NEOPLASIA
The histone deacetylase inhibitor AN-9 has selective toxicity to acute leukemia
and drug-resistant primary leukemia and cancer cell lines
Ayse Batova, Li-en Shao, Mitchell B. Diccianni, Alice L.Yu, Tetsuya Tanaka, Ada Rephaeli, Abraham Nudelman, and John Yu
The novel prodrug of butyric acid, pivaloyloxymethyl butyrate (AN-9), a histone
deacetylase inhibitor, shows great promise as an effective and relatively nontoxic
anticancer agent for solid malignancies.
However, little is known about its effects
on hematopoietic malignancies. In this
study, we show that 21 primary samples
of acute leukemia were sensitive to the
antiproliferative effects of AN-9, with a
50% inhibitory concentration (IC50) of
45.8 ⴞ 4.1 ␮M. In colony-forming assays,
primary T-cell acute lymphoblastic leukemia (T-ALL) cells were 3-fold more sensitive to AN-9 than the normal hematopoi-
etic progenitors, erythroid burst-forming
units and granulocyte/monocyte colonyforming units. AN-9 induced apoptosis in
the T-ALL cell line CEM. A common problem
with cancer is chemoresistance, which is
often typical of relapsed cancers. Remarkably, a T-ALL sample at diagnosis and an
acute myeloid leukemia sample at relapse
that were resistant to doxorubicin in vitro
were sensitive to AN-9, with an IC50 of 50 ␮M
for both samples. More strikingly, samples
from 2 infants with t(4;11) ALL obtained at
diagnosis and relapse each were the most
sensitive to AN-9, with IC50 values of 25 ␮M
and 17 ␮M, respectively. Furthermore, a
doxorubicin-resistant clone of HL60, HL60/
ADR, obtained by the transfection of the
MDR-1 gene, was equally sensitive to AN-9
cytotoxicity as the parental cells. AN-9 induced the expression of p21 in an infant
leukemia sample with 11q23 rearrangement,
but not in T- or B-precursor ALL. Collectively, our results suggest that AN-9 is a
selective agent for hematopoietic malignancies that can circumvent the mechanisms of
chemoresistance limiting most conventional
chemotherapy. (Blood. 2002;100:3319-3324)
© 2002 by The American Society of Hematology
Introduction
The leukemias account for the largest number of cases of childhood
cancer. Acute lymphoblastic leukemia (ALL) represents approximately three fourths, whereas acute myeloid leukemia (AML)
represents one sixth of the cases. Despite recent advances in the
treatment of childhood cancers, leukemias remain the primary
cause of cancer-related mortality among children in the United
States. Although ALL and AML are potentially curable diseases,
with a 5-year survival of 70% for ALL and 40% for AML, once
leukemia recurs, the outcome is dismal. Therefore, there is a need
for novel antileukemia agents, especially those that are effective for
relapsed leukemias resistant to existing chemotherapeutic agents.
Histone acetylation plays a key role in the regulation of
transcription by modulating chromatin structure.1-3 In general,
histone acetylation is associated with activation of transcription,
whereas histone deacetylation is associated with repression of
transcription. It has recently been established that many malignancies, particularly leukemias, are associated with aberrant recruitment of histone deacetylases (HDACs) or with mutations in histone
acetyl transferases such as EP300.4-6 For example, several investigators reported that the oncoprotein promyelocytic leukemia/
retinoic acid receptor–␣, generated as a result of a translocation in
acute promyelocytic leukemia (APL), suppressed transcription by
recruiting HDACs and thus interfering with normal cell growth and
differentiation.7-9 Furthermore, resistance to the differentiating
actions of all-trans retinoic acid in APL-derived cells could be
overcome by cotreatment with the HDAC inhibitor sodium phenyl
butyrate.10 There is in fact an increasing body of evidence that
HDAC inhibitors are effective therapeutic agents for a variety of
cancers that are refractory to conventional anticancer agents.11,12
Pivaloyloxymethyl butyrate (AN-9), a butyric acid prodrug, is a
relatively new member of an established family of acyloxyalkyl ester
prodrugs of carboxylic acids that undergo rapid hydrolysis. Upon
hydrolysis, the resulting products are butyric acid, pivalic acids, and
formaldehyde (Figure 1). The anticancer effect of AN-9 is assumed to
stem primarily from the release of butyric acid, an HDAC inhibitor.13 It
is important to note, however, that AN-9 is 10-fold more potent than
butyric acid in vitro and has anticancer effects in vivo not observed with
butyric acid even at 10-fold higher concentrations.14 The increased
potency of AN-9 over butyric acid is most likely due to its increased
permeability across cell membranes, allowing for efficient delivery of
butyric acid to subcellular targets.15 AN-9 inhibits cell proliferation and
soft agar colony formation of a variety of cancer cell lines and primary
human solid tumor cells, including those of colon, breast, ovary, lung,
kidney, and bladder.13,16,17 In addition to being an antiproliferative agent,
AN-9 has been shown to induce differentiation and apoptosis in HL60
cells.18 Earlier studies in mouse tumor models have demonstrated that
From the Division of Pediatric Hematology/Oncology, Department of
Pediatrics, University of California San Diego; Department of Immunology, The
Scripps Research Institute, La Jolla, CA; Felsenstein Medical Research
Center, Sackler School of Medicine, Tel Aviv University, Beilinson Campus,
Petach Tikva, Israel; and the Chemistry Department, Bar Ilan University, Ramat
Gan, Israel.
part by grant MO1 RR00827 from the General Clinical Research Center
program. In addition, it is supported by grant 542/0 (to A.R.) from the Israel
Science Foundation.
