Affiliations of authors: L. M. Leoni, D. Genini, H. Shih, C. J. Carrera, H. B. Cottam, D. A. Carson, Department of Medicine and The Sam and Rose Stein Institute for Research on Aging, University of California San Diego, La Jolla; E. Hamel, Laboratory of Drug Discovery Research and Development, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, MD.
Correspondence to: Lorenzo M. Leoni, Ph.D., Department of Medicine 0663, University of California San Diego, 9500 Gilman Dr., La Jolla, CA 92093 (e-mail: lleoni{at}ucsd.edu).
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ABSTRACT |
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INTRODUCTION |
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The multidrug-resistant phenotype, although not strictly specific for cancer cells, is an
attractive
target for anticancer drugs because it develops during chemotherapy with bulky hydrophobic
antineoplastic agents, limiting their efficacy (10). Several mechanisms
may
contribute to intrinsic and acquired cross-resistance to multiple antineoplastic agents (clinical
drug
resistance). They include decreased drug accumulation due to overexpression of the
P-glycoprotein
drug efflux pump encoded by the mdr1 gene (11,12), the multidrug
resistance-associated protein (MRP) (13), and the p110 major vault
glycoprotein (14). In addition, multidrug resistance has been linked to
decreased expression of topoisomerase II (15), to altered expression
of
drug-metabolizing enzymes and drug-conjugate export pumps (16,17),
and to
modification of the apoptotic machinery (18,19).
Various hydrophobic drugs with low toxicity for tumor cells can partially reverse multidrug resistance in vitro and in vivo. In contrast, cytotoxic compounds that preferentially target multidrug-resistant cells are not well described, but such agents should be very useful in the treatment of cancer. The National Cancer Institute's Developmental Therapeutics Program has identified indanocine, a newly synthesized indanone, as a compound with antiproliferative activity.
In this article, we investigate the action of indanocine on cultured multidrug-resistant cancer cells and their corresponding parental (wild-type) cells.
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MATERIALS AND METHODS |
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Indanocine, NSC 698666 (Fig. 1, A), is one of a series of synthetic
indanones with antiproliferative activity (Shih H, Deng L, Carrera CJ, Adachi S, Cottam HB,
Carson
DA: unpublished data). Solid idanocine is a white powder that is stable when stored dry at room
temperature or when dissolved in dimethyl sulfoxide or in water containing cyclodextrins.
Paclitaxel,
vinblastine sulfate, and nocodazole were from Calbiochem (San Diego, CA). Electrophoretically
homogeneous bovine brain tubulin was prepared as described previously (20).
Media and tissue culture supplies were purchased from Irvine Scientific (Santa Ana, CA) and
Fisher
Scientific (San Diego, CA). All radiochemicals were from NEN-Dupont (Boston, MA). Unless
otherwise indicated, all other reagents were obtained from Sigma Chemical Co. (St. Louis, MO).
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Cell lines from the American Type Culture Collection (Manassas, VA), propagated
according to
the instructions of the supplier, were as follows: MES-SA (human uterine sarcoma) and its
multidrug-resistant variant MES-SA/DX5 raised against doxorubicin (21),
monkey COS-1, and Hep-G2 (human hepatocellular carcinoma). KB-3-1 (human carcinoma) and
KB-GRC-1 (a transfectoma expressing high levels of the MDR1-encoded 170-kd P-glycoprotein)
were provided by Dr. Stephen Howell (University of California San Diego, La Jolla) and have
been
described previously (22). Dr. Michael J. Kelner (University of California
San
Diego) provided the following cell lines: MV522 (human metastatic lung carcinoma) and
MV522/Q6 (a
transfectoma expressing high levels of the MDR1 gene-encoded 170-kd P-glycoprotein);
MCF-7/ADR, a human breast adenocarcinoma multidrug-resistant line selected against
doxorubicin
(expressing both gp170 and the embryonic glutathione transferase isoform), and
MCF-7/wt, the
parental (wild-type) line; MDA-MB-231, a human breast adenocarcinoma line, and
MDA3-1/gp170+,
the doxorubicin-resistant daughter line expressing the 170-kd P-glycoprotein; and HL-60, a
human
acute promyelocytic leukemia line, and HL-60/ADR, the multidrug-resistant variant line selected
against
doxorubicin and expressing the MRP/gp180 protein. Dr. William T. Beck (Cancer Center,
University
of Illinois at Chicago) provided CEM, a human lymphoblastoid line, and CEM/VLB100, a
multidrug-resistant line selected against vinblastine and expressing the 170-kd P-glycoprotein.
