Sodium metaarsenite (NaAs2O3: code name KML001) is an orally bioavailable arsenic compound with potential anti-cancer activity. However, the effect of KML001 has not been studied in acute myeloid leukemia (AML). We investigated the anti-leukemic effect of KML001 in AML, and determined the mode of action of KML001. KML001 inhibited the cellular proliferation in all AML cell lines and primary AML blasts as well as HL-60R (cytosine arabinoside-resistant HL-60) cells, while As2O3 was not effective in primary AML blasts and AML cell lines including HL-60R cells. KML001 induced G1 arrest and apoptosis in HL-60 and HL-60R cells. KML001 inhibited the activation of STAT (signal transducer and activator of transcription) 1, 3, 5, NF-κB, AKT and PI3K, while phosphorylated PTEN was upregulated. In addition, activation of ERK, p38 and JNK was observed in KML001-induced growth inhibition of HL-60 and HL-60R cells. Furthermore, KML001 induced telomeric terminal restriction fragment (TRF) length shortening in a time-dependent manner in HL-60 and HL-60R cells. Real-time PCR with RNA extracted from KML001-treated HL-60 and HL-60R cells showed a significant reduction of catalytic subunit of telomerase, hTERT, in a time-dependent manner. Additionally, γ-H2AX, a sensitive molecular marker of DNA damage, in HL-60 and HL-60R cells was induced by KML001. These results suggest that KML001 inhibits the proliferation of AML cells including cytosine arabinoside-resistant AML cells via various mechanisms such as cell cycle arrest, induction of apoptosis, inhibition of JAK/STAT and PI3K pathways, activation of MAPK pathway and telomere targeting.
Arsenic compounds have been used in traditional medicine as anti-tumor and anti-inflammatory agents over 2,400 years, however, their clinical use is strikingly decreased because of the carcinogenic and toxic effects (
KML001, sodium metaarsenite, is a water-soluble, therefore, orally bioavailable, trivalent arsenical compound (
In the present study, we investigated the effect of KML001 on acute myeloid leukemia (AML) cell lines. KML001 inhibited the proliferation of all AML cell lines including Ara-C (cytosine arabinoside)-resistant HL-60 (HL-60R) cells by cell cycle arrest and induction of apoptosis. In addition, KML001 shortened the telomere length without affecting telomerase activity. All together, KML001 seems to be a candidate agent for clinical investigation in the treatment of refractory acute myeloid leukemia.
Human myeloid leukemia cell lines examined in the present study were HL-60, BV173, HEL, K562, KCL22, KG1, KU812, MegO1, ML1, NB4 and U937, and they were kindly presented by Dr H.P. Koeffler (Cedars-Sinai Medical Center/University of California, Los Angeles, CA, USA). The cells were cultured in tissue flasks in RPMI-1640 medium (Gibco-BRL, Gaithersburg, MD, USA) supplemented with 10% (vol/vol) fetal bovine serum (FBS; Gibco-BRL), 100 U/ml penicillin and 100 μg/ml streptomycin (Sigma Chemical, St. Louis, MO, USA) and maintained in a humidified atmosphere, 5% CO2 at 37°C. The culture medium was changed every 3–4 days. The Ara-C-resistant cell line HL-60R was established by stepwise increase of concentrations of cytosine arabidoside in medium. Leukemic blasts were harvested from bone marrow aspirates of 4 AML patients (2 AML M1 and 2 AML M2), who were initially diagnosed when the blasts counts were high (>85%) at presentation and gave written informed consent. Mononuclear cells (MNCs) from bone marrow were collected by separation on Ficoll-Paque (Amersham Biosciences AB, Uppsala, Sweden) gradients at a density of 1.077, washed twice in phosphate-buffered saline (PBS), and suspended in Iscove’s modified Dulbecco’s medium (IMDM; Gibco-BRL) containing 10% heat-inactivated FBS (Gibco-BRL).
