[Cancer Research 60, 1002-1008, February 15, 2000]
© 2000 American Association for Cancer Research
Experimental Therapeutics |
Inhibition of Human Prostate Cancer Proliferation in Vitro and in a Mouse Model by a Compound Synthesized to Block Ca2+ Entry1
Doris M. Haverstick,
Tiffany N. Heady,
Timothy L. Macdonald and
Lloyd S. Gray2
Departments of Pathology [D. M. H., L. S. G.] and Chemistry [T. N. H., T. L. M.], University of Virginia, Charlottesville, Virginia 22908
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ABSTRACT
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Accelerated Ca2+ entry may be one component of the pathway
regulating the proliferative phenotype of some types of cancer. Thus, a
pharmacological agent with the ability to retard Ca2+
influx in susceptible cancers might inhibit proliferation of them by a
cytostatic mechanism rather than by inducing cytotoxicity. We have
developed a chemical synthetic scheme that has produced a small library
of novel compounds that block Ca2+ entry induced by
occupancy of the P2 receptor in two prostate cancer cell lines and
inhibit proliferation of these cells in vitro. One of
the agents, named TH-1177, was used to treat severe combined
immunodeficient mice inoculated with the human prostate cancer line
PC-3. Although the doses used and treatment schedule were chosen
arbitrarily, treatment extended the mean life span of mice bearing
tumors by up to 38%. Treatment of mice without cancer at doses 18
times that used in mice with tumors was not associated with any obvious
toxicity, either grossly or on histological examination. These results
suggest that novel cytostatic agents with efficacy against human
prostate cancer cells can be developed by chemical synthesis of agents
directed at the Ca2+ entry pathway.
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INTRODUCTION
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Malignant transformation is often associated with acquisition of a
phenotype that is consistent with an abnormally high sensitivity to
ambient concentrations of growth factors. In prostate cancer, the
source of these growth factors can be autocrine, from the cancer cells
themselves, or paracrine, from the surrounding stroma (1
, 2)
. The molecular role of growth factors and their corresponding
receptors in malignant transformation and prostate cancer progression
is not wholly clear but is likely to be complex (2)
. This
complexity derives at least in part from the ability of various growth
factors to either promote or inhibit cellular proliferation
(3, 4, 5)
. Complexity is compounded by the ability of
proteins other than growth factor receptors to participate in and
modify the final expression of the malignant phenotype in a particular
tumor (6)
. Despite the intricacy of growth regulation in
prostate cancer, the mitogenic properties of growth factors and their
receptors make them a tempting target for therapeutic intervention.
Growth factor receptors are often linked to the pathway that regulates
Ca2+ homeostasis. As a general rule, engagement
of a growth factor receptor by an appropriate ligand results in the
activation of phospholipase C by tyrosine phosphorylation
(7)
. Activated phospholipase C metabolizes
phosphatidylinositol bisphosphate to produce diacylglycerol and
inositol 1,4,5-trisphosphate (7
, 8) . Via engagement of a
specific receptor, inositol trisphosphate releases
Ca2+ from an internal storage depot, and this
release of intracellular Ca2+ triggers the influx
of extracellular Ca2+ (8)
. Thus, the
mitogenic interaction of a growth factor with its receptor can activate
a pathway that includes enhancement of the entry of extracellular
Ca2+, which in turn is a component of the
proliferative cascade (9)
.
What role enhanced Ca2+ entry might play in the
proliferation of prostate cancer cells is not well understood. It has
been shown, however, that proliferation of at least some cancer cell
lines can be slowed or stopped at specific points in the cell cycle by
removal of extracellular Ca2+ (9
, 10)
. Consistent with this is the observation that a drug that
blocks Ca2+ entry can retard the metastasis of
human melanoma cells in immune-deficient mice (11)
. It is
unlikely that blockade of Ca2+ entry could
reverse the malignant phenotype, even if it could ameliorate excessive
proliferation. However, inhibition of prostate cancer proliferation
in vivo would be a therapeutically appealing goal by, for
example, lengthening remission duration or treating early, limited
disease. With these ideas in mind, we have taken as our working
hypothesis the idea that a pharmacological agent capable of inhibiting
receptor-linked Ca2+ entry in vitro
might also slow prostate cancer proliferation in vivo.
