Go Taq DNA polymerase, M-MLVreverse transcriptase and recombinant RNasin ribonucleaseinhibitor were purchased from Promega (Madison, USA).Trizol reagent was purchased from Invitrogene (Carlsbad, CA,

ells. Cells were incubated with RNPs at specified concentrations for 72 h. (A)

assay. Results are mean� standard deviations of triplicate determinations from

A content and degraded DNA were measured by flow cytometric assay after

experiments.

Fig. 2. Morphological changes of HL-60 cells induced by 6 mM of RNPs for 72 h. Cells were stained with Hoechst 33258. Digital photomicrographs were taken of

the control (right) and differentiated cells (left). These results are representative of 3 independent experiments.

1499N. Wang et al. / Cell Biology International 32 (2008) 1497e1505

USA). PE-conjugated CD11b antibody and mouse IgG1 iso-type control were products of eBioscience Inc. (San Diego,CA, USA). Other chemicals used were commercially availableproducts of the highest purity.

2.2. Preparation of RNPs

The nano-sized realgar particles were made in a grindingequipment apparatus (QM-BP planet-type ball mill, NanjingUniversity, China) in the media of double-distilled water. Theproduced dispersion was ultrasonicated for 1 min, and thenpassed through a 0.22 mm membrane. The particle sizedistribution of RNPs in the filtrate was measured on witha Nano ZS90 (Malvern Instruments Ltd, Worcestershire, UK),and the arsenic content was determined with arseno-molyb-denum blue photometry (Lenoble et al., 2003). The filtrate waskept at 4 �C under light-free conditions.

The z-average diameter of the RNPs was 143 nm, with92.8% of the realgar particles in the range of 68e220 nm. Thequantity of RNPs used in this study is given by the arsenicconcentration in micromolarity, the content of the stocksolution of RNPs being 61.73 mg As/l, i.e. As 824 mM.

2.3. Cell culture

HL-60 cells obtained from Peking University HealthSciences Center were cultured in RPMI 1640 medium sup-plemented with 10% heat-inactivated fetal bovine serum at37 �C in a humidified 5% CO2 in air incubator. Controlcultures received the same amount of RPMI 1640 withoutRNPs. The amount of DMSO in the cell culture medium neverexceeded a final concentration of 0.1%. Cells were subculturedevery 2 days.

Fig. 3. Effect of RNP concentration on NBT reducing activity in HL-60 cells.

Cells were incubated with various concentrations of RNPs for 72 h. Differ-

entiation of HL-60 cells was determined by the increase in NBT absorbance at6

2.4. Determination of cell proliferation with trypanblue exclusion

590 nm/10 cells. The experiment was performed 3 times with similar results.

Each column represents the mean� standard deviations of triplicate deter-

minations from one experiment. *The difference is significant (P< 0.05) from

the control.

Cells grown in culture bottles were treated with indicatedconcentrations of RNPs for 72 h. At the end of each period,

trypan blue solution (0.4% in phosphate buffer saline, PBS)and cell suspension were mixed in equal volumes (0.1 ml).Cell number was estimated using a hemocytometer. Cellsstained blue were scored as dead.

2.5. Flow cytometric analysis of cell cycle

Cell cycle distribution was quantified by flow cytometry.HL-60 cells were treated for 72 h with RNPs at indicatedconcentrations. At the end of the incubation, cells werecollected, resuspended in 70% ice-cold ethanol. After fixationin �20 �C overnight, the cells were treated with 1 mg/mlRNase for 30 min at 37 �C. Propidium iodide (PI) was addedto the solution at a final concentration of 50 mg/ml. Data wereanalyzed on FACScan flow cytometer with CELL Questsoftware (BD Biosciences, USA).

1500 N. Wang et al. / Cell Biology International 32 (2008) 1497e1505

2.6. Flow cytometric analysis of cell differentiation

The expression of cell surface markers CD11b was exam-ined by flow cytometry. Samples of 1� 106 cells were washedtwice with FACS buffer, resuspended in 100 ml FACS buffer andincubated for 30 min at 4 �C with 10 ml PE-conjugated anti-human CD11b. Cells were washed twice with PBS and resus-pended in 0.5 ml PBS, and analyzed on FACS Calibur flowcytometer with CELL Quest software (BD Biosciences, USA).Isotypic mouse IgG1 was used to set threshold parameters.