Submitted February 25, 2002; accepted April 30, 2002. Prepublished online as
Blood First Edition Paper, July 12, 2002; DOI 10.1182/blood-2002-02-0567.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
Supported by grants from the Leukemia & Lymphoma Society 6125 (to A.L.Y.)
and 6226 (to J.Y.), CA79951 (to J.Y.), The Cindy Matters Fund (to A.L.Y.), and in
BLOOD, 1 NOVEMBER 2002 䡠 VOLUME 100, NUMBER 9
Reprints: Alice L.Yu, Division of Pediatric Hematology/Oncology, University of
California San Diego Medical Center, 200 West Arbor Dr, San Diego, CA
92103-8447.
© 2002 by The American Society of Hematology
3319
3320
BLOOD, 1 NOVEMBER 2002 䡠 VOLUME 100, NUMBER 9
BATOVA et al
mined by Wright stain, was generally 80% or higher. Leukemia cells were
cultured in RPMI 1640 supplemented with 10% fetal calf serum, 2 mM
glutamine, and 1% penicillin/streptomycin (complete medium).
3H-thymidine
incorporation assay
Primary leukemia and normal bone marrow cells were plated in complete RPMI
at 75 ⫻ 103 cells per well in a 96-well plate. Leukemia and neuroblastoma cell
lines were plated at 10 and 5 ⫻ 103 cells per well, respectively. Cells were treated
with increasing concentrations of AN-9 or with 0.1% dimethyl sulfoxide
(DMSO; control) for 3 days. Cells were then pulsed with 3H-thymidine at 1.6
␮Ci/well (0.0592 MBq) for 6 hours. Incorporation of 3H-thymidine was
determined in a scintillation counter (Beckman Coulter, Fullerton, CA) after the
cells were washed and deposited onto glass microfiber filters using cell harvester
M-24 (Brandel, Gaithersburg, MD).
Figure 1. Structure of AN-9 and the products released upon metabolic
hydrolysis.
AN-9 is an effective antitumor agent and displays low toxicity.19
Currently, AN-9 is being studied in a phase II clinical trial for non–small
cell lung cancer. To date, all in vitro and in vivo studies of AN-9 have
been confined to solid tumors, with the exception of a murine monocytic
leukemia and the HL60 cell line. In this study, we investigated the in
vitro therapeutic efficacy of AN-9 in primary human acute leukemias,
including doxorubicin-resistant and/or clinically refractory acute leukemias. Our findings demonstrate the selective toxicity of AN-9 to acute
leukemias, including drug-resistant relapsed leukemias, and thus provide the rationale for the initiation of clinical trials of AN-9 in relapsed
acute leukemias.
Colony-formation assays
Primary T-ALL and normal bone marrow cells were plated at 5 ⫻ 105 per
35-mm dish in methylcellulose medium with increasing concentrations of
AN-9 for 14 days. An erythroid burst-forming unit (BFU-E) was identified
as a large aggregate of more than 64 hemoglobinized cells or as clusters of 3
or more subcolonies consisting of 8 or more hemoglobinized cells per
subcolony. The granulocyte/monocyte colony-forming unit (CFU-GM) was
enumerated as a group of more than 50 granulocytic/monocytic translucent
cells. T-ALL colonies were identified as clusters of more than 50 cells.
Cytotoxicity assays
Primary leukemia and CEM cells were plated in complete RPMI at 75 ⫻ 103
cells per well and 10 ⫻ 103 cells per well (96-well plate), respectively. Cells were
treated with increasing concentrations of AN-9 or 0.1% DMSO for 3 days. The
number of viable cells was determined by trypan blue exclusion.
Apoptosis assays
Patients, materials, and methods
Antibodies and reagents
AN-9 was prepared as described previously and determined to be 99% pure
by nuclear magnetic resonance spectroscopy.13 doxorubicin was obtained
from Bedford Laboratories (Bedford, OH). The p21 and p27 antibodies
were purchased from Pharmingen (San Diego, CA), and the ␤-actin
antibody was from Sigma (St Louis, MO). Ficoll-Hypaque was purchased
from Pharmacia (Piscataway, NJ).