Previously, we identified a Ca2+ current named
IT in a malignant T lymphocyte cell line that is
augmented by a mitogenic stimulus (12)
. Guided by the
general biophysical features of this current, we surveyed the
literature for compounds that inhibited Ca2+
currents with roughly similar characteristics in other systems. Several
known compounds were identified and tested for their ability to inhibit
receptor-linked Ca2+ entry and proliferation.
Although this structurally diverse group of agents was not strikingly
effective, they nonetheless suppressed receptor-linked
Ca2+ influx and proliferation with a high degree
of concordance. From the structures of these compounds and their
relative efficacy, we constructed a structure-activity series. This
series was used to direct the synthesis of novel compounds with the
intent of blocking Ca2+ entry and proliferation.
In vitro testing showed that many of these compounds,
including one named TH-1177, were more effective at blocking
Ca2+ influx and proliferation of prostate cancer
cells than those already known. We also examined TH-1177 in a murine
model of human prostate cancer. Our results suggest that TH-1177 is
cytostatic for prostate cancer cells in vitro and can
significantly prolong the life of immune-deficient
SCID3
mice inoculated with the human prostate cancer line PC-3.
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MATERIALS AND METHODS
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Synthesis of TH-1177.
TH-1177 was synthesized in three simple steps (Fig. 1)
. L-Proline methyl ester was coupled with
4-methoxyphenylacetic acid using
benzotriazol-1-yl-oxytripyrrolidinephosphonium to generate methyl
1-[2-(4-methoxyphenyl)acetyl]pyrrolidine-2-carboxylate, a yellowish
oil. The resulting amide was subsequently reduced to the amino alcohol
with LiAlH4 and AlCl3 in
tetrahydrofuran. The resulting colorless oil was coupled with
4-chlorobenzhydrol under Williamson conditions with catalytic
p-toluenesulfonic acid in refluxing toluene. The final
brownish oil was isolated by column chromatography, and its structure
was confirmed by nuclear magnetic resonance and mass spectrometry.
TH-1177 was dissolved in DMSO for use in vitro and in
ethanol for use in vivo. Details of the synthesis of TH-1177
and related compounds will be presented
elsewhere.4
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Fig. 1. Chemical synthesis of TH-1177. TH-1177 was synthesized as
outlined in "Materials and Methods." The chemical steps and
structures are indicated in the figure. THF,
tertahydrofuran; NMM, 4-methylmorpholine;
p-TSOH, p-toluenesulfonic acid;
PyBOP, benzotriazol-1-yl-oxytripyrrolidinephosphonium.
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Cell Lines and Maintenance.
Hormone-responsive LNCaP-FRG and hormone-resistant PC-3 prostate cancer
cells were obtained from the American Type Culture Collection
(Manassas, VA). Both cell lines were maintained in RPMI 1640 and
supplemented with glutamine and 5% fetal bovine serum containing
SerXtend (Irvine Scientific). The fetal bovine serum used for culture
was heat-inactivated by maintaining the serum at 56°C for 1 h.
Measurement of the [Ca2+]i
Concentration.
Cells were incubated in growth media containing 1 µM of
the acetoxy-methyl ester of the Ca2+-sensitive
fluorescent dye indo-1 (indo-1/AM; Molecular Probes, Eugene, OR) for
1 h at 37°C. Cells were washed three times in buffer A [10
mM HEPES (pH 7.4), 1 mM
MgCl2, 3 mM KCl, 1 mM
CaCl2, 140 mM NaCl, 0.1% glucose,
and 1% fetal bovine serum] and suspended to a final concentration of
106 cells/ml. Before stimulation, cells were
warmed to 37°C. Changes in [Ca2+]i
were monitored in an SLM 8100C spectrofluorometer (SLM/Aminco; Urbana,
IL) using previously published methods (13
, 14)
.
Measurement of Cellular Proliferation.
