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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 4 (February 15), 1999:
pp. 1308-1318
Increased Sensitivity of Acute Myeloid Leukemias to
Lovastatin-Induced Apoptosis: A Potential Therapeutic Approach
By
Jim Dimitroulakos,
Dana Nohynek,
Karen L. Backway,
David W. Hedley,
Herman Yeger,
Melvin H. Freedman,
Mark D. Minden, and
Linda Z. Penn
From the Departments of Cellular and Molecular Biology and
Experimental Therapeutics, the Ontario Cancer Institute, Toronto,
Ontario, Canada; the Departments of Paediatric Laboratory Medicine and
Haematology, The Hospital for Sick Children, Toronto, Ontario, Canada;
and the Departments of Medical Biophysics and Laboratory Medicine and
Pathobiology, University of Toronto, Toronto, Ontario, Canada.
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ABSTRACT |
We recently demonstrated that 3-hydroxy-3-methylglutaryl coenzyme A
(HMG-CoA) reductase, the rate-limiting enzyme of de novo cholesterol
synthesis, was a potential mediator of the biological effects of
retinoic acid on human neuroblastoma cells. The HMG-CoA reductase
inhibitor, lovastatin, which is used extensively in the treatment of
hypercholesterolemia, induced a potent apoptotic response in human
neuroblastoma cells. This apoptotic response was triggered at lower
concentrations and occurred more rapidly than had been previously
reported in other tumor-derived cell lines, including breast and
prostate carcinomas. Because of the increased sensitivity of
neuroblastoma cells to lovastatin-induced apoptosis, we examined the
effect of this agent on a variety of tumor cells, including leukemic
cell lines and primary patient samples. Based on a variety of
cytotoxicity and apoptosis assays, the 6 acute lymphocytic leukemia
cell lines tested displayed a weak apoptotic response to lovastatin. In
contrast, the majority of the acute myeloid leukemic cell lines (6/7)
and primary cell cultures (13/22) showed significant sensitivity to
lovastatin-induced apoptosis, similar to the neuroblastoma cell
response. Of significance, in the acute myeloid leukemia, but not the
acute lymphocytic leukemia cell lines, lovastatin-induced cytotoxicity
was pronounced even at the physiological relevant concentrations of
this agent. Therefore, our study suggests the evaluation of HMG-CoA
reductase inhibitors as a therapeutic approach in the treatment of
acute myeloid leukemia.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
MEVALONATE IS A CRITICAL component of a
complex biochemical pathway whose products are vital for a variety of
key cellular functions, including membrane integrity, cell signaling,
protein synthesis, and cell cycle progression.1 Regulation
of mevalonate synthesis is complex, involving multiple feedback
mechanisms in which the endproducts of this pathway can regulate the
activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme of this metabolic pathway.1 The
endproducts of the mevalonate pathway include sterols, especially
cholesterol, involved in membrane structure and steroid production;
ubiquinone, involved in electron transport; farnesyl and geranylgeranyl
isoprenoids involved in covalent binding of proteins such as ras to
membranes; dolichol, which is required for glycoprotein synthesis; and
retinoic acid precursors.1,2 This diverse array of critical
metabolic endproducts strongly suggests that physiological regulation
of HMG-CoA reductase is essential for the maintenance of cellular homeostasis.
Lovastatin is a specific, nonreversible competitive inhibitor of
HMG-CoA reductase, whose ability to block this critical metabolic pathway has led to its extensive clinical use as a treatment for hypercholesterolemia.3,4 Lovastatin is also a potent
inducer of growth arrest at the G1/S boundary in a wide variety of
normal and tumor-derived cell lines. This effect can be reversed by the addition of mevalonate,5 the immediate endproduct of the
reaction catalyzed by HMG-CoA reductase. The ability of lovastatin to
cause growth arrest and its reversibility has led to its use as a
cell-synchronizing agent in vitro.1,5 Prolonged exposure of
various tumor-derived cell lines to relatively high concentrations of
lovastatin (30 to 100 µmol/L) can lead to cellular
apoptosis.3,5-7 The growth arrest and apoptotic properties
of lovastatin has led to its evaluation as a potential therapeutic
agent in the treatment of cancers that included breast and prostate
carcinomas.8 The therapeutic dose for treatment of
hypercholesterolemia is approximately a 1 mg/kg/d oral dose that
produces serum levels in the order of 0.1 µmol/L lovastatin.8,9 In the phase I study of lovastatin as a
therapeutic,8 doses up to 25 mg/kg/d were generally well
tolerated; however, at these doses, the achievable peak serum levels of
lovastatin were only in the range of 0.1 to 3.92 µmol/L and, as such,
had little effect in reducing tumor load.8 Therefore,
lovastatin did not appear to be a viable therapeutic alternative in the
treatment of human cancer.