Cell lines
The leukemia cell lines HL60 and HL60/ADR, derived by transfection of HL60
with the mdr1 gene, were generously provided by Dr Michael Kelner.20,21 The
T-cell acute lymphoblastic leukemia (T-ALL) cell line CEM was obtained from
American Type Culture Collection (Rockville, MD). The neuroblastoma cell
lines Be2c and Be2c/ADR, derived by selection in doxorubicin-containing
media, were generously provided by J. Biedler.22
Patient population and isolation of primary leukemia cells
Heparinized bone marrow or peripheral blood samples were obtained at
diagnosis and relapse from patients with AML or B-precursor ALL at the
University of California in San Diego. T-ALL samples were obtained from
patients enrolled in Pediatric Oncology Group protocols 9900 (ALL
Biology Study) and 9673 (Relapsed T-ALL Study) and were shipped
overnight to the University of California San Diego for biology studies.
Bone marrow cells obtained from patients in complete remission who
underwent diagnostic bone marrow examination were used as normal
controls. Only excess samples obtained for clinical purposes were analyzed.
These samples were collected under a protocol approved by the Institutional
Review Board. Mononuclear cells were isolated by density gradient
centrifugation through Ficoll-Hypaque (specific gravity 1.077 g/mL) at
400g for 30 minutes, followed by 2 washes in RPMI 1640 (Ammersham
Biosciences, Uppsala, Sweden). The content of lymphoblasts, as deter-
CEM cells were plated at 1.2 ⫻ 106 cells per well (6-well plate) in complete
RPMI and treated with 0.1% DMSO or 60 and 75 ␮M AN-9 for 20 and 45
hours. Cells were then visualized and photographed using a Nikon Eclipse
TE-300 inverted microscope and the SPOT camera (SPOT Diagnostic
Instruments, Sterling Heights, MI). Alternatively, cells were stained with
Alexa Fluor 488 annexin V and propidium iodide using the Vybrant
Apoptosis Assay Kit (Molecular Probes, Eugene, OR) and then analyzed by
flow cytometry using a FACScan flow cytometer and CellQuest 3.2.1
software (Becton Dickinson).
Western blot analysis
Primary leukemia cells (10-20 ⫻ 106) were treated with 10, 25, 50, and 100
␮M AN-9 for 24 and 48 hours. As a control, cells were treated with 0.1%
DMSO. Protein was extracted with 30 ␮L sodium dodecyl sulfate (SDS)
lysis buffer (50 mM Tris-HCl, pH 6.8, 2% SDS, 0.1% bromophenol blue,
10% glycerol, 5 mM dithiothreitol). Samples were heated in boiling water
for 10 minutes, and then the lysate was passed through a 28-gauge needle to
shear chromosomal DNA. Supernatants were then collected after spinning
in a microfuge at 13 000 rpm for 10 minutes at 4°C. Proteins were resolved
on an 8% SDS-polyacrylamide gel and transferred to Immobilon-P nylon
membranes (Millipore, Bedford, MA). Membranes were probed with p21
and p27 antibodies at 0.5 ␮g/mL. Membranes were also probed with a
␤-actin antibody at 2 ␮g/mL as a control for protein loading. Antibodybound proteins were detected by chemifluorescence and analyzed using the
Storm 840 Phosphorimager (Molecular Dynamics).
Results
Primary ALL cells are more sensitive than normal
bone marrow cells to AN-9
The effect of AN-9 on DNA synthesis in both primary ALL and
normal bone marrow was examined by 3H-thymidine incorporation
BLOOD, 1 NOVEMBER 2002 䡠 VOLUME 100, NUMBER 9
assays. The results demonstrated that ALL cells were more
sensitive than normal bone marrow cells to inhibition of 3Hthymidine uptake by AN-9 (Figure 2).
The mean 50% inhibitory concentration (IC50) of AN-9 in 6 normal
bone marrow cell samples was 87 ⫾ 5 ␮M. In contrast, the mean AN-9
IC50 in 15 primary T-ALL samples was 50 ⫾ 5 ␮M, and that in 2
B-precursor ALL samples was 44 ␮M and 54 ␮M (data not shown). The
finding that both T- and B-cell ALL have similar sensitivity to AN-9
suggests that the effects of AN-9 are not lineage specific. Remarkably,
however, primary leukemias obtained from 2 infants with an 11q23
rearrangement were the most sensitive to AN-9. One of these samples
was obtained from a patient who suffered from leukemia relapse shortly
after bone marrow transplantation and became refractory to reinduction
chemotherapy. On 2 separate occasions during his relapse, the IC50 of
AN-9 was only 17 ␮M (Figure 2) and 13 ␮M (data not shown),
respectively. The second infant ALL sample, collected at the time of
diagnosis, had an AN-9 IC50 of 25 ␮M (Figure 2). To confirm the results
obtained by 3H-thymidine incorporation on the selectivity of AN-9 for
leukemia, we performed colony-formation assays using normal bone
marrow cells and primary T-ALL cells. The mean IC50 of AN-9 in 6
primary T-ALL samples was 46.6 ⫾ 1.1 ␮M, and those of 5 normal
hematopoietic progenitors of BFU-E and CFU-GM were 159.7 ⫾ 2.7
␮M and 159.5 ⫾ 2.9 ␮M, respectively (Figure 3). The difference in the
AN-9 IC50 between T-ALL and normal hematopoietic progenitors was
highly significant, with P ⬍ .001. Thus, the results of colony-formation
assays confirm the selectivity of AN-9 for ALL rather than normal bone
marrow cells.