KML001 (sodium metaarsenite) was obtained from Komipharm International, 10−3 M drug stocks were prepared in distilled water and aliquots were stored at 4°C. The stock solution was stable for >1 year. Working concentrations were freshly prepared daily by diluting the stock with RPMI-1640. As2O3 was purchased from Sigma-Aldrich, 5×10−2 M stock solution was prepared in 5 M NaOH, and aliquots were stored at 4°C. SP600125, a specific JNK inhibitor, and SB202190, specific inhibitor of p38 protein were purchased from Calbiochem (San Diego, CA, USA) (
Cellular growth inhibition effect of KML001 was determined by measuring the MTT [3- [4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide dye absorbance of living cells as previously described (
Cells were fixed with methanol for 1 h and stained with 50 μg/ml of propidium iodide (PI) containing 50 μg/ml of RNaseA. The DNA contents of the cells (10,000 cells/experimental group) were analyzed using a FACStar flow cytometer (Becton-Dickinson, San Jose, CA, USA) equipped with a ModFit LT program (Lysis II, CellFit). The percentage of cell population in each cell cycle phase (G1, S or G2/M) was calculated from DNA content histograms, excluding the population in the sub-G1 phase.
Apoptosis was determined by staining cells with Annexin V-FITC (BD Biosciences, San Diego, CA, USA) and PI (
Cells were suspended in lysis buffer containing 50 mM Tris (pH 7.5), 1% NP-40, 2 mM EDTA, 10 mM NaCl, 20 μg/ml aprotinin, 20 μg/ml leupeptin and 1 mM phenylmethylsulphonyl fluoride and were placed on ice for 20 min. Samples containing 20–100 μg of total protein were resolved in SDS-polyacrylamide denaturing gel, transferred to nitrocellulose membranes, and probed with antibodies. The blots were developed using the ECL kit (Intron Biotechnology, Gyeonggi-do, Korea).
Cells were suspended in an extraction buffer [50 mM Tris-Cl (pH 7.5), 250 mM NaCl, 0.1% NP-40, 5 mM EDTA, 50 mM NaF, 0.1 mM NaVO4, 100 mM phenylmethylsulfonyl fluoride, 0.2 mM leupeptin, 10 μg/ml aprotinin, 0.1 mM pepstatin A) and incubated on ice for 15 min. After centrifugation at 13,000 rpm for 20 min, the supernatant was collected and protein concentration was determined using a Bio-Rad assay kit. Two micrograms of each antibody (CDK2, CDK4 and CDK6) were added to 200 μg of each cell extract in 500 μl of extraction buffer and incubated for 4 h at 4°C with continuous agitation. To collect immune complexes, 30 μl of protein A/G-agarose was added to the mixture, which was then incubated for 2 h. Immune complexes were centrifuged at 1,200 rpm for 2 min and the precipitates were washed three times with extraction buffer and twice with kinase reaction buffer [50 mM Tris-Cl (pH 7.5), 10 mM MgCl2 and 1 mM DTT]. CDK 2 kinase assays on histone H1 was performed by mixing the respective immune complexes with 5 μg of histone H1 and 1 μCi of [γ-32P]-ATP in 35 μl of kinase reaction buffer. CDK 4 and CDK 6 kinase assays on Rb-c residue were performed in the same way. Kinase reactions were performed at 37°C for 30 min and were terminated with 2X SDS-PAGE loading buffer. The reaction mixtures were resolved by SDS-PAGE. The extent of phosphorylation was determined by autoradiography.
For the measurement of telomere length, Roche Diagnostics TeloTAGG telomere restriction fragment (TRF) length kit was used according to manufacturer’s instructions (Roche Diagnostics GmbH, Mannheim, Germany).
Total RNA was isolated with TRI reagent (Molecular Research Center, Inc., Cincinnati, OH, USA) and cDNA was synthesized from 1 μg of total RNA using ImProm-II Reverse Transcriptase (Promega Corp., Madison, WI, USA) and random hexamers. Quantitative PCR was performed using SYBR-Green I as a double-strand DNA-specific binding dye on iCycler IQ detection system (Bio-Rad Laboratories). Thermocycling was performed in a final volume of 20 μl containing; 4 μl cDNA sample, 10 pM of each primer, 0.125 mM dNTP mixture, 0.25 mg/ml BSA, 0.05% Tween-20, 1X rTaq reaction buffer containing 1.5 mM MgCl2 (Takara, Shiga, Japan), 1 unit rTaq DNA polymerase (Takara), and 1X SYBR-Green I (Molecular Probes, Sunnyvale, CA, USA). After an initial denaturation at 95°C for 10 min, 35 cycles of 94°C for 30 sec, 53°C for 30 sec, and 72°C for 30 sec were carried out.