LNCaP cells at 2.5 x 104
cells/well or PC-3 cells at 5 x 104
cells/well, both in a final volume of 100
µl, were plated in triplicate in standard flat-bottomed 96-well
tissue culture plates in the presence of drug or vehicle (DMSO). Unless
otherwise indicated, cells were grown for 48 h at 37°C in a
CO2 incubator. Relative cell growth was
determined with the CellTiter 96 aqueous cell proliferation assay
(Promega, Madison, WI) as described by the manufacturer using an
automated plate reader. Results were calculated in a blinded fashion
and are the means of triplicate determinations.
Determination of Apoptosis.
Cells at 2.5 x 105
cells/ml
(LNCaP) or 5 x 105
cells/ml
(PC-3) were cultured in growth medium for 24 h in the presence or
absence of 100 µM TH-1177 on chambered microscope slides
that were coated with 0.01% poly-L-lysine to promote cell
adhesion. Apoptotic cells were identified by the terminal
deoxynucleotidyl transferase-mediated nick end labeling method using a
commercially available kit (Promega, Madison, WI). Staining with
propidium iodide identified all cells, both living and apoptotic.
Slides were scored in a blinded fashion, and a minimum of 200 cells
under each condition were counted.
Animal Studies.
The Animal Care Committee of the University of Virginia approved all
protocols. SCID mice were housed in a barrier isolation facility of the
University of Virginia Department of Comparative Medicine, and all
personnel observed sterile technique when entering the facility and
handling animals. TH-1177 was dissolved in ethanol and diluted 10-fold
in sterile buffered NaCl solution [PBS; 150 mM NaCl, 20
mM sodium phosphate (pH 7.4)] immediately before each
day’s injection. Injection volumes of 0.5 ml/animal were used. Vehicle
consisted of PBS-diluted ethanol. PC-3 cells for injection were
prepared by washing in sterile PBS three times before suspension to
2 x 106 cells/ml. Each animal
received 0.5 ml of cells by i.p. injection on day 0 of the experiment
shown in Fig. 10
. Each animal received a daily i.p. injection of
vehicle or drug beginning on day 1.
Statistical Methods.
In vivo survival data were analyzed with Prism 2.01
(GraphPad Software, San Diego, CA). The results of the Kaplan-Meier
analysis are presented as one-tailed probabilities because there was no
reasonable expectation that drug treatment would cause the mice to
succumb more rapidly to the implanted cancer than would control
animals.
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RESULTS
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General Overview of Chemical Synthesis of TH-1177
Design.
It is thought that in dihydropyridines, orientation and
ortho, meta, para substitution of the
aryl ring(s) affect the activity, as do varying degrees of
hydrophobicity in benzothiazepines. The efficacy of phenylalkylamine
and derivatives varies with the enantiomer as well as with the type of
group substituted on the aryl rings (15)
. Calcium channel
blockers used in preliminary experiments designed to test our
hypothesis included dihydropyridines, benzothiazepines, and
phenylalkylamines. This survey of known blockers provided structural
features and functionality thought to be important for activity. These
include a basic tertiary nitrogen, varying regions of hydrophobicity,
core heterocycles, substituted aryl rings, and ether linkages. Our
proline (specifically, TH-1177) and nipecotate targets incorporate
these features and are typically N- and O-
dialkylated.
Nature of the Interaction with a Potential Target.
Dihydropyridines, phenylalkylamines, and benzothiazepine are classified
by their binding to specific sites on the four-subunit calcium channel
protein. Phenylalkylamines are known to covalently incorporate into the
1 subunit, specifically the 42-residue segment from
Glu1349 to Trp1391
(16)
. The dihydropyridine receptor site is thought to be
formed by the extracellular ends of transmembrane segments in two
separate domains also on the 1 subunit. As with the phenylalkylamine
and dihydropyridines, the receptor site for the benzothiazepines is
also located on the 1 subunit. Because the design of TH-1177
incorporates basic structural features of all three classes and because
the 1 subunit of L-type calcium channels is highly
conserved across many cell types, it is reasonable to assume that it
shares the same receptor or a similar receptor (16)
.
Criteria Used to Select TH-1177.