AN-9 is cytotoxic to ALL
To distinguish between cytostatic and cytotoxic effects of AN-9, we
performed cell-viability assays using both a T-ALL cell line, CEM,
as well as primary T-ALL cells. The results of trypan blue exclusion
assays demonstrated that AN-9 was cytotoxic to CEM and primary
T-ALL in a dose-dependent manner (Figure 4).
The IC50 of AN-9 was 47 ␮M in CEM and 41 ␮M in primary
T-ALL after 3 and 5 days of AN-9 treatment, respectively (Figure
4). At 75 ␮M AN-9, cytotoxic effects on CEM were evident as
early as after 24 hours of incubation (data not shown).
Figure 2. Inhibition of 3H-thymidine incorporation by AN-9 in primary ALL cells
and normal bone marrow cells. Normal bone marrow cells and primary ALL cells
were treated with increasing concentrations of AN-9 for 3 days. Cells were then
pulsed with 3H-thymidine for 6 hours. F indicates T-ALL; f, B-precursor ALL;
䉬, relapsed infant ALL; 䉫, diagnosed infant ALL; Œ, normal bone marrow. All
experimental conditions were performed in triplicate. Error bars represent standard
error of the mean for data obtained with normal bone marrow (n ⫽ 6) and primary
T-ALL (n ⫽ 16). For data obtained with infant ALL (n ⫽ 1) and for a representative
B-precursor ALL (n ⫽ 1), error bars represent standard deviation.
SELECTIVE TOXICITY OF AN-9 TO ACUTE LEUKEMIA
3321
Figure 3. Inhibition of colony formation by AN-9 of primary T-ALL and normal
hematopoietic progenitors. Primary T-ALL (n ⫽ 6) and normal bone marrow cells
(n ⫽ 5) were cultured in methylcellulose medium and increasing concentrations of
AN-9 for 14 days. The BFU-E was identified as a large aggregate of more than 64
hemoglobinized cells, or as clusters of 3 or more subcolonies consisting of 8 or more
hemoglobinized cells per subcolony. The CFU-GM was enumerated as a group of
more than 50 granulocytic/monocytic translucent cells. f indicates T-ALL; Œ, CFU-GM;
E, BFU-E. Error bars represent standard error of the mean.
Induction of apoptosis by AN-9
To determine whether cytotoxicity of AN-9 involved the induction of
apoptosis, we treated CEM cells with 0.1% DMSO (negative control),
60 or 75 ␮M AN-9, or with 5% ethanol (positive control) for 20 and 45
hours, and then examined them by flow cytometry after staining with
Alexa Fluor 488 annexin V and propidium iodide. As shown in Figure 5,
AN-9 induced apoptosis in CEM cells in a dose-dependent manner. For
instance, treatment with 60 ␮M and 75 ␮M AN-9 for 45 hours resulted
in 3.5% and 8.3% apoptotic cells, respectively. Percentage apoptosis in
negative control cells was 0.5%, and that in the positive control cells was
10%. Late-stage apoptosis or necrosis was also evident after treatment
with 60 and 75 ␮M AN-9 (data not shown).
To further confirm the results presented in Figure 5, CEM cells
treated with 75 ␮M AN-9 for 20 hours were photographed in a
12-well plate in the absence of cell manipulations to avoid the loss
of fragile apoptotic cells. Results of a representative experiment as
presented in Figure 6B revealed numerous cells with fragmented
Figure 4. Cytotoxicity of AN-9 in CEM and primary T-ALL cells. CEM and T-ALL
cells were treated with increasing concentrations of AN-9 for 3 and 5 days,
respectively. Viable cells were then determined by trypan blue exclusion. F indicates
primary T-ALL; Œ, CEM. Results obtained with primary T-ALL are representative of 2
independent experiments with 2 different patient samples. Data obtained with CEM
cells are an average of 2 independent experiments. All experimental conditions were
performed in triplicate. Error bars represent standard deviation.
3322
BLOOD, 1 NOVEMBER 2002 䡠 VOLUME 100, NUMBER 9
BATOVA et al
T-ALL (data not shown). These results suggest that the effects of
AN-9 on T-ALL are independent of p21 or p27.
Drug-resistant primary leukemia and cancer cell
lines are sensitive to AN-9
Figure 5. Induction of apoptosis by AN-9 in CEM cells. CEM cells were treated
with 60 or 75 ␮M AN-9 for 45 hours. Positive and negative control cells were treated
with 5% ethanol and 0.1% DMSO, respectively. Cells were then stained with Alexa
Fluor 488 annexin V and propidium iodide using the Vybrant Apoptosis Kit (Molecular
Probes). Apoptotic cells were then determined by flow cytometry. Results are
representative of 3 independent experiments.
nuclei as well as clusters of membrane-bound bodies (see arrows),
which are characteristic of apoptosis.