All cDNA samples were synthesized in parallel and PCR reactions were run in triplicate. The mRNA levels were derived from standard curves and are expressed as relative changes after normalization vs. β-actin mRNA levels.
HL-60 and HL-60R cells were treated with KML001 at a concentration of 10−7 M for 24, 48 and 72 h. Chromatin immunoprecipitation (ChIP) assays for studying the specific association of γ-H2AX with telomeric repeat sequences were performed as described by d’Adda di Fagagna
The data represent mean values ± SEM (error bars) of experiments repeated at least 3 times. Statistical significance was determined using the Student’s t-tests. P-values <0.05 were considered statistically significant.
KML001 inhibited the cellular proliferation in all AML cell lines in a dose-dependent manner with IC50 of 5×10−8 M, while arsenic trioxide (As2O3) did not (
The effect of KML001 on the cell cycle was determined in HL-60 and HL-60R cells by FACS analysis. Three independently performed DNA flow cytometric analyses indicated that KML001 induced a G1 arrest in HL-60 and HL-60R cells at a concentration of 1×10−7 M during 72 h of exposure; population of HL-60 cells in G1 phase was 46.21% at 0 h and 64.56% at 72 h (
Cell cycle arrest was associated with downregulation of CDKs of CDK4, CDK6, as well as cyclins of cyclin D1 and cyclin E. In addition, the expression of p21 and p27 proteins, and cyclin-dependent kinase inhibitors (CDKIs), was increased in HL-60 and HL-60R cells. In contrast, no significant change of CDKs and cyclins was observed and the expression of CDKIs was decreased in arsenic trioxide-treated HL-60 and HL-60R cells (
Since western blot analysis showed that KML001 induced a marked accumulation of p27 protein, we investigated whether the KML001-induced p27 protein existed in complexes with CDKs active in the G1 phase of the cell cycle in HL-60 and HL-60R cells. As shown in
To determine whether the increased CDKIs and the changed cell cycle-regulatory proteins result in the inhibition of CDK activity in KML001-treated HL-60 and HL-60R cells,
Taken together, these results suggest that p27 protein could play a critical role in a G1 arrest via its increased binding to CDK2, CDK4 and CDK6 proteins and subsequent inhibition of CDK2-, CDK4-, CDK6-associated kinase activities in KML001-treated HL-60 and HL-60R cells.
In contrast, arsenic trioxide did not reduce the activity of CDK2-, CDK4- and CDK6-associated kinase in HL-60 and HL-60R cells (
In order to determine whether KML001 treatment could induce apoptosis in HL-60 and HL-60R cells,
STATs are cytoplasmic transcription factors and key mediators of cytokine and growth factor signaling pathways. Many malignant cells have shown to consistently express STAT molecules. In order to investigate the role of STAT molecules in anti-leukemic effect of KML001, HL-60 and HL-60R cells were treated with KML001 for 2 h at indicated concentrations.
The expression of p-STAT1, p-STAT3 and p-STAT5 was downregulated in HL-60 and HL-60R cells following treatment with KML001, while no significant expressional change was noted in As2O3-treated HL-60 and HL-60R cells (
Additionally, pre-treatment of HL-60 and HL-60R cells for 12 h with 20 μM of SB202190, specific inhibitor of p38 protein, blocked KML001-induced p-p38, and subsequently abrogated the effect of KML001 on STAT1 (
Additional study showed that the expression of P65 and P50 subunits of NF-κB was decreased in KML001-treated HL-60 and HL-60R cells (
The maintenance of telomere lengths is critical for chromosomal integrity in cells. It has been shown that KML001 targets the telomere (
Uncapping the telomere signal can induce cellular growth arrest/apoptosis via telomere length shortening. It accelerates the double-strand break-mediated DNA damage signaling pathways, including phosphorylation of γ-H2AX at Ser139, which represents an early event of DNA damage signaling (
In the present study, we demonstrated for the first time that KML001 potently inhibited the proliferation of HL-60 and HL-60R cells by inducing cell cycle arrest and triggering apoptosis. KML001 induced a dose-dependent inhibition of cellular proliferation of all AML cell lines including HL-60R cells as well as primary leukemic blasts from AML patients. The cell cycle analysis revealed that KML001 was able to prominently induce a G1 growth arrest of HL-60 and HL-60R cells at a concentration of 10−7 M during 72-h exposure, in contrast, cell cycle arrest was not observed in As2O3-treated HL-60 and HL-60R cells. Among CDKs that regulate the cell cycle, CDK4 and CDK6 are activated in association with cyclin D during G1 progression, whereas CDK2 is activated primarily in association with cyclin E in the late G1 phase (
Our data demonstrated that KML001 markedly induced the apoptosis in HL-60 and HL-60R cells, showing the downregulation of Bcl-2 and effector procaspase-3 in a dose-dependent manner in HL-60 and HL-60R cells, in contrast, As2O3 did not. We confirmed apoptosis using Annexin/PI double staining in which apoptotic cells were increased in a time-dependent manner in HL-60 and HL-60R cells.