2.7. Examination of cell morphology

Cells were washed with cold PBS, and fixed with 4%paraformaldehyde. After washing twice with PBS, the cellswere stained with Hoechst 33258 (2.5 mg/ml). Cell

Fig. 4. RNP-induced expression of cell differentiation marker CD11b and its mRNA

CD11b mRNA was measured by RT-PCR analysis. (B) CD11b positive cells were

mouse IgG1 isotype-specific antibodies. (C) CD11b expression presented as geo

experiments are shown.

differentiation was examined morphologically using a fluores-cence microscope (TCSNT, LEICA, Germany).

2.8. Determination of cell differentiation with NBTreduction assay

Cells (2� 105 cells/ml) were cultured with indicatedconcentrations of RNPs in RPMI 1640 medium containing10% FBS for 72 h, and the NBT reducing activity determined.Briefly, the cells (1� 106) were washed twice and incubatedfor 30 min at 37 �C in 1 ml PBS containing 1 mg/ml NBT and100 ng/ml PMA. After centrifugation, 600 ml DMSO wereadded to the cell pellets to solubilize the formazan deposits.The amount of formazan was determined by reading theabsorbance at 560 nm.

. Cells were incubated with the specified concentrations of RNPs for 72 h. (A)

examined by flow cytometry. As controls, cells were also analyzed by using

metry mean fluorescence intensity (MFI). Typical results of 3 independent

1501N. Wang et al. / Cell Biology International 32 (2008) 1497e1505

2.9. Reverse transcription-polymerase chain reaction(RT-PCR)

Semiquantitative reverse transcription-PCR (RT-PCR) wasperformed to examine the mRNA expression of CD11b. Cellswere cultured with inducing agents for indicated periods oftime. Total cellular RNA was extracted using TRIzol reagent.Then cDNA was synthesized with the use of 2 mg total RNAprimed with oligod (T) (deoxy-thymidine) in 25 ml reactionsolution. The resulting total cDNA was used in the polymerasechain reaction. PCR products were subjected to 1.5% agarosegel electrophoresis. The bands were visualized by ethidium-bromide staining and observed under ultraviolet light. Theprimers sequences were as follows:

(1) CD11b upper primer 50-CAGAGCGTGGTCCAGCTTCA-30 and lower primer 30-CCTTCATCCGCCGAAAGTCA-50 define an amplicon of 406 bp.

(2) b-Actin upper primer 50-TCACCCACACTGTGCCCATCTACGA-30 and lower primer 30-CAGCGGAACCGCTCATTGCCAATGG-50 define an amplicon of271 bp.

3. Results

3.1. RNPs induce growth inhibition of HL-60 cells

RNPs inhibited cell growth in a concentration-dependentmanner. After treatment with 6 mM of RNPs for 72 h, cellnumber was half of that of the control group (Fig. 1A).However, as long as RNP concentration was <10 mM, cellviability (Fig. 1B) did not change significantly. Apoptosis wasalso negligible at this concentration (Fig. 1C). The sub-G1population increased and viability decreased at higherconcentrations of RNPs. G0/G1 arrest was observed in cellstreated with 30 mM of RNPs for 72 h. Therefore, 6 mM ofRNPs was used in the following experiment to study therelationship between cell differentiation and MAPK signalingpathways.

3.2. RNPs induce differentiation in HL-60 cells

Fig. 5. Effects of MAPK inhibitors on RNP-induced differentiation in HL-60

cells. After pre-treatment with the MEK inhibitor PD098059 (20 mM), the p38

inhibitor SB202190 (5 mM), or the JNK inhibitor SP600125 (10 mM) for 2 h

respectively, cells were incubated with 6 mM RNPs for 72 h. (A) NBT

reducing activity of the cells. Results are mean� standard deviations of

triplicate determinations from one experiment. *The difference is significant

(P< 0.05) from the control. (B) CD11b mRNA expression. These results are

representative of 3 independent experiments.

After staining with Hoechst 33258, cell morphology wasexamined. Cells treated with medium had relatively large andround nuclei (Fig. 2). In contrast, the RNP-treated cells wereshrunken, with relatively small and deformed nuclei, charac-teristic of mature neutrophils (Lu et al., 2002; Suzuki et al.,2005).

RNP-induced differentiation was also assessed by NBTreduction assay. The NBT positive cells greatly increased after72 h treatment, with a maximal at the concentration of 6 mM(Fig. 3). The lower NBT reductivity induced by 8 and 10 mMin Fig. 3 can be explained by the reductive ability of realgarthat can react with the induced oxidative species, thus causinga lower NBT reduction value. This becomes more obviouswhen there is excess realgar at higher concentrations.