These findings demonstrate that apoptosis occurs within 20 hours
and appears to be more extensive than the flow cytometry results
indicate because no apoptosis was evident by flow cytometry at 20 hours
(data not shown). This result is most likely due to the loss of apoptotic
cells during the staining procedure before flow cytometry.
AN-9 induces p21 in infant ALL, but not in T-ALL
or B-precursor ALL
Previous studies have reported the induction of p21 by butyrate and
its correlation with cell-cycle arrest and apoptosis.23,24 To determine whether p21 is involved in AN-9 action, we examined the
expression of p21 in response to AN-9 by Western blot analysis. As
shown in Figure 7A, p21 was induced in a primary infant ALL
sample within 24 hours of AN-9 treatment, and the levels declined
by 48 hours, indicating a transient induction. In contrast, as
presented in a representative experiment (Figure 7), induction of
p21 by AN-9 was not observed in any of the 2 B-precursor ALL
(Figure 7B) or the 4 primary T-ALL (Figure 7C, data shown for one
patient) cells examined, even at AN-9 concentrations that effectively arrest DNA synthesis in ALL (Figure 2). The effects of AN-9
on the levels of p27, a homolog of p21, were also examined. The
levels of p27 were not induced by AN-9 in B-precursor ALL or
The finding that clinically refractory infant ALL cells are sensitive to
AN-9 prompted us to determine whether AN-9 is cytotoxic to other
drug-resistant malignant cells. The effect of AN-9 was examined in a
doxorubicin-resistant clone of the myeloid leukemia cell line HL60,
HL60/ADR, obtained by the transfection of the MDR-1 gene; and a
doxorubicin-resistant neuroblastoma cell line, Be2c/ADR, induced by
repeated exposure of Be2c to doxorubicin. The doxorubicin-sensitive
(IC50 ⫽ 0.002 ␮M) and doxorubicin-resistant HL60 cells (IC50 ⬎ 2.0
␮M) were equally sensitive to AN-9, with IC50 values of 52.0 ⫾ 5.6 ␮M
and 57.3 ⫾ 10.7 ␮M, respectively (Table 1).
Similarly, the doxorubicin-sensitive (IC50 ⫽ 0.007 ␮M) and
-resistant neuroblastoma cell lines (IC50 ⬎ 2.0 ␮M) were equally
sensitive to AN-9, with IC50 values of 69.4 ⫾ 8.4 ␮M and
70.7 ⫾ 6.2 ␮M, respectively (Table 1). More important, a doxorubicin-resistant primary T-ALL and a relapsed AML that was
refractory to chemotherapy were sensitive to the cytotoxicity of
AN-9, each with an IC50 of 50 ␮M (Table 1), an IC50 similar to that
obtained in doxorubicin-sensitive primary ALL (Figure 2). Taken
together, these findings, in addition to the finding of heightened
sensitivity to AN-9 of relapsed infant leukemia (Figure 2), suggest
that AN-9 may be a promising agent for acute leukemias, even in
cases of relapsed or refractory leukemias.
Discussion
In the present study of 21 primary samples of acute leukemias, we
demonstrated that AN-9 has antiproliferative and cytotoxic effects
on leukemia cells. AN-9 arrested DNA synthesis in both T-ALL and
B-precursor ALL at similar concentrations, indicating that its
effects on leukemia cells were not lineage specific. More important,
leukemia cells were 2- and 3-fold more sensitive than normal
hematopoietic progenitors to the antiproliferative effects of AN-9
in 3H-thymidine incorporation and colony-formation assays, respectively. These findings indicate that AN-9 is less toxic to normal
Figure 6. Morphology of CEM cells treated with AN-9. CEM cells were treated with either 75 ␮M AN-9 (A) or with 0.1% DMSO (B; control) for 20 hours and then
photographed using the SPOT camera. Magnification was ⫻10 for both samples. Arrows indicate fragmented nuclei and clusters of membrane-bound bodies characteristic of
apoptosis.
BLOOD, 1 NOVEMBER 2002 䡠 VOLUME 100, NUMBER 9
Figure 7. Western blot analysis of p21 in primary ALL. Protein was extracted by
the SDS/boiling method from (A) infant ALL; (B) B-precursor ALL; and (C) T-ALL cells
treated with AN-9 as indicated. SJ SA-1 osteosarcoma cells, known to express p21,
were used as the positive control. Western blot analysis was performed using a p21
antibody at 0.5 ␮g/mL. Equal amounts of protein in each lane were verified by probing
the blot with a ␤-actin antibody at 2 ␮g/mL. U indicates untreated (0.1% DMSO);
C, positive control for p21; pt, patient. AN-9 IC50 in the infant ALL was 13 ␮M in a
3H-thymidine uptake assay.