Signal transduction pathways regulate cellular differentiation, division and cell death. Therefore, we investigated the effect of KML001 on the signal transduction pathways. STATs are latent cytoplasmic transcription factors activated by JAK which are constitutively associated with cytokines and growth factor receptors, and frequently activated in many types of human malignancies including leukemia (
Phosphatidylinositol 3-kinase (PI3K)/Akt pathway is crucial in signaling of diverse biological functions, including cell proliferation, survival and metastasis (
NF-κB is a heterodimer composed of the p65 and p50 subunits that contains the transcriptional activation domain required for initiation of gene transcription (
Arsenic has been found to target telomere by inhibition of the transcription of the human telomerase reverse transcriptase (hTERT) and alteration of telomere length, telomerase activity, and telomere binding proteins (
Uncapping the telomere signal can induce cellular growth arrest/apoptosis via telomere length shortening. It accelerates the double-strand break-mediated DNA damage signaling pathways, including p-γ-H2AX at Ser139, which represents an early event of DNA damage signaling (
In summary, KML001 inhibited the cellular proliferation of human primary leukemic blasts as well as AML cell lines including HL-60R via induction of the cell cycle arrest, triggering apoptosis and modulation of STATs, MAPK, PI3K and NF-κB signaling. In addition, KML001 targeted the telomere. Therefore, KML001 might be a candidate agent for the treatment of
The present study was conducted in the Hanyang University Hospital Clinical Laboratory, and it was supported in part by Dong-A Socio Holdings, Inc.
Effect of KML001 and As2O3s on cellular proliferation. (A) Effect of KML001 in AML cell lines. Cellular proliferation of AML cell lines was determined by MTT assay after treatment of KML001 for 72 h. (B) Effect of As2O3 in AML cell lines. Cellular proliferation of AML cell lines was determined by MTT assay after treatment of As2O3 for 72 h. (C) Effect of KML001, Ara-C and As2O3 on HL-60 cells. Proliferation of HL-60 cells was determined by MTT assay after treatment of agents for 72 h. (D) Effect of KML001, Ara-C and As2O3 on HL-60R cells. Proliferation of HL-60R cells was determined by MTT assay after treatment of agents for 72 h. (E) Effect of KML001 on AML blasts isolated from AML patients. Cellular proliferation of AML blasts was determined by MTT assay after treatment of KML001 for 72 h. (F) Effect of As2O3 on AML blasts isolated from AML patients. Cellular proliferation of AML blasts was determined by MTT assay after treatment of As2O3 for 72 h.
Effect of KML001 and As2O3 on cell cycle. (A) Modulation of cell cycle-regulatory proteins in HL-60 and HL-60R. Cells were harvested at the indicated times after incubation with KML001 (10−7 M) or As2O3 (10−7 M). Cells were then lysed, and the supernatants were subjected to western blot analysis. (B) Association of p27 with CDKs in HL-60 and HL-60R cells. Cells were treated with KML001 (10−7 M) for 72 h. Total lysates were immunoprecipitated with anti-CDK2, anti-CDK4 and anti-CDK6 antibodies. The bound p27 in each immunocomplex was determined by western blot analysis. (C) Effect of KML001 and As2O3 on CDK-associated kinase activities in HL-60 and HL-60R cells. Cells were treated with KML001 (10−7 M) or As2O3 (10−7 M) for 72 h. CDK activity assays on substrates were performed using immunoprecipitation with indicated antibodies.