In a previous study, we identified HMG-CoA reductase as a potential
mediator of the biological effects of retinoic acid on human
neuroblastoma cells.10 Retinoic acid, derived from
mevalonate metabolites,2 is a potent cell-differentiating
and growth-inhibitory agent during embryogenesis that has demonstrated
efficacy in the prevention and therapy of specific
cancers.11,12 Exposure of a variety of neuroblastoma cell
lines to lovastatin induced extensive apoptosis10 that was
triggered with relatively low concentrations (<10 µmol/L) and rapid
kinetics, compared with other tumor-derived cell lines previously
reported.3,5-7 Therefore, a re-evaluation of lovastatin to
induce apoptosis in tumor derived cell lines was undertaken. We
included a variety of retinoic acid responsive cancers that had not
been adequately surveyed previously. In this study, we evaluated the
apoptotic response of acute myeloid leukemic (AML) and acute
lymphocytic leukemic (ALL) cells, retinoic acid responsive12-14 and nonresponsive13 leukemias,
respectively, to lovastatin.
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MATERIALS AND METHODS |
Cell culture.
The ALL cell lines B1, C1, W1, G2, KK, and NGR were derived from
primary patient tumors from children with pre-B ALL at the Hospital for
Sick Children (Toronto, Ontario, Canada) as previously described.15 The AML cell lines OCI-AML-1, OCI-AML-2,
OCI-AML-3, OCI-AML-4, and OCI-AML-5 were established from patients from
the Ontario Cancer Institute (Toronto, Ontario, Canada).16
For presentation purposes, the OCI designation has been omitted when
referring to these cell lines in this study. The AML cell lines NB-4
and HL60 were kindly provided by Dr E.A. McCulloch (Ontario Cancer Institute). Primary cultures were derived from the peripheral blood of
the AML patients with informed consent. The fresh leukemic samples
tested in this study were drawn from consecutive patients presenting at
the Ontario Cancer Institute/Princess Margaret Hospital in Toronto.
Normal bone marrow and cord blood cells were obtained with informed
consent for the use of this material for research purposes and kindly
provided by Drs H. Messner (Ontario Cancer Institute) and J.E. Dick
(The Hospital for Sick Children), respectively. Mononuclear cell
fractions from patient material were obtained by Ficoll-Hypaque
centrifugation as previously described17 and regularly
consisted of 95% to 98% blasts as determined by morphological examination. The ALL cell lines AML-2, AML-3, NB-4, and HL60 were maintained in  -minimal essential media (-MEM;
Princess Margaret Hospital Media Services) supplemented with 10% fetal
calf serum (Sigma, St Louis, MO). AML-1, AML-4, and AML-5 cell lines as
well as the bone marrow and the leukemic primary cultures were
maintained in -MEM supplemented with 10% fetal calf serum and 10%
5637 conditioned media.16 Cells were cultured in liquid
suspension and treated with 1 to 150 µmol/L lovastatin (generously
provided by Merck Research Laboratories, Rahway, NJ; diluted from a 10 mmol/L stock in ethanol prepared as previously described5)
and processed for 3-4,5-dimethylthiazolyl-2,2,5-diphenyl tetrazolium
bromide (MTT), colony growth, flow cytometric, and electron microscopic analysis.
MTT assay.
In a 96-well flat-bottom plate (Nunc, Naperville, IL) approximately
10,000 cells/150 µL of cell suspension was used to seed each well.
After 2 days of lovastatin treatment (0 to 150 µmol/L), 50 µL of a
5 mg/mL solution in phosphate-buffered saline of the MTT tetrazolium
substrate (ICN, Toronto, Ontario, Canada) was added and incubated for 6 hours at 37°C. The resulting violet formazan precipitate was
solubilized by the addition of 100 µL of a 0.01 mol/L HCl/10% sodium
dodecyl sulfate (SDS; Sigma) solution overnight at
37°C.18 The plates were then analyzed on an SLT Labinstruments 340 ATTC enzyme-linked immunosorbent assay (ELISA) plate
reader (SLT Labinstruments, Crailsheim, Germany) at 450 nm
to determine the optical density of the samples.