hematopoietic progenitors and thus has selectivity for leukemia
cells. In line with these results, earlier studies in a mouse tumor
model as well as a phase I clinical trial of AN-9 conducted in adults
with solid tumors have shown that AN-9 has efficacy and is
relatively nontoxic.19,25 Notably, in vitro data from solid tumors13,16,17 and Table 1 indicate that the IC50 of AN-9 is about
2-fold higher than that observed by us in leukemia, suggesting that
leukemia cells are particularly sensitive to the cytotoxicity of
AN-9. Collectively, our results indicate that AN-9 is a selective
anticancer agent and may have an advantage over standard
chemotherapeutic agents such as daunorubicin and doxorubicin,
which are equally toxic to leukemia cells and hematopoietic
progenitors in vitro.26-28
The anticancer effects of several HDAC inhibitors, including
butyrate, were found to be dependent on their ability to inhibit
HDACs and correlated with their capability to modulate cell-cycle
and apoptosis-regulatory genes.12,23,24 A recent study reported that
induction of p21 expression by butyrate was required for its effect
in arresting cell growth in colon carcinoma cells.23 However,
SELECTIVE TOXICITY OF AN-9 TO ACUTE LEUKEMIA
3323
another study reported that butyrate arrested cell growth in 3T3
fibroblasts derived from p21 knockout mice, indicating that p21
was not required for the antiproliferative effects of butyrate.29
Interestingly, p21 induction by AN-9 in this study was observed
only in an infant ALL sample that was the most sensitive to AN-9
cytotoxicity. The levels of p21 were not induced by AN-9 in any of
the primary T-ALL or B-precursor ALL cells examined, even at
AN-9 concentrations that completely inhibited DNA synthesis.
Thus, it appears that AN-9 may exert its antiproliferative effects
through different pathways depending on the cell type.
Our results demonstrate that in addition to being an antiproliferative agent, AN-9 induces apoptosis, as observed in CEM cells.
Zimra et al18 previously reported that AN-9 induced apoptosis in
HL60 cells and that this was accompanied by the reduction of Bcl-2
expression. However, another study reported that butyrate activated caspase 3 and induced apoptosis in lymphoid and colorectal
cancer cells that was dependent on the inhibition of HDACs but
independent of changes in the levels of Bcl-2 and Bax.30 As these
authors suggested, it is possible that other members of the
Bcl-2/Bax family may influence butyrate-induced apoptosis. Alternatively, apoptosis could be induced through the death receptors,
including CD95, tumor necrosis factor (TNF), and the receptor for
the TNF-related apoptosis-inducing ligand. Although the hybrid
polar HDAC inhibitor, M-carboxycinnamic acid bishydroxamide,
was found to induce CD95/CD95 ligand expression, butyrate did
not alter the expression of either the ligand or receptor, but did alter
cell sensitivity to CD95-mediated apoptosis.31 The repression of
Bcl-218 and the induction of Bax expression (A.R., unpublished
observations, 1997) by AN-9 suggest that the intrinsic pathway of
apoptosis that does not involve the death receptors may be the
pathway responsible for AN-9–induced apoptosis. Because the
precise mechanisms underlying the effects of HDAC inhibitors
remain unknown, future work is aimed at further examining the
pathways and key components involved in AN-9–induced growth
arrest and apoptosis in ALL.
Unfortunately, as with many cancers, relapses are common in
ALL and are associated with multidrug resistance. In this study,
AN-9 was effective in inhibiting the growth of HL60 myeloid and
neuroblastoma clones that harbored multiple genetic alterations32
and displayed a multidrug-resistant phenotype.20-22 Moreover,
AN-9 was equally effective in arresting proliferation of all the
primary leukemia cells tested, including a doxorubicin-resistant
T-ALL, a clinically refractory relapsed AML, and a relapsed infant
ALL characterized by an 11q23 rearrangement and a very poor
prognosis. Thus, our finding that AN-9 arrested the growth of these
different cancer cell types indicates that AN-9 is a unique agent
with potential advantage over standard chemotherapeutic agents.
Table 1. AN-9 sensitivity of doxorubicin-resistant tumor cells
Cells
HL60
HL60/ADR*
AN-9 IC50, ␮M
doxorubicin IC50, ␮M
52 ⫾ 5.6
0.002
57.3 ⫾ 10.7
⬎ 2.0
Diagnosed T-ALL
50
⬎ 2.0
Relapsed AML
50
⬎ 5.0
Be2c
69.4 ⫾ 8.4
0.007
Be2c/ADR†
70.7 ⫾ 6.2
⬎ 2.0
AN-9 IC50 in cell lines is an average from 2 independent experiments. Errors
represent standard error of the mean. doxorubicin IC50 in cell lines is representative of
that determined in at least 2 independent experiments. All conditions were performed
in triplicate.
*HL60/ADR was obtained by transfection of HL60 with the MDR-1 gene.
†Be2c/ADR was obtained by selection of Be2c/ADR in doxorubicin-containing
medium.