TH-1177 was chosen because of its low IC50
proliferation values and its ability to halt the influx of calcium in
both prostate cancer cell lines.
Synthesis of TH-1177.
Fig. 1
outlines the three-step synthesis of TH-1177 that allows for a
large number of additional target compounds to be made easily and
efficiently. Details of the synthetic strategy for these other
compounds will be presented elsewhere (3)
. All steps
provided yields of greater than 75%. Efficient reduction of both the
amide and the ester is achieved in one step using
LiAlH4 and AlCl3 in a 1:3
molar ratio. Three different syntheses of TH-1177 were used for the
completion of these studies, with no differences in nuclear magnetic
resonance or mass spectroscopy characteristics among the batches. Each
batch was assessed for its ability to inhibit PC-3 and LNCaP prostate
cancer cell proliferation in vitro (see below), and the
IC50 values for each batch were within the
variance of the assay.
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TH-1177 Blocks Capacitative Ca2+ Entry in Human
Prostate Cancer Cells
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In electrically nonexcitable cells, Ca2+
influx is triggered by release of Ca2+ from its
internal storage depot by a phenomenon that has been called
capacitative Ca2+ entry (17, 18, 19)
.
Capacitative entry can be initiated by treatment of cells with
thapsigargin. Thapsigargin inhibits the
Ca2+-ATPase of the endoplasmic reticulum,
allowing uncompensated leak of Ca2+ from this
compartment into the cytosol, thereby causing
Ca2+ entry in the absence of engagement of a
specific receptor (20
, 21)
. As shown in Fig. 2A
, addition of TH-1177 to a suspension of LNCaP human
prostate cancer cells before thapsigargin resulted in a dose-dependent
inhibition of the increase in [Ca2+]i.
TH-1177 also reduced the increase in
[Ca2+]i initiated by thapsigargin when
added after the stimulus (Fig. 2B)
. The elevated
[Ca2+]i seen at 100 s in Fig. 2B
is generally attributable to
Ca2+ entry alone (18)
, suggesting
that the effect of TH-1177 is mediated at least in part by inhibition
of Ca2+ entry.
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Fig. 2. Effect of TH-1177 on Ca2+ entry stimulated by
thapsigargin in LNCaP cells. LNCaP cells were stimulated with 300
nM thapsigargin to initiate Ca2+ entry in a
receptor-independent manner. The indicated concentrations of TH-1177
were added before stimulation with thapsigargin (A) or
after the influx pathway had been opened (B). Times of
addition are indicated on the figure.
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To confirm an inhibitory action of TH-1177 on capacitative
Ca2+ entry, we performed the experiment outlined
in Fig. 3, A and B
. The
[Ca2+]i of a suspension of LNCaP
prostate cancer cells was monitored while the extracellular
Ca2+ concentration was reduced by the addition of
EGTA. The intracellular Ca2+ storage pool was
depleted by thapsigargin, which is a conventional means of initiating
capacitative Ca2+ entry (21)
.
Capacitative Ca2+ entry was then monitored after
the readdition of Ca2+ to the extracellular
medium. TH-1177 was added immediately before or shortly after
readdition of Ca2+. As shown in Fig. 3
, TH-1177
caused a concentration-dependent inhibition of
Ca2+ entry under both circumstances, suggesting
that its effects are on capacitative Ca2+ entry.
When added after readdition of Ca2+, the
IC50 for TH-1177 was 3.2 µM, which
is comparable to the value of 3 µM for the experiment
shown in Fig. 2
. When the experiment shown in Fig. 3
was performed with
PC-3 cells, the IC50 for TH-1177 was 17
µM, whereas it was 16 µM for the experiment
shown in Fig. 2
when using the same cell line (data not shown). Taken
together, these results strongly suggest that TH-1177 inhibits
capacitative Ca2+ entry in these two prostate
cancer cell lines.
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Fig. 3. Effect of TH-1177 on capacitative Ca2+ entry.