Trypan blue exclusion assay.
A total of 5 × 105 leukemic cells in 1 mL of media
were seeded in a 24-well plate (Falcon; Fisher Scientific, Mississauga, Ontario, Canada) and exposed to either solvent control or 1, 10, or 20 µmol/L lovastatin for up to 4 days in triplicate. Lovastatin was
replenished after 2 days of treatment. Cell counts were evaluated using
a 1:1 dilution of cell suspension in trypan blue (GIBCO-BRL, Mississauga, Ontario, Canada). Viable and nonviable cells were counted
using a hemocytometer as trypan blue excluded or stained cells,
respectively, as previously described.19
Colony growth assay.
Mononuclear cell cultures from normal bone marrow were either seeded at
106 cells/mL of cell suspension in a 24-well plate and
treated for 2 days or plated directly in methylcult (Stem Cell
Technologies, Vancouver, British Columbia, Canada) with 0 to 150 µmol/L lovastatin following the manufacturer's instructions.
Incubation in methylcult was for 14 days and aggregates of more than 50 cells were scored as colonies. AML primary cultures were seeded at 5 × 104 cells/150 µL of cell suspension to each well
in a 96-well plate and treated for 2 days with 0 to 150 µmol/L
lovastatin. The clonogenic assay used here has been well
described.20 Briefly, approximately 2 × 104 of the lovastatin-treated cells were plated in 0.1 mL
of growth medium containing 10% fetal calf serum, 10% 5637 conditioned medium, and 0.8% methylcellulose (GIBCO-BRL) in triplicate
in a 96-well plate. Incubation was for 7 days and aggregates of more
than 50 cells were scored as colonies.
Flow cytometry and electron microscopy.
Cell-cycle parameters were determined by flow cytometry using propidium
iodide labeling of single cells. The method used was described
previously.10,21 Single-cell suspensions were labeled with
50 µg/mL propidium iodide (Sigma), and approximately 106
cells in 100 µL were analyzed by flow cytometry. Ten thousand cells
were evaluated and the percentage of cells in pre-G1 phase was
determined using the Modfit LT program (Verity Software House, Topsham,
ME). Reduced glutathione (GSH) was measured using monobromobimane and
mitochondrial membrane potential (MMP) was measured using the cyanine
dye DiIC1(5).22 A combined labeling method using triple
laser excitation was used as previously described22 to examine the associations between cellular GSH and MMP. Ultrathin sections of cultured cell pellets were cut and prepared for electron microscopy as previously described.10,21 Cultured cell
pellets were fixed in phosphate-buffered 2% glutaraldehyde and 1%
osmium tetroxide, dehydrated through acetone, and embedded in epon araldite.
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RESULTS |
Increased sensitivity of AML-derived cell lines to lovastatin.
The potential growth-inhibitory and cytotoxic effects of targeting
HMG-CoA reductase function using lovastatin in leukemia-derived cell
lines was first evaluated using the MTT assay. The MTT assay is a
measure of mitochondrial dehydrogenase activity in viable cells; this
assay has been used extensively to evaluate the cytotoxic effects of
chemotherapeutics on tumor cells in vitro.18 The 6 ALL and
the 7 AML cell lines used in this study were treated with various
concentrations of lovastatin ranging from 1 to 150 µmol/L for 2 days
(Fig 1). In our previous work, this
timepoint and the lovastatin concentrations given above clearly
delineated the apoptotic sensitive neuroblastoma cells.10
Whereas the ALL cell lines tested consistently displayed a gradual
decline in MTT activity with increasing concentrations of lovastatin,
the AML cell lines tested were significantly more sensitive to
lovastatin as determined by MTT activity (compare Fig 1A and Fig 1B).
An analysis of the concentration of lovastatin to induce a decrease in
MTT activity by 50% (MTT50) clearly highlighted the different responses to lovastatin in these cell lines. The MTT50 of the ALL cell
lines was in the range of 70 to 125 µmol/L lovastatin, whereas in the
AML cell lines, the range was 1 to 28 µmol/L, a significant
difference of P = .0012 (Fig 1C). In 6 of 7 AML cell lines, the
MTT50 values obtained were less than 5.3 µmol/L lovastatin, indicating a greater than 10-fold increased sensitivity to this agent
in comparison to the ALL cell lines. Clear demarcation between the ALL
and the AML apoptotic response groups was distinguished by the latter
showing a decrease of MTT activity to less than 30% (MTT30) under
these experimental conditions (Fig 1A and B).