CD11b is a myeloid marker which appears later in mono-cytic or granulocytic differentiation (Drayson et al., 2001). Toconfirm the RNP-induced differentiation, RT-PCR analysis ofthe CD11b mRNA expression was performed. After treatmentwith RNPs, the mRNA expression of CD11b was elevated(Fig. 4A). The marker on cell surface was also examined withimmunofluorescence analysis. CD11b was increased aftertreatment with RNPs, as revealed by FASC analysis, both interms of the percentage of CD11b-positive cells (Fig. 4B) andgeometric mean fluorescence intensity (Fig. 4C). These resultsconfirmed the RNP-induced differentiation in HL-60 cells.

3.3. Inhibition of ERK MAPK blocks the RNP-induceddifferentiation in HL-60 cells

The role of MAPK signaling pathway in the RNP-induceddifferentiation was assessed with specific inhibitors. PD98059is considered specific for MEK, and as a consequence, selec-tively inhibits ERK activation (Alessi et al., 1995). Thisinhibitor blocked the RNP-induced differentiation, as indi-cated by NBT reduction ability (Fig. 5A), the expressions ofmRNA (Fig. 5B) and protein (Fig. 6) of CD11b. The datasuggest a positive role of the pathway in mediating the RNP-induced differentiation in HL-60 cells.

1502 N. Wang et al. / Cell Biology International 32 (2008) 1497e1505

3.4. Inhibition of JNK MAPK potentiates the RNP-induced differentiation in HL-60 cells

SP600125 is a specific inhibitor to JNK activation. It didnot induce cell differentiation, but remarkably enhanced theRNP-induced differentiation, as indicated by NBT reductionability (Fig. 5A), the expressions of mRNA (Fig. 5B) andprotein (Fig. 7) of CD11b. The data suggest a negative role ofJNK pathway in mediating the RNP-induced differentiation inHL-60 cells.

3.5. RNPs and p38 MAPK inhibitor synergizedifferentiation in HL-60 cells

SB202190 is a pyridinyl imidazole compound that isgenerally accepted as a specific inhibitor of p38 MAPK.Although the inhibitor alone induced remarkable cell differ-entiation in the absence of RNPs, the differentiation wasgreatly increased after pre-treatment with the inhibitor fol-lowed by RNP incubation, as indicated by NBT reductionability (Fig. 5A), the expressions of mRNA (Fig. 5B) andprotein (Fig. 8) of CD11b. The percentage of CD11b-positive

Fig. 6. Effect of the MEK inhibitor PD98059 on RNP-induced CD11b expression

incubated with 6 mM RNPs for 72 h. (A) CD11b positive cells were examined by

isotype-specific antibodies. (B) CD11b expression presented as geometry mean fl

shown.

cells was 96.8%, much greater than the sum of those inducedby the inhibitor (38.8%) and RNPs (50.7%).

4. Discussion

Differentiation therapy, a potentially less toxic approachthan apoptosis induction, provides an alternative treatment ofcancer. All-trans retinoic acid (ATRA) has now been estab-lished as an effective differentiation inducer in APL treatment(Degos, 2003; Sanz, 2006). Realgar has also shown efficacy inpatients with newly diagnosed and relapsed APL. When givenorally for remission induction at 50 mg/kg of body weight perday (Lu et al., 2002). We have demonstrated that RNPs at lowconcentrations (�10 mM) markedly induce differentiation andgrowth inhibition in HL-60 cells, and that this event is asso-ciated with MAPK signaling pathways. These findings mayconstitute, at least in part, the cellular basis of the clinicalaction of realgar.

MAPK signaling pathways are known to be involved indifferentiation of HL-60 cells, but the exact way varies withthe stimulus used. In ATRA-induced granulocytic differenti-ation, the ERK2, but not the JNK or the p38 MAPK, is

in HL-60 cells. After pre-treatment with 20 mM PD098059 for 2 h, cells were

flow cytometry. As controls, cells were also analyzed by using mouse IgG1

uorescence intensity (MFI). Typical results of 3 independent experiments are

Fig. 7. Effect of the JNK inhibitor SP600125 on RNP-induced CD11b expression in HL-60 cells. After pre-treatment with 10 mM SP600125 for 2 h, cells were

incubated with 6 mM RNPs for 72 h. (A) CD11b positive cells were examined by flow cytometry. As controls, cells were also analyzed by using mouse IgG1

isotype-specific antibodies. (B) CD11b expression presented as geometry mean fluorescence intensity (MFI). Typical results of 3 independent experiments are

shown.