The effect of TH-1177 on the capacitative influx of Ca2+
was examined by inducing Ca2+ entry with thapsigargin in
the absence of extracellular Ca2+. Readdition of
Ca2+ to the extracellular medium revealed the influx
component of changes in [Ca2+]i dissociated from
the release of Ca2+ from the internal storage pool. LNCaP
cells were incubated with indo-1 to measure changes in
[Ca2+]i as described in "Materials and
Methods." EGTA (5 mM) was added at 30 s, as
indicated by the arrow, to remove extracellular
Ca2+. Thapsigargin (1 µM) was added at
60 s, as indicated by the arrow, to release
Ca2+ from the internal storage pool. A,
TH-1177 at 1, 3, and 10 µM was added at 180 s.
Ca2+ (5 mM) was added at 260 s.
B, Ca2+ (5 mM) was added at
180 s, and TH-1177 at 1, 3, and 10 µM was added at
250 s.
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TH-1177 Inhibits Receptor-linked Ca2+ Entry
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The P2 purinergic receptor is linked to activation of the
Ca2+ entry pathway in many types of cells
including prostate cancer cells (22)
. The ability of
TH-1177 to block capacitative Ca2+ entry induced
by thapsigargin suggested that this compound might also block
Ca2+ entry triggered by the engagement of a
specific receptor. The P2 receptor binds extracellular ATP, inducing
multiple biochemical events including Ca2+ entry
(22)
. Addition of ATP to LNCaP prostate cancer cells
resulted in a rapid rise in [Ca2+]i
that was inhibited by the prior addition of TH-1177 (Fig. 4A)
. As shown in Fig. 4B
, TH-1177 added after ATP
also caused a reduction in the [Ca2+]i
that had been augmented by P2 receptor engagement.
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Fig. 4. Effect of TH-1177 on Ca2+ entry stimulated by
ATP in LNCaP cells. Ten µM TH-1177 was added to a
suspension of LNCaP cells either before stimulation with 1
mM ATP (A) or after the influx pathway had
been opened by the addition of ATP (B). Times of
addition are indicated on the figure.
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PC-3 prostate cancer cells also demonstrated an increase in
[Ca2+]i when stimulated by ATP (Fig. 5
; Ref. 22
). In the experiment depicted in Fig. 5A
, TH-1177 was added before the cells were stimulated with
ATP. TH-1177 caused a concentration-dependent inhibition of the
increase in [Ca2+]i that was otherwise
induced by engagement of the purinergic receptor. By 70 s after
ATP addition, release of Ca2+ from the internal
storage pool is largely over (for example, see Fig. 6
), and the maintenance of elevations of
[Ca2+]i over baseline is dependent on
Ca2+ entry from the extracellular compartment. As
shown in Fig. 5B
, addition of TH-1177 to cells treated
previously with ATP caused a reduction in
[Ca2+]i. This suggests that TH-1177 is
interacting with the Ca2+ influx pathway
initiated by P2 receptor stimulation.
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Fig. 5. Effect of TH-1177 on Ca2+ entry stimulated by
ATP in PC-3 cells. The indicated concentrations of TH-1177 were added
before stimulation of PC-3 with 300 µM ATP
(A) or after the influx pathway had been opened
(B) at the times indicated on the figure.
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Fig. 6. Effect of TH-1177 on release of Ca2+ from the
internal storage depot. The effect of TH-1177 on inositol
1,4,5-triphosphate-mediated release of Ca2+ from the
internal storage pool was monitored by chelating extracellular
Ca2+ with the addition of 5 mM EGTA to uncover
the release component. The indicated concentrations of TH-1177 were
added at 30 s, followed by 2.5 mM EGTA at 60 s,
and receptor stimulation was accomplished by treating with 1
mM ATP (LNCaP cells) or 300 µM ATP (PC-3
cells) at 90 s. A, PC-3 cells; B,
LNCaP cells.
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The efficiency of TH-1177 in blocking Ca2+ entry
was assessed by comparison to the effect of chelation of extracellular
Ca2+ with EGTA, which was considered 100%
inhibition of Ca2+ entry. For LNCaP cells, the
IC50 for TH-1177 was 3 µM, and for
PC-3 cells, the IC50 was 16 µM
under these conditions.