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| Fig 1.
Evaluating the cytotoxic effects of lovastatin on
leukemic cell lines using the MTT assay. (A and B) MTT enzyme activity,
after exposure to 0 to 150 µmol/L lovastatin for 2 days, of 6 representative ALL and 7 AML cell lines, respectively.
The dotted lines are at the level of MTT30. Results
shown are the average of two independent experiments performed in
quadruplicate, where the error bars represent the standard
deviation of the mean. The values obtained were normalized to the
solvent controls set at 100 for clarity of presentation. (C) Histogram
showing the concentration of lovastatin required to achieve a decrease
of MTT activity by 50% (MTT50) in the ALL and AML cell lines examined.
MTT50 values for the ALL and AML cell lines were determined by
regression analysis by the method of Chou-Talalay as previously
described.49 The significant difference (P = .0012) in the MTT50 values between the ALL and AML cell lines was
determined by the Wilcoxon test.
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To determine if the decrease in MTT activity was due to
lovastatin-induced cytotoxicity, we measured the effect of this agent on cell viability using trypan blue exclusion. Viable cells can exclude
the dye trypan blue, whereas nonviable cells lose this ability, retain
dye, and stain blue.19 Treatment of the ALL cell lines G2
and KK with either solvent control or 1, 10, or 20 µmol/L lovastatin
displayed decreases in viable cell counts and an increase in cell
cytotoxicity after prolonged exposure (3 to 4 days) to 10 and 20 µmol/L lovastatin (Fig 2A). In the AML
cell lines, AML-2 and AML-5, these concentration of lovastatin had
dramatic effects on cell viability with extensive cytotoxicity. The
effect of this agent on cell counts and viability was more extensive
and occurred more rapidly than within the ALL cell lines tested (Fig
2B). Importantly, effects on cell viability were evident in the AML
cell lines at 1 µmol/L lovastatin, at which, after 4 days of
treatment, approximately 50% of the cells were nonviable (Fig 2B).
Therefore, the AML cell lines were significantly more sensitive to
lovastatin-induced cytotoxicity than the ALL cell lines.
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| Fig 2.
The effect of lovastatin on leukemic cell viability
determined by trypan blue exclusion. Five hundred thousand cells from
two ALL cell lines KK and G2 (A) and two AML cell lines AML-2 and AML-5
(B) were seeded in triplicate and exposed to solvent control or 1, 10, or 20 µmol/L lovastatin for 4 days. The number of viable cells that
can exclude the dye trypan blue and the percentage of nonviable cells
that stained blue were evaluated daily by microscopy using a
hemocytometer. Results shown are the average of the triplicate counts
at each timepoint, where the error bars represent the standard
deviation of the mean. This experiment was repeated with similar
results.
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Lovastatin-induced apoptosis of AML cell lines.
To better understand the mechanism of lovastatin-induced cytotoxicity,
we further analyzed cells during the death process. To determine
whether the decrease in MTT activity and the increase in cytotoxicity
observed by trypan blue staining after exposure to lovastatin was due
to an apoptotic response, flow cytometric, ultrastructural, and
biochemical alterations characteristic of this response were evaluated.
Cells undergoing apoptosis typically show a pre-G1 peak due to nuclear
fragmentation.23,24 Flow cytometric analysis indicated that
the G2 and KK ALL cell lines lacked significant evidence of apoptosis
upon 20 µmol/L lovastatin exposure for 2 days
(Fig 3). By contrast, under identical
experimental conditions, the AML cell lines showed a significant
apoptotic response highlighted by the presence of a prominent pre-G1
peak after lovastatin exposure (Fig 3).
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| Fig 3.
Representative flow cytometric analysis of ALL (G2 and
KK) and AML (AML-1, -2, -3, and -5) cell lines after exposure to
lovastatin. The percentage of cells in the pre-G1 (apoptotic) fraction
is shown in the upper left quadrant of the individual histograms. Cells
were exposed to solvent control (left column), 20 µmol/L lovastatin
treatment for 1 day (center column), or 20 µmol/L lovastatin
treatment for 2 days (right column).