1503N. Wang et al. / Cell Biology International 32 (2008) 1497e1505

activated (Yen et al., 1999). It can be inhibited by specificinhibitors of MEK, such as U0126 and PD98059 (Mirandaet al., 2002; Battle et al., 2001). Differently, the monocyticdifferentiation induced by 1,25-(OH)2D3 is associated withactivation of the JNK/c-jun pathway, while ERK1/2 only hasa transient effect. And the JNK/c-jun activity can be enhancedby the inhibition of the other arm of the stress-activatedpathways, the p38 MAPK pathway (Wang et al., 2000; Wangand Studzinski, 2001; Wang et al., 2003). Our data show thatthe inhibition of ERK pathway with PD98059 significantlysuppressed the RNP-induced differentiation, suggestinga positive role of ERK in mediating the differentiation.

In contrast, the JNK inhibitor enhanced the RNP-induceddifferentiation in HL-60 cells. JNK signaling is not essentialfor ATRA-induced differentiation (Wang and Studzinski,2001), but may participate in the monocytic differentiation ofHL-60 cells induced by 1,25-(OH)2D3 and D3 derivatives (Jiet al., 2002). In ATO-induced apoptosis, JNK MAPK signalingpathway is also activated (Lau et al., 2004; Potin et al., 2007).Therefore, the negative regulatory effect of the JNK pathwayfound in this study implies a different mechanism under theRNP-induced differentiation in HL-60 cells.

Most impressively, the p38 inhibitor exhibited significantsynergistic effect on the RNP-induced differentiation in HL-60cells. The inhibitor itself can also induce CD11b expression.This is probably due to the upregulation of ERK pathway byinhibition of p38 MAPK pathway (Wang et al., 2000). The p38inhibitor SB202190 is a tritiated pyridinyl imidazole, and hasa similar binding affinity to the unphosphorylated and phos-phorylated forms of p38 (Frantz et al., 1998). Another pyr-idinyl imidazole compound and inhibitor to p38, SB203580,was also effective in inducing differentiation of HL-60 cells(Ishii et al., 2001). These p38 inhibitors are effective in animalmodels of arthritis and some inflammatory diseases (Lee et al.,2000; Goldstein and Gabriel, 2005; Underwood et al., 2000;English and Cobb, 2000). Inhibition of both JNK and p38pathways enhanced the RNP-induced differentiation in HL-60cells, indicating a similar negative feedback control on cellularactivity. A positive outcome of inhibition of MAPK pathwayshas occurred in clinical trials (Saklatvala, 2004; Kaminska,2005; Mayer and Callahan, 2006).

In conclusion, the treatment of HL-60 cells with RNPs atconcentrations of micromolarity induces differentiation andgrowth inhibition accompanied with G0/G1 arrest. The

Fig. 8. Effect of the p38 inhibitor SB202190 on RNP-induced CD11b expression in HL-60 cells. After pre-treatment with 5 mM SB202190 for 2 h, cells were

incubated with 6 mM RNPs for 72 h. (A) CD11b positive cells were examined by flow cytometry. As controls, cells were also analyzed by using mouse IgG1

isotype-specific antibodies. (B) CD11b expression presented as geometry mean fluorescence intensity (MFI). Typical results of 3 independent experiments are

shown.

1504 N. Wang et al. / Cell Biology International 32 (2008) 1497e1505

inhibition of p38 MAPK pathway greatly synergized the RNP-induced differentiation, and the inhibition of ERK and JNKpathways had inhibitory and enhancing effects respectively. Thefindings demonstrate that MAPK signaling pathways are closelyrelated to the RNP-induced differentiation in HL-60 cells.

Acknowledgments

This study was supported by National Natural ScienceFoundation of China (Grants 20271005 and 20571006), NaturalScience Foundation of Beijing (Grant 2062007), BeijingMunicipal Education Commission and Beijing Talent Project.

References

Alessi DR, Cuenda A, Cohen P, Dudley DT, Saltiel AR. PD098059 is

a specific inhibitor of the activation of mitogen-activated protein kinase in

vitro and in vivo. J Biol Chem 1995;270:27489e94.

Battle TE, Roberson MS, Zhang T, Varvayanis S, Yen A. Retinoic acid-induced blr1

expression requires RAR-alpha, RXR, and MAPK activation and uses ERK2 but

not JNK/SAPK to accelerate cell differentiation. Eur J Cell Biol 2001;80:59e67.