The data presented above suggest that TH-1177 was inhibiting stimulated
increases in [Ca2+]i by blockade of
Ca2+ entry. In Fig. 6
, extracellular
Ca2+ was markedly reduced by the addition of EGTA
to the extracellular medium. Under these conditions, addition of ATP to
either PC-3 (Fig. 6A)
or LNCaP (Fig. 6B)
cells
resulted in a rise in [Ca2+]i that is
more transient and of smaller magnitude than in the presence of
extracellular Ca2+. This increase represents
release of Ca2+ from the internal storage pool.
Addition of TH-1177 had no effect on the size of this change in
[Ca2+]i. Similar results were obtained
when Ca2+ release was induced by treatment with
thapsigargin (data not shown). This indicates that TH-1177 does not
interfere with Ca2+ release and suggests that
TH-1177 has no influence on the biochemical events upstream from
release of Ca2+ from the internal pool.
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TH-1177 Inhibits Prostate Cancer Cell Proliferation in
Vitro by a Cytostatic Mechanism
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Inhibition of Ca2+ entry has been shown to
limit proliferation of cancer cells in vitro
(11)
. The ability of TH-1177 to block
Ca2+ entry induced by release of
Ca2+ from internal stores such as that stimulated
by engagement of the P2 purinergic receptor suggested the possibility
that this agent could inhibit proliferation of prostate cancer cells
in vitro. As shown in Fig. 7
, TH-1177 caused a concentration-dependent inhibition of the
proliferation of both LNCaP and PC-3 cells. The
IC50 for inhibition of LNCaP proliferation was 4
µM (Fig. 7A)
, whereas the value for
PC-3 prostate cancer cells was 14 µM (Fig. 7B)
. When compared to the IC50 values
for inhibition of Ca2+ entry of 3 and 16
µM for LNCaP and PC-3 cells, respectively, it
is clear that TH-1177 inhibits proliferation at a concentration similar
to that which blocks Ca2+ entry in these two cell
types.
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Fig. 7. Effect of TH-1177 on cellular proliferation. LNCaP cells
(A) at 2.5 x 104 cells in
100 µl or PC-3 cells (B) at 5 x 104 cells in 100 µl were grown for 48 h in the
absence (100% cell growth) or presence of the indicated concentrations
of TH-1177. Relative cell growth was measured as outlined in
"Materials and Methods." Results are the mean of four
determinations.
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Most conventional cancer chemotherapeutic drugs are cytotoxic and exert
their therapeutic benefit by killing cancer cells. On the other hand,
agents that act by inhibition of Ca2+ entry would
likely arrest cell proliferation rather than induce cell death
(9)
. To address this possibility, LNCaP prostate cancer
cells were allowed to grow unimpeded or exposed to TH-1177 for 2 or 3
days before the agent was washed away (Fig. 8, A and B)
. As predicted, both cell lines grew in
the absence of TH-1177, whereas growth was stopped when the compound
was present. Removal of TH-1177 after exposure for 2 or 3 days was
associated with resumption of a rate of growth that was similar to that
seen in cells never exposed to TH-1177 (Fig. 8)
. Similar results were
seen with PC-3 prostate cancer cells (data not shown). This effect was
not due to the induction of apoptosis by TH-1177. In the presence of
100 µM TH-1177 for 24 h, 8.4 ± 0.4%
of LNCaP cells and 9.8 ± 0.4% of PC-3 cells were
apoptotic, as compared to 10.1 ± 0.4% of cells in
control cultures. Taken together, these observations suggest that
prostate cancer cells were quiescent in the presence of TH-1177
relative to cells that were cultured in the absence of this agent. We
do not know whether TH-1177 blocks proliferation at a specific point in
the cell cycle. However, Ca2+ influx is required
at several specific transition points (23
, 24)
, and it is
possible that TH-1177 acts at each of these.
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Fig. 8. TH-1177 inhibits prostate cancer cell proliferation
cytostatically. LNCaP cells were plated at 2.5 x 105 cells/ml in the absence of TH-1177 or with 1
µM (A) or 3 µM
(B) of this agent on day 1 in triplicate flasks, and the
number of viable cells was determined in each flask on days 2–5. On
days 2, 3, and 4, all flasks were centrifuged, and fresh media were
added with or without TH-1177 as indicated. An experiment
representative of three experiments is shown.