We incubated cells for 72 hours in 96-well plates with the test compounds and then measured cell proliferation by reduction of the yellow dye MTT [i.e., 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] to a blue formazan product. The cleavage is performed by the "succinate-tetrazolium reductase" system, which belongs to the respiratory chain of the mitochondria and is active only in viable cells. Therefore, the amount of formazan dye formed is a direct indication of the number of metabolically active cells in the culture. The optical density of the blue formazan product was measured at 570 nm with a ThermoMax (Molecular Devices, Sunnyvale CA) and analyzed with the Vmax Program (BioMetallics, Princeton, NJ).
Cell Cycle Analysis
Cells were harvested, fixed in ice-cold 70% ethanol, treated with ribonuclease A at 100 µg/mL, and stained with propidium iodide at 50 µg/mL for 1 hour at 37 °C. The DNA content of the cells was analyzed by flow cytometry (FACScalibur; Becton Dickinson Immunocytometry Systems, San Jose, CA), and the cell cycle distribution was calculated with the ModFit LT 2.0 Program (Verity Software House, Topsham, ME).
Caspase Analysis
Extracts were prepared by the suspension of 5 x 106 cells in 100 µL of a lysis buffer (i.e., 25 mM Tris-HCl [pH 7.5], 150 mM KCl, 5 mM EDTA, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate), incubation on ice for 10 minutes, and then centrifugation at 14 000g for 5 minutes at 4 °C. The resulting supernatants were collected and frozen at -80 °C or used immediately. Lysates (20 µL containing 5-10 µg of total protein) were mixed with 30 µL of assay buffer [50 mM piperazine-N,N'-bis(2-ethanesulfonic acid), 50 mM KCl, 5 mM ethylene glycol bis(ß-aminoethyl ether) N,N,N',N'-tetraacetic acid, 2 mM MgCl2, 1 mM dithiothreitol, and 0.1 mM phenylmethylsulfonyl fluoride], containing 100 µM of Z-DEVD-AFC (where DEVD is Asp-Glu-Val-Asp, Z is benzyloxycarbonyl, and AFC is 7-amino-4-trifluoromethyl coumarin). Caspase-3-like protease activity was measured at 37 °C with a spectrofluorometric plate reader (LS50B; The Perkin-Elmer Corp., Foster City, CA) in the kinetic mode with excitation and emission wavelengths of 400 nm and 505 nm, respectively. Activity was measured by the release of 7-amino-4-methyl coumarin (AMC) from the synthetic substrate Z-DEVD-AFC (Biomol, Plymouth Meeting, PA).
Mitochondrial Analysis
Cells were treated with the indicated amount of drug and 10 µM Ac-DEVD-fmk (N-acetyl-Asp-Glu-ValAsp-fluoromethylketone; Enzyme System Products, Livermore, CA), the cell-permeable caspase-3/caspase-7-selective inhibitor. Cells were then incubated for 10 minutes at 37 °C in culture medium containing 40 nM 3,3'-dihexyloxacarbocyanine iodide (DiOC6; Molecular Probes, Inc., Eugene, OR), followed by immediate analysis in a FACScalibur cytofluorometer. Fluorescence at 525 nm was recorded.
Immunofluorescence Assays
Hep-G2 (human hepatocellular carcinoma) cells were grown on glass coverslips in the presence or absence of drugs for 16 hours. Cells were fixed in paraformaldehyde, permeabilized in Triton X-100, and stained with an anti-ß-tubulin monoclonal antibody, followed by tetramethyl rhodamine B isothiocyanate-conjugated anti-mouse immunoglobulin G (IgG). To visualize filamentous actin filaments, we stained the cells with fluorescein isothiocyanate-conjugated phalloidin, as described previously (23). The type 1 nuclear mitotic apparatus protein was detected with monospecific human autoantibodies, as described