Birnie GD. The HL60 cell line: a model system for studying human myeloid

cell differentiation. Br J Cancer 1998;(Suppl. 9)::41e5.

Chang L, Karin M. Mammalian MAP kinase signaling cascades. Nature 2001;

410:37e40.

Degos L. The history of acute promyelocytic leukaemia. Br J Haematol 2003;

122:539e53.

Deng Y, Xu HB, Huang K, Yang X, Xie C, Wu J. Size effects of realgar

particles on apoptosis in a human umbilical vein endothelial cell line:

ECV-304. Pharmacol Res 2001;44:513e8.

Drayson MT, Michell RH, Durham J, Brown G. Cell proliferation and CD11b

expression are controlled independently during HL60 cell differentiation

initiated by 1,25a-dihydroxyvitamin D3 or all-trans-retinoic acid. Exp Cell

Res 2001;266:126e34.

English JM, Cobb MH. Pharmaceutical inhibitors of MAPK pathways. Trends

Pharmacol Sci 2000;23:40e5.

Evens AM, Tallman MS, Gartenhaus RB. The potential of arsenic trioxide in

the treatment of malignant disease: past, present, and future. Leuk Res

2004;28:891e900.

Frantz B, Klatt T, Pang M, Parsons J, Rolando A, Williams H, et al. The

activation state of p38 mitogen-activated protein kinase determines the

efficiency of ATP competition for pyridinylimidazole inhibitor binding.

Biochemistry 1998;37:13846e53.

Goldstein DM, Gabriel T. Pathway to the clinic: inhibition of p38 MAP kinase.

A review of ten chemotypes selected for development. Curr Top Med

Chem 2005;5:1017e29.

Huang SY, Chang CS, Tang JL, Tien HF, Kuo TL, Huang SF, et al. Acute and

chronic arsenic poisoning associated with treatment of acute promyelo-

cytic leukaemia. Br J Haematol 1998;103:1092e110.

1505N. Wang et al. / Cell Biology International 32 (2008) 1497e1505

Ishii Y, Sakai S, Honma Y. Pyridinyl imidazole inhibitor SB203580 activates

p44/42 mitogen-activated protein kinase and induced the differentiation of

human myeloid leukemia cells. Leuk Res 2001;25:813e20.

Ji Y, Kutne A, Verstuyf A, Verlinden L, Studzinski GP. Derivatives of vitamins

D2 and D3 activate three MAPK pathways and upregulate pRb expression

in differentiating HL60 cells. Cell Cycle 2002;1:410e5.

Kaminska B. MAPK signalling pathways as molecular targets for anti-

inflammatory therapy e from molecular mechanisms to therapeutic

benefits. Biochim Biophys Acta 2005;1754:253e62.

Kim SH, Oh SM, Kim TS. Induction of human leukemia HL-60 cell differ-

entiation via a PKC/ERK pathway by helenalin, a pseudoguainolide

sesquiterpene lactone. Eur J Pharmacol 2005;511:89e97.

Lau A, Li M, Xie R, He QY, Chiu JF. Opposed arsenite-induced signalling

pathways promote cell proliferation or apoptosis in cultured lung cells.

Carcinogenesis 2004;25:21e5.

Lee JC, Kumar S, Griswold DE, Underwood DC, Votta BJ, Adams JL. Inhi-

bition of p38 MAP kinase as a therapeutic strategy. Immunopharmacology

2000;47:185e201.

Lenoble V, Deluchat V, Serpaud B, Bollinger J. Arsenite oxidation and arsenate

determination by the molybdene blue method. Talanta 2003;61:267e76.

Lu DP, Qiu JY, Jiang B, Wang Q, Liu KY, Liu YR, et al. Tetra-arsenic tetra-

sulfide for the treatment of acute promyelocytic leukemia: a pilot report.

Blood 2002;99:3136e43.

Mayer RJ, Callahan JF. p38 MAP kinase inhibitors: a future therapy for

inflammatory diseases. Drug Disc Today 2006;3:49e54.

Miller Jr WH, Schipper HM, Lee JS, Singer J, Waxman S. Mechanisms of

action of arsenic trioxide. Cancer Res 2002;62:3893e903.

Miranda MB, McGuire TF, Johnson DE. Importance of MEK-1/-2 signaling in

monocytic and granulocytic differentiation of myeloid cell lines. Leukemia

2002;16:683e92.