3324
BLOOD, 1 NOVEMBER 2002 䡠 VOLUME 100, NUMBER 9
BATOVA et al
Similar observations have been reported using other HDAC
inhibitors.10-12 For instance, the HDAC inhibitor MS-27-275 strongly
inhibited the growth of solid tumor implants in nude mice that did
not respond to the standard chemotherapeutic agent 5-fluorouracil.11 Furthermore, the more familiar HDAC inhibitor trichostatin A
was shown to inhibit proliferation and induce apoptosis in gastric
and oral carcinoma cell lines resistant to 9-cis–retinoic acid and
interferon-␤.12 Another advantage of AN-9 is its ability to synergize with anthracyclines such as daunomycin, commonly used in
the treatment of leukemia patients.19,33 The molecular basis for the
synergistic actions of doxorubicin and AN-9 was reported to be the
ability of formaldehyde, released upon cellular hydrolysis of AN-9,
to facilitate the formation of DNA adducts with doxorubicin.34,35
Concurrently, butyrate, also released upon cellular hydrolysis,
inhibits HDACs, resulting in histone hyperacetylation and relaxation of the chromatin structure, allowing greater accessibility of
DNA for the formation of doxorubicin–DNA adducts. The synergistic action of AN-9 with doxorubicin would allow the use of
significantly lower doses of these agents in treatment and consequently would greatly improve the therapeutic index.
Thus, AN-9, an HDAC inhibitor with low toxicity and selectivity toward cancer cells, may provide a novel therapeutic strategy
for cancers refractory to traditional antitumor agents. The present
study provides strong evidence to warrant clinical trials in relapsed
ALL using AN-9 as a single agent or in combination with standard
chemotherapeutic agents.
Acknowledgments
We gratefully acknowledge the investigators from the Pediatric
Oncology Group for providing T-ALL samples. We also thank
Louis Bridgeman, Ruby Gribi, and Dr Sigrun Gebauer for technical
assistance. We are very grateful to Dr Denis Sasaki for assistance
with flow cytometry.
References
1. Grunstein M. Histone acetylation in chromatin
structure and transcription. Nature. 1997;389:
349-352.
2. Allfrey VG, Faulknere R, Mirsky AE. Acetylation
and methylation of histones and their possible
role in the regulation of RNA synthesis. Proc Natl
Acad Sci U S A. 1964;61:786-794.
14. Aviram A, Rephaeli A, Shaklai M, et al. Effect of
butyric acid pro-drug, pivaloyloxymethyl butyrate,
on the tumorigenicity of cancer cells. J Cancer
Res Clin Oncol. 1997;123:267-271.
3. Pazin MJ, Kadonaga JT. What’s up and down
with histone deacetylation and transcription? Cell.
1997;89:325-328.
15. Zimra Y, Maron L, Shaklai M, Nudelman A,
Rephaeli A. Pivaloyloxymethyl butyrate (AN-9)
exhibits higher anticancer activity than butyric
acid (BA): it penetrates faster than the acid and
induces apoptosis in HL60 cells. Blood. 1994;84:
579a.
4. Marks PA, Richon VM, Rifkind RA. Histone
deacetylase inhibitors: inducers of differentiation
or apoptosis of transformed cells. J Natl Cancer
Inst. 2000;92:1210-1216.
16. Rephaeli A, Rabizadeh E, Aviram A, Shaklai M,
Ruse M, Nudelman A. Derivatives of butyric acid
as potential anti-neoplastic agents. Int J Cancer.
1991;49:66-72.
5. Brehm A, Nielsen SJ, Miska EA, et al. The E7 oncoprotein associates with Mi2 and histone
deacetylase activity to promote cell growth.
EMBO J. 1999;18:2449-2458.
17. Siu LL, Von Hoff DD, Rephaeli A, et al. Activity of
pivaloyloxymethyl butyrate, a novel anticancer
agent, on primary human tumor colony-forming
units. Invest New Drugs. 1998;16:113-119.
6. Gayther SA, Batley SJ, Linger L, et al. Mutations
truncating the EP300 acetylase in human cancers. Nat Genet. 2000;24:300-303.
18. Zimra Y, Wasserman L, Maron L, Shaklai M,
Rephaeli A, Nudelman A. Butyric acid and pivaloyloxymethyl butyrate, AN-9, a novel butyric
acid derivative, induce apoptosis in HL60 cells.
J Cancer Res Clin Oncol. 1997;123:152-160.
7. Grignani F, De Matteis S, Nervi C, Tomassoni L,
Gelmetti V, Cioce M. Fusion proteins of the retinoic acid receptor-alpha recruit histone deacetylase in promyelocytic leukemia. Nature. 1998;
391:815-818.
8. Lin RJ, Nagy L, Inoue S, Shao W, Miller WH Jr,
Evans RM. Role of the histone deacetylase complex in acute promyelocytic leukemia. Nature.
1998;391:811-814.
9. He LZ, Guidez F, Tribioli C, Peruzzi D, Ruthardt
M, Zelent A. Distinct interactions of PML-RARalpha
and PLZF-RARalpha with corepressors determine differential responses to RA in APL. Nat
Genet. 1998;18:126-135.
10. Warrel RP, He L-Z, Richon V, Calleja E, Pandolfi
PP. Therapeutic targeting of transcription in acute
promyelocytic leukemia by use of an inhibitor of
histone deacetylase. J Natl Cancer Inst. 1998;90:
1621-1625.