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Ultrastructural features of apoptotic cell death include chromatin and
cytoplasmic condensation, followed by nuclear and cellular fragmentation.25 Electron microscopic analysis of the
effect of 20 µmol/L lovastatin for 2 days on AML blasts included an
examination of the AML-5 cell line. Lovastatin-treated AML-5 cells
displayed dramatic morphological changes to their nuclei, with a
bilobed-indented nucleus present in cells before apoptosis. However,
the majority of cells showed characteristic ultrastructural features of
apoptosis, including nuclear and cytoplasmic condensation
(Fig 4A).
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| Fig 4.
(A) Ultrastructural features of the AML-5 cell line after
2 days of exposure to solvent control or 20 µmol/L lovastatin. (a)
Control AML-5 cells; (b and c) lovastatin treatment-induced nuclear
morphology changes in AML-5; (d) characteristic features of apoptosis
were observed in the majority of lovastatin-treated AML-5 cells. The
bar represents 1 µm. (B) Multilaser flow cytometric analysis of
reduced GSH and MMP. AML-5 cells were exposed to solvent control or 20 µmol/L lovastatin for 2 days and analyzed. Dual-parameter dot plots
of GSH and MMP for each sample are also presented.
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In addition to the ultrastructural changes, several metabolic
alterations also form part of the common phase of the apoptotic process.22,26-28 Apoptosis is typically accompanied by a
depletion of intracellular reduced GSH, which lowers the capacity of
cells to buffer against endogenous oxidants.26,28 In all
cell types and in response to all inducers of apoptosis studied thus
far, cells display a collapse of their MMP that precedes the nuclear signs of apoptosis.27 In this study, we used multilaser
flow cytometry methods22 to analyze the cellular content of
GSH and measurements of MMP coincident with lovastatin treatment.
Analysis was limited to viable cells that retained surface membrane
integrity identified by their ability to exclude the membrane
impermeable fluorescent DNA stain propidium iodide. AML-5 cells exposed
to 20 µmol/L lovastatin for 2 days displayed significant reductions in both GSH levels and MMP on exposure to lovastatin (Fig 4B). The
dual-parameter dot plots of GSH content versus MMP clearly demonstrated
that, in the responsive blast cells, lovastatin-induced GSH depletion
occurred in the same population of cells showing a decrease in MMP (Fig
4B). Similar results were demonstrated in the AML-2 and AML-3 cell
lines; however, no changes in GSH or MMP levels were observed in the KK
ALL cell line treated with lovastatin under the same experimental
conditions (data not shown). Thus, lovastatin induced the flow
cytometric, ultrastructural, and metabolic alterations characteristic
of apoptosis in AML cell lines.
Lovastatin targets primary AML cultures.
After noting the dramatic response of lovastatin on cell viability in
the AML cell lines, we next evaluated its effect on normal bone marrow
as well as primary leukemic cells from a variety of AML patients. The
responsiveness of this primary patient material to lovastatin-induced
cytotoxicity was first evaluated by MTT analysis. Sensitivity to
lovastatin-induced cytotoxicity was evaluated as nonresponsive, weak,
and sensitive using the MTT50 and MTT30 values obtained after 2 days of
exposure. Based on the responsiveness of the cell lines to
lovastatin-induced cytotoxicity, the primary patient samples showing
both MTT50 and MTT30 values greater than 100 µmol/L were considered
nonresponsive and samples with an MTT50 less than 100 µmol/L and an
MTT30 greater than 100 µmol/L lovastatin showed a weak response,
whereas primary patient samples with both MTT50 and MTT30 values less
than 100 µmol/L were considered sensitive. The effects of HMG-CoA
reductase inhibitors, including lovastatin, have been evaluated
extensively both in vitro and in vivo and have shown negligible effects
on normal bone marrow progenitors and hematopoiesis.8,29
Indeed, MTT analysis of three bone marrow samples and one from cord
blood demonstrated that normal hematopoietic cells do not undergo
significant cytotoxicity and were nonresponsive to lovastatin
(Fig 5A). However, the MTT analysis of 22 AML patient samples (Table 1) indicated
that 2 were nonresponsive, 7 showed a weak response, and 13 were
sensitive to lovastatin-induced cytotoxicity (Fig 5B and Table 1). Age,
gender, French-American-British (FAB) subtype, or total white blood
cell and blast counts in peripheral blood at presentation were not
predictive of lovastatin responsiveness in these AML primary cell
cultures (Table 1).
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| Fig 5.