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The experiment depicted in Fig. 8
is consistent with the idea that
TH-1177 does not induce any long-lasting alteration in the
proliferative phenotype of prostate cancer cells. The experiment
depicted in Fig. 9
was performed to further evaluate this possibility. LNCaP cells were
grown in the continuous presence of TH-1177 at one of three
concentrations or in the absence of the compound for 48 h. The
cell culture medium was then replaced with medium free of TH-1177 and
maintained for an additional 48 h. TH-1177 was then added to all
cell cultures at the concentrations indicated in Fig. 9
, such that
previously treated cell cultures received an identical second
treatment. Prior treatment with TH-1177 had no effect on the response
to the second exposure to this compound (Fig. 9)
. This observation is
clearly consistent with the idea that TH-1177 must be present to
inhibit proliferation and that TH-1177 does not alter the
drug-sensitive phenotype of human prostate cancer cell lines under
these conditions.
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Fig. 9. Prostate cancer cells do not become acutely refractory to
the effects of TH-1177. LNCaP cells were grown in the absence (naive
cells) or presence (prior treatment) of the indicated concentrations of
TH-1177 for 48 h. The media was removed, and drug-free media were
added for 48 h. The drug-free medium was then removed, and medium
containing the drug was added to all cells for 72 h. The data
shown indicate the relative number of cells at the end of this
process.
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TH-1177 Slows Prostate Cancer Progression in Vivo
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To begin an investigation of the possibility that TH-1177
possesses in vivo activity against prostate cancer, SCID
mice were inoculated with PC-3 cells by i.p. injection. One day later,
daily i.p. injections of TH-1177 or vehicle alone were begun. TH-1177
was administered at doses of either 3 or 10 mg/kg. These doses were
selected arbitrarily, based on the general range of doses for
Ca2+ channel blockers given p.o. to patients for
the treatment of hypertension. Daily dosing was also selected
arbitrarily, based on the general desirability of a once-a-day
treatment regimen. As shown in Fig. 10
, there was a dose-dependent increase in longevity associated with
TH-1177 administration. Life span was increased by 34%
(P = 0.047) by TH-1177 at a dose of 3
mg/kg/day and by 38% (P = 0.0044) at a dose
of 10 mg/kg/day.
Although the experiment shown in Fig. 10
suggested a lack of toxicity
associated with TH-1177 administration, the drug was further examined
specifically for possible toxicity. Four SCID mice without tumors were
given TH-1177 at a dose of 180 mg/kg/day by daily i.p. injection for 22
days. Treated mice were all well groomed and active during treatment,
and no gross abnormalities were noted at necropsy (data not shown).
Samples of kidney, adrenal gland, heart, and liver were within normal
limits on histological examination including active hepatic
hematopoiesis.5
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DISCUSSION
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Antimetabolic, cytotoxic therapies for cancer have achieved much
success in extending the lives of people with this disease. The goal of
this approach to cancer treatment, at its limit, is the complete
eradication of cancer cells. Elimination of all residual cancer cells
results in cure, although emergence of drug resistance or of disease
that is more aggressive not infrequently hampers this outcome. The goal
of cytostatic cancer therapies is to retard cellular proliferation
rather than eliminate all cancer cells. This should not diminish
interest in such an approach because controlling the growth of cancer
would, at the limit, render the disease effectively impotent. Failure
to achieve this limit could be nonetheless valuable clinically if, for
example, cytostatic therapy significantly extended the duration of
remissions induced by cytotoxic agents.
A role for Ca2+ entry in controlling the overall
rate of cellular proliferation has been speculated about for some time
(9
, 23
, 25) . However, the function of
Ca2+ entry is paradoxical; although it is
critical for cell division (9
, 23)
, it is equally
necessary for apoptotic cell death (25)
. It has, for
example, been shown that the normally occurring rate of apoptosis in
prostate cancer cell lines is reduced by transfection of the cytosolic
Ca2+-binding protein calbindin (26)
.