Effect of KML001 and As2O3 on apoptosis. (A) Induction of apoptosis by KML001 in HL-60 and HL-60R cells. Cells were stained with Annexin V-FITC and PI and analyzed by FACS. (B) Modulation of apoptosis-related proteins in HL-60 and HL-60R cells. Cells were treated with KML001 or As2O3 at indicated doses for 72 h. Total protein was resolved in SDS-polyacrylamide denaturing gel, transferred to nitrocellulose membranes, and probed with indicated antibodies.
Effect of KML001 and As2O3 on cell signaling. (A) Modulation of STAT signaling in HL-60 and HL-60R cells. Cells were treated with KML001 or As2O3 for 2 h at indicated doses. (B) Modulation of MAPK signaling in HL-60 and HL-60R. Cells were treated with KML001 or As2O3 for 2 h at indicated doses. (C) Blocking of JNK phosphorylation by SP600125, a specific JNK inhibitor in HL-60 and HL-60R cells. Cells were pre-treated with 10 μM of SP600125 for 1 h and further cultured with KML001 for 2 h at the indicated doses. (D) Blocking of p38 by SB202190, specific inhibitor of p38 protein in HL-60 and HL-60R cells. Cells were pre-treated with 20 μM of SB202190 for 1 h and further cultured with KML001 for 2 h at the indicated doses.
Modulation of PI3K/Akt signaling by KML001 and As2O3 in HL-60 and HL-60R cells. Cells were treated with KML001 or As2O3 for 2 h at indicated doses. Cells were lysed and proteins were separated by SDS-PAGE. Western blot analysis was performed using indicated antibodies.
Effect of KML001 on telomere length and telomere gene expression. (A) Effect of KML001 on telomere length in HL-60 and HL-60R cells. Cells were treated with KML001 (10−7 M) for indicated times, and telomere length was calculated using Roche Diagnostics TeloTAGG telomere restriction fragment (TRF) length kit. The experiments were performed in triplicates. (B) Effect of KML001 on hTERT mRNA in HL-60 and HL-60R cells. Cells were treated with KML001 (10−7 M) for indicated times, and hTERT mRNA expression was measured by quantitative RT-PCR. The experiments were performed in triplicates.
Effect of KML001 on telomere-associated DNA damage. The expression of p-γ-H2AX, a marker for DNA damage checkpoint response in telomere-initiated senescence, was evaluated in HL-60 and HL-60R cells by chromatin immunoprecipitation (ChIP) assay. ChIP with γ-H2AX revealed its very strong association with telomeres after KML001 treatment in HL-60 and HL-60R cells for 12 h.
Cell cycle analysis in KML001-treated HL-60.
0 h | 12 h | 18 h | 24 h | 48 h | 72 h | |
---|---|---|---|---|---|---|
G0–G1 | 46.21 | 48.02 | 41.61 | 42.33 | N.D | 64.56 |
S | 40.48 | 38.76 | 29.05 | 34.52 | N.D | 23.77 |
G2/M | 12.31 | 13.22 | 29.33 | 21.15 | N.D | 11.67 |
SubG1 | 2.10 | 11.22 | 70.79 | 51.30 | 74.58 | 48.04 |
Debris | 2.04 | 11.33 | 90.77 | 66.07 | N.D | 50.36 |
N.D, not detected.
Cell cycle analysis in KML001-treated HL-60R.
0 h | 12 h | 18 h | 24 h | 48 h | 72 h | |
---|---|---|---|---|---|---|
G0–G1 | 28.28 | 36.34 | 50.90 | 35.13 | N.D | 59.67 |
S | 56.11 | 46.13 | 38.48 | 18.96 | N.D | 36.95 |
G2/M | 15.07 | 17.53 | 10.62 | 15.90 | N.D | 3.37 |
SubG1 | 3.99 | 21.81 | 11.13 | 3.08 | 63.71 | 57.86 |
Debris | 6.27 | 21.59 | 11.89 | 4.37 | N.D | 66.34 |
N.D, not detected.