Potin S, Bertoglio J, Breard J. Involvement of a Rho-ROCK-JNK pathway in

arsenic trioxide-induced apoptosis in chronic myelogenous leukemia cells.

FEBS Lett 2007;581:118e24.

Saklatvala J. The p38 MAP kinase pathway as a therapeutic target in

inflammatory disease. Curr Opin Pharmacol 2004;4:372e7.

Sanz MA. Treatment of acute promyelocytic leukemia. Hematology 2006:147e55.

Shen ZX, Chen GQ, Ni JH, Li XS, Xiong SM, Qiu QY, et al. Use of arsenic

trioxide (As2O3) in the treatment of acute promyelocytic leukemia (APL):

II. Clinical efficacy and pharmacokinetics in relapsed patients. Blood

1997;89:3354e60.

Soignet SL, Frankel SR, Douer D, Tallman MS, Kantarjian H, Calleja E, et al.

United States multicenter study of arsenic trioxide in relapsed acute

promyelocytic leukemia. J Clin Oncol 2001;19:3852e60.

Suzuki K, Adachi R, Hirayama A, Watanabe H, Otani S, Watanabe Y, et al.

Indirubin, a Chinese anti-leukaemia drug, promotes neutrophilic differ-

entiation of human myelocytic leukeamia HL-60 cells. Br J Haematol

2005;130:681e90.

Underwood DC, Osborn RR, Bochnowicz S, Webb EF, Rieman DJ, Lee JC,

et al. SB 239063, a p38 MAPK inhibitor, reduces neutrophilia, inflam-

matory cytokines, MMP-9, and fibrosis in lung. Am J Physiol Lung Cell

Mol Physiol 2000;279:895e902.

Wang Q, Harrison JS, Uskokovic M, Kutner A, Studzinski GP. Translational

study of vitamin D differentiation therapy of myeloid leukemia: effects of

the combination with a p38 MAPK inhibitor and an antioxidant. Leukemia

2005;19:1812e7.

Wang Q, Wang X, Studzinski GP. Jun N-terminal kinase pathway enhances

signaling of monocytic differentiation of human leukemia cells induced by

1,25-dihydroxyvitamin D3. J Cell Biochem 2003;89:1087e101.

Wang X, Rao J, Studzinski GP. Inhibition of p38 MAP kinase activity

up-regulates multiple MAP kinase pathways and potentiates 1,

25-dihydroxy vitamin D3-induced differentiation of human leukemia

HL60 cells. Exp Cell Res 2000;258:425e37.

Wang X, Studzinski PG. Activation of extra cellular signal-regulated kinases

(ERKs) defines the first phase of 1,25-dihydroxyvitamin D3-induced

differentiation of HL-60 cells. J Cell Biochem 2001;80:471e82.

Wang XB, Gao HY, Hou BL, Huang J, Xi RG, Wu LJ. Nanoparticle realgar

powders induce apoptosis in U937 cells through caspase MAPK and

mitochondrial pathways. Arch Pharm Res 2007;30:653e8.

Wang Z, Chen Z. Differentiation and apoptosis induction therapy in acute

promyelocytic leukaemia. Oncology 2000;1:101e6.

Waxman S, Anderson KC. History of the development of arsenic derivatives in

cancer therapy. Oncologist 2001;6(Suppl. 2):3e10.

Wu JZ, Ho PC. Evaluation of the in vitro activity and in vivo bioavailability of

realgar nanoparticles prepared by cryo-grinding. Eur J Pharm Sci 2006;29:

35e44.

Ye HQ, Gan L, Yang XL, Xu HB. Membrane toxicity accounts for apoptosis

induced by realgar nanoparticles in promyelocytic leukemia HL-60 cells.

Biol Trace Elem Res 2004;103:117e32.

Yen A, Robertson MS, Varvayanis S. Retinoic acid selectively activates the

ERK2 but not JNK/SAPK or p38MAP kinases when inducing myeloid

differentiation. In Vitro Cell Dev Biol Anim 1999;35:527e32.

Yin T, Wu YL, Sun HP, Sun GL, Du YZ, Wang KK, et al. Combined effects of

As4S4 and imatinib on chronic myeloid leukemia cells and BCR-ABL

oncoprotein. Blood 2004;104:4219e25.

Zhang TD, Cheng GQ, Wang ZG, Wang ZY, Chen SJ, Chen Z. Arsenic

trioxide, a therapeutic agent for APL. Oncogene 2001;20:7146e53.