The effect of lovastatin on primary cell cultures. (A and
B) MTT enzyme activity after exposure to 0 to 150 µmol/L lovastatin
for 2 days in bone marrow and cord blood samples as well as a
representative sampling of the 22 AML primary cultures analyzed (see
Table 1), respectively. The results are the average of six replicates
of a single experiment, where the error bars represent the standard
deviation of the mean. The MTT activity values were normalized to the
solvent controls for each sample and set at 100 for clarity of
presentation. (C) Colony growth potential of bone marrow progenitor
cells after exposure to 0 to 150 µmol/L lovastatin for 2 days in
vitro (BM 4 and BM 5a) or added to the methylcellulose at time of
plating (BM 5b) and incubation for 14 days; colonies were scored where
the values represent the mean of duplicate readings and error bars as
the standard deviation of the mean. (D) Colony growth potential of
various AML primary cultures after exposure to 0 to 150 µmol/L
lovastatin for 2 days; cells were then plated in methylcellulose and
incubated for 7 days. Blast colonies of greater than 50 cells were
scored and the values represent the mean of triplicate readings, where
the error bars are the standard deviation of the mean.
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The ability of leukemic cells to grow and form colonies in a semisolid
media such as methylcellulose is an indicator of their growth and
malignant potential.17,29 Using this approach, previous studies have demonstrated that HMG-CoA reductase inhibitors, including lovastatin, can significantly affect the colony-forming ability of AMLs
with minimal effects on normal bone marrow progenitor cells29,30; however, this effect was attributed to growth
arrest of the leukemic blasts. In line with these previous reports,
lovastatin had minimal effects on the colony-forming ability of normal
bone marrow progenitor cells when treated for 2 days in vitro (Fig 5C,
BM 4 and BM 5a) or when plated directly (Fig 5C, BM 5b) in the
methylcellulose with 0 to 150 µmol/L lovastatin. A representative sampling of the AML primary cultures was also treated for 2 days with
the same concentrations of lovastatin used in the MTT analysis and
subsequently plated in methylcellulose. Of the 12 samples tested, the 4 that demonstrated a plating efficiency of greater than 10% are
presented (Fig 5D). The colony growth potential of these AML primary
cultures paralleled their responsiveness to lovastatin-induced
cytotoxicity as determined by MTT analysis. For example, the
nonresponsive AML primary sample A showed a marginal decrease in
colony-forming ability and the intermediate responsive patient I
displayed a partial response, whereas the sensitive primary cultures S
and V dramatically reduced their colony-forming potential (compare Fig
5B to 5D). Thus, in the majority of AML patient samples tested, a
significant lovastatin-induced cytotoxic response was observed.
Analysis of the apoptotic response of lovastatin in AML primary
samples.
Similar to its effect on the cell lines, the potential apoptotic
response in the primary samples after lovastatin exposure was
investigated by analyzing characteristic flow cytometric, ultrastructural, and biochemical changes. The primary cultures were
exposed to ethanol control or to low (10 µmol/L), intermediate (50 µmol/L), or high (100 µmol/L) concentrations of lovastatin for 2 days and then analyzed for the appearance of a pre-G1 peak indicative
of cells undergoing nuclear fragmentation.23,24 Lovastatin
did not induce significant apoptosis in normal bone marrow cells and
the leukemic cells of two patients (A and B). Flow cytometric analysis
of one of the bone marrow samples and patient A are representative and
are shown in Fig 6. By comparison, the weak
responding patient samples as determined by MTT analysis displayed weak
apoptotic responses only at the highest concentration of lovastatin
used (patients C through I), with patient I shown in Fig 6. Finally,
patient samples J through V showed a dramatic induction of apoptosis
visualized by the presence of an abundant pre-G1 peak (representative
patient samples K, L, and O are shown in Fig 6).
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| Fig 6.
Flow cytometric analysis of normal bone marrow and AML
primary cultures exposed to solvent control or to low (10 µmol/L),
intermediate (50 µmol/L), and high (100 µmol/L) concentrations of
lovastatin for 2 days. Representative primary cultures from each of the
three lovastatin response groups are shown, including a nonresponsive
sample A, an intermediate responsive sample I, and sensitive samples K,
L, and O. The percentage of cells in the pre-G1 (apoptotic) fraction of
the cell cycle is shown in the upper left corner of the individual
histograms.