11. Saito A, Yamashita T, Mariko Y, et al. A synthetic
inhibitor of histone deacetylase, MS-27-275, with
marked in vivo anti-tumor activity against human
tumors. Proc Natl Acad Sci U S A. 1999;96:45924597.
12. Suzuki T, Yokozaki H, Kuniyasu H, et al. Effect of
trichostatin A on cell growth and expression of cell
cycle and apoptosis-related molecules in human
gastric and oral carcinoma cell lines. Int J Cancer.
2000;88:992-997.
13. Nudelman A, Ruse M, Aviram A, et al. Novel anticancer prodrugs of butyric acid. J Med Chem.
1992;35:687-694.
19. Kasukabe T, Rephaeli A, Honma Y. An anticancer derivative of butyric acid (pivaloyloxymethyl butyrate) and daunorubicin cooperatively
prolong survival of mice inoculated with monocytic leukemia cells. Br J Cancer. 1997;75:850854.
20. Kelner MJ, McMorris TC, Montoya MA, et al.
Characterization of cellular accumulation and toxicity of illudin S in sensitive and nonsensitive tumor cells. Cancer Chemother Pharmacol. 1997;
40:65-71.
21. Marsh W, Center MS. doxorubicin resistance in
HL60 cells and accompanying modification of a
surface membrane protein contained in drugsensitive cells. Cancer Res. 1987;47:5080-5086.
22. LaQuaglia MP, Kopp EB, Spengler BA, Meyers
MB, Biedler JL. Multidrug resistance in human
neuroblastoma cells. J Pediatr Surg. 1991;26:
1107-1112.
23. Archer SY, Meng S, Shei A, Hodin RA. p21waf1 is
required for butyrate-mediated growth inhibition
of human colon cancer cells. Proc Natl Acad Sci
U S A. 1998;95:6791-6796.
24. Chai F, Evdokiou A, Young GP, Zalewski PD. Involvement of p21(Waf1/Cip1) and its cleavage by
DEVD-caspase during apoptosis of colorectal
cancer cells induced by butyrate. Carcinogenesis. 2000;21:7-14.
25. Eckhardt SG, Villalona-Calero MA, Hammond L,
et al. A phase I study of AN-9 (pivaloyloxymethyl
butyrate, PivanexTM), a lipophilic butyric acid analog, in patients with advanced solid tumors. Proc
Am Assoc Cancer Res. 1998;39:507.
26. Singer CRJ, Linch DC. Comparison of the sensitivity of normal and leukaemic myeloid progenitors to in-vitro incubation with cytotoxic drugs: a
study of pharmacological purging. Leuk Res.
1987;11:953-959.
27. Spiro TE, Mattelaer M-A, Efira A, Stryckmans P.
Sensitivity of myeloid progenitor cells in healthy
subjects and patients with chronic myeloid leukemia to chemotherapeutic agents. J Natl Cancer
Inst. 1981;66:1053-1059.
28. Linssen P, Brons P, Knops G, Wessels H, de
Witte T. Plasma and cellular pharmacokinetics of
m-AMSA related to in vitro toxicity towards normal and leukemic clonogenic bone marrow cells
(CFU-GM, CFU-L). Eur J Haematol. 1993;50:
149-154.
29. Vaziri C, Stice L, Faller DV. Butyrate-induced G1
arrest results from p21-independent disruption of
retinoblastoma protein-mediated signals. Cell
Growth Differ. 1998;9:465-474.
30. Medina V, Edmonds B, Young GP, James R,
Appleton S, Zalewski PD. Induction of caspase-3
protease activity and apoptosis by butyrate and
trichostatin A (inhibitors of histone deacetylase).
Dependence on protein synthesis and synergy
with a mitochondrial/cytochrome c-dependent
pathway. Cancer Res. 1997;57:3697-3707.
31. Bonnotte B, Favre N, Reveneau S, et al. Cancer
cell sensitization to fas-mediated apoptosis by
sodium butyrate. Cell Death Differ. 1998;5:480487.
32. Diccianni MB, Omura-Minamisawa M, Batova A,
Le T, Bridgeman L, Yu AL. Frequent deregulation
of p16 and the p16/G1 cell cycle-regulatory pathway in neuroblastoma. Int J Cancer.
1999;80:145-154.
33. Niitsu N, Kasukabe T, Yokoyama A, et al. Anticancer derivative of butyric acid (pivalyloxymethyl
butyrate) specifically potentiates the cytotoxicity
of doxorubicin and daunorubicin through the suppression of microsomal glycosidic activity. Mol
Pharmacol. 2000;58:27-36.
34. Cutts SM, Rephaeli A, Nudelman A, Hmelnitsky I,
Phillips DR. Molecular basis for the synergistic
interaction of doxorubicin with the formaldehydereleasing prodrug pivaloyloxymethyl butyrate
(AN-9). Cancer Res. 2001;61:8194-8202.
35. Zeman SM, Phillips DR, Crothers DM. Characterization of covalent doxorubicin-DNA adducts.
Proc Natl Acad Sci U S A. 1998;95:11561-11565.