On the other hand, a drug that blocks Ca2+ entry
into human melanoma cells was shown to retard the metastasis of these
cells in immune-deficient mice (11)
. Using malignant T
lymphocytes, we have shown that a previously known and relatively
simple organic compound blocks receptor-linked
Ca2+ entry and that this blockade is accompanied
by inhibition of proliferation (14)
. Based on these
observations, we undertook the directed synthesis of chemical agents
with the intent of developing compounds that, in vitro,
block growth factor receptor-linked entry of Ca2+
and, in association, growth factor-driven cellular proliferation. The
synthetic scheme was designed based on a structure-activity analysis of
known compounds that we have shown to be effective inhibitors of both
Ca2+ entry and tumor cell proliferation (data not
shown). The resulting synthetic strategy yielded a small library of
agents that were effective for the purpose intended. We have no
information yet on the identity of the molecular target of TH-1177.
However, conventional Ca2+ channel blockers, upon
which TH-1177 is roughly based, bind to the 1 subunit of the target
Ca2+ channel (16)
, and it is
possible that TH-1177 acts at a structurally similar site.
The ubiquity of Ca2+ as a second messenger could
lead to the speculation that systemic administration of a
Ca2+ entry antagonist would have numerous,
unwanted consequences. However, this was not observed in mice treated
with a dose of TH-1177 that was 18-fold higher than that which showed
efficacy in slowing prostate cancer progression. It is possible that
polymorphisms among Ca2+ entry pathways may allow
relatively specific targeting of drugs to one pathway or another,
similar to the strategy used in the treatment of hypertension. The
success of therapy for the prevention of cardiovascular disease, as
well as our own observations, suggests that certain
Ca2+ entry antagonists can be administered
without poorly tolerated systemic toxicity.
Whereas a possible role of Ca2+ entry in cancer
cell proliferation has been known for some time, the use of directed
Ca2+ entry antagonists for the potential
treatment of this disease has not yet been developed. Based on our
characterization of the Ca2+ entry pathway in one
malignant cell type (14)
, we developed a strategy for the
identification of pharmacological agents that could act as
Ca2+ entry antagonists in cancer cells more
generally. From this assortment of known compounds, we constructed a
hypothetical chemical structure to pharmacologically inhibit
Ca2+ entry and hence proliferation in the types
of cancer cells included in our test panel, namely hormone-sensitive
and -insensitive breast and prostate cancer. A library of candidate
chemical agents was created based on this hypothetical structure. From
this library of compounds, TH-1177, which showed good correlation
between inhibition of Ca2+ entry and
proliferation of prostate cancer cells in vitro, was chosen
for further testing in a murine model of human prostate cancer. In this
initial study, administration of TH-1177 to mice significantly retarded
the progression of lethal human prostate cancer and did so without
apparent toxicity. This observation raises the possibility that
directed synthesis of novel Ca2+ channel blockers
could be useful in the therapy of cancer by minimizing effects on
unintended organs and cells and by maximizing the cytostatic efficacy
for the malignant tissue of clinical concern.
|
FOOTNOTES
|
|---|
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 Supported in part by a grant to the University
of Virginia Cancer Center from CaPCURE. 
2 To whom requests for reprints should be
addressed, at Box 214 HSC, Department of Pathology, University of
Virginia Health System, Charlottesville, VA 22908. Phone: (804)
924-8128; Fax: (804) 924-8060; E-mail: lsg5m{at}virginia.edu
3 The abbreviations used are: SCID, severe
combined immunodeficient; [Ca2+]i, intracellular
Ca2+.
4 T. N. Heady, M. M. Rotier, D. M.
Haverstick, L. S. Gray, T. Miller, and T. L. Macdonald. Novel
calcium channel blockers as cytostatic cancer agents, manuscript in
preparation.
5 Dr. Bruce Williams, personal communication.
Received 6/11/99.
Accepted 12/16/99.
|
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Top
ABSTRACT
INTRODUCTION
) or TH-1177 at 3 (•) or 10 mg/kg (
)
beginning on day 1. The survival curves were analyzed by the
Kaplan-Meier method as described in "Materials and Methods."