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Electron microscopic and biochemical analyses of the effect of 20 µmol/L lovastatin for 2 days on the AML primaries included an
examination of a nonresponsive (patient A,
Fig 7A) and a sensitive (patient O, Fig 7B)
primary culture. In the AML primary samples, the most striking effect
of lovastatin in both the nonresponsive and the sensitive primary
cultures examined was the presence of numerous, large lysosomal or
granular structures within the cytoplasm of these cells. In the
ethanol-treated control cells, only a small proportion of cells
possessed these structures. Ultrastructural features of apoptosis were
not evident in the nonresponsive sample but constituted a prominent
feature of the sensitive primary sample examined. Primary cells from
this nonresponsive patient (patient A) exposed to 20 µmol/L
lovastatin for 2 days showed no significant changes to GSH or MMP
measurements compared with ethanol-treated controls. By contrast,
primary cells from the sensitive patient (patient O) displayed dramatic
reduction in both GSH levels and MMP on exposure to lovastatin. As
shown in the AML-5 cell line, the dual-parameter dot plots of GSH
content versus MMP clearly demonstrated that, in the sensitive blast
cells, lovastatin-induced GSH depletion occurred in the same population
of cells showing a decrease in MMP. Thus, as demonstrated in the cell
lines examined, lovastatin-induced cytotoxicity possessed the flow
cytometric, ultrastructural, and metabolic alterations typical of
apoptosis in sensitive AML blasts.
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| Fig 7.
Ultrastructural and biochemical features of AML leukemic
blast cells after 2 days of exposure to solvent control or lovastatin.
(A) A nonresponsive primary sample (patient A) and (B) a sensitive
primary sample (patient O) were evaluated. (A) (a and b) Control blast
cells; (c and d) 20 µmol/L lovastatin-treated blast cells from
patient A displayed a number of large lysosomal granules. (B) (a and b)
Control blast cells; (c) 20 µmol/L lovastatin-treated blast cells
from patient O also displayed a number of large lysosomal granules in
the few surviving cells; (d) 20 µmol/L lovastatin induced a prominent
apoptotic response in patient O. The bar represents 1 µm except in
(B) (b), where it represents 3 µm. Multilaser flow cytometric
analysis of reduced GSH and MMP in the AML primary cultures A and O
exposed to 20 µmol/L lovastatin for 2 days and analyzed.
Dual-parameter dot plots of GSH and MMP for each sample are also
presented.
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DISCUSSION |
Present therapeutic regimens for the treatment of patients with AML are
toxic and often ineffective.31 Despite initial favorable responses to a variety of chemotherapeutics, most patients relapse, develop drug resistance, and quickly succumb to their
disease.14,31-33 Clearly, novel therapeutic approaches are
urgently needed. In this study, we demonstrated that the targeting of
HMG-CoA reductase, the rate-limiting enzyme of de novo cholesterol
synthesis,1 represents a potential novel therapeutic
approach in the treatment and control of AML. Inhibition of enzyme
function with lovastatin induced a significant apoptotic response in
the majority of the AML samples tested. Apoptosis is a well-defined
mechanism of programmed cell death that is distinguished by
characteristic morphological and metabolic features.25-27
Lovastatin treatment of sensitive AML cells demonstrated these
hallmarks of apoptosis, including nuclear and cytoplasmic condensation
and fragmentation, depletion of GSH, and a decrease in MMP. Serum
levels of approximately 4 µmol/L are achievable with oral
administration of lovastatin.8 This concentration in vitro
significantly affected cell viability of the majority of AML blast
cells tested but had little effect on ALL blasts or normal bone marrow
cells, indicating specificity in response to this agent.
We have now identified two retinoic acid-responsive tumors,
neuroblastoma10 and AML, that demonstrate increased
sensitivity to lovastatin-induced apoptosis. The mechanism by which
lovastatin triggers apoptosis and the determinants of tumor cell
sensitivity and specificity to this response remains unknown.
Lovastatin's ability to suppress proliferation is thought to be
mediated by its ability to block receptor signaling, particularly from
the insulin-like growth factor-I and the platelet-derived growth factor receptors.34-37 Because mitogenic receptors such as
insulin-like growth factor-1 are also mediators of cell
survival,38,39 lovastatin-induced apoptosis may result from
abrogation of cell survival signals. The dependency of cells for
specific survival factors is tissue and developmental stage
restricted.40 This phenomenon may account for the tumor
specificity of lovastatin-induced apoptosis observed in our studies.
Further work is ongoing to delineate the mechanism of action of
lovastatin-induced apoptosis. Whether the retinoic acid responsiveness
documented in the lovastatin-sensitive tumor types is a phenotypic
marker or mechanistically linked to the mevalonate pathway remains to
be elucidated.
Understanding the mechanism of lovastatin-induced apoptosis in the
sensitive tumor types will likely require an evaluation of the
potential roles of the various endproducts of the mevalonate pathway.
Because lovastatin inhibits HMG-CoA reductase, critical mevalonate
metabolites such as farnesyl and geranylgeranyl isoprenoids, dolichol,
ubiquinone, and cholesterol are reduced,1,41 possibly contributing to lovastatin-induced apoptosis. The addition of these
endproducts or other inhibitors further along this pathway that may
modulate the apoptotic response of lovastatin may delineate its
mechanism of action. For example, the mevalonate metabolite dolichol,
involved in N-linked glycosylation,1 plays a role in the
translocation of cell survival factor receptors to the cell
membrane.34,35 In fact, treatment with lovastatin can result in the diminished trafficking of a number of receptors to the
cell surface.34,35 Furthermore, essential regulatory proteins such as ras are dependent on isoprenylation for their proper
localization to cellular membranes and their function.1,41 Ras has been demonstrated to be a key effector of mitogenic and cell
survival stimuli,42 with constitutive activation of ras through mutation as a common feature of many human
cancers.43 As such, targeting of ras isoprenylation through
farnesyl transferase inhibitors has been evaluated as a potential
therapeutic target.41,44 The growth arrest properties of
lovastatin have been shown to be independent of ras mutational
status.45 Similarly, the extent of ras mutations are
approximately equal in AML and ALL samples46,47; yet, our
work shows that AML, but not ALL, is sensitive to lovastatin-induced apoptosis. However, because the role of ras in mediating cell survival
signals is an independent signaling pathway to its mitogenic effects,43,48 a formal evaluation of ras family of
signaling molecules and their downstream targets in the apoptotic
response triggered by lovastatin is required.
Our work suggests that lovastatin has potential as an immediate, novel
therapeutic approach in the treatment of AML. First, lovastatin can
induce a specific apoptotic response in AML blast cells within its
therapeutic range. Second, it has a proven record in the clinic as a
safe and effective drug.3,4,8 The well-documented potent
growth arrest property of HMG-CoA reductase inhibitors5 has
led to their evaluation as potential chemotherapeutics in human
cancers.8 In a phase I clinical trial of lovastatin, this
inhibitor displayed minimal adverse side effects at high doses;
however, lovastatin had little effect in reducing tumor-load in these
patients.8 The therapeutic potential of lovastatin on the
apoptotic sensitive neuroblastoma10 and AML cells
identified in our studies were not evaluated in this phase I trial.
HMG-CoA reductase inhibitors have been used extensively in the
treatment of hypercholesterolemia and as a result have well-defined
pharmacokinetics.3,4,8,9 There is no evidence of acquired
resistance to HMG-CoA reductase inhibitors with respect to their
ability to lower plasma cholesterol levels, even with extended
use.3,4,9 The tumor-specific apoptosis induction as well as
the biological properties of the HMG-CoA reductase inhibitors suggests
that they are potentially ideal therapeutic agents in AML. The efficacy
of lovastatin alone or in combination with other therapeutics in the
treatment of AML can be evaluated in clinical trials.
| |
ACKNOWLEDGMENT |
The authors are grateful to Dr S. Minkin for the statistical
analysis;to Dr J.E. Dick and the Penn lab for critically reviewing this
manuscript; to Dr J. Squire, A. Pandita, and J. Bayani for helpful
discussions; to J. Kao, N. Jamal, J. Sheldon, and J. Hwang for
technical assistance; and to Drs H. Messner and J.E. Dick for providing
the bone marrow and cord blood cells used in this study, respectively.
Our thanks is extended to Merck and Frost (Montreal, Quebec, Canada)
for generously supplying the lovastatin used in this study.
| |
FOOTNOTES |
Submitted December 18, 1997; accepted October 5, 1998.
Supported by funds from The Medical Research Council of Canada (L.Z.P.)
as well as a joint industry grant from MRC/Apotex Inc (L.Z.P.).
Fellowship support from OCI/Amgen (J.D.) is gratefully acknowledged.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Linda Z. Penn, PhD, Department of Molecular
and Cellular Biology, Ontario Cancer Institute, the Princess Margaret
Hospital, 610 University Ave, Toronto, Ontario, Canada, M5G 2M9.
| |
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Abstract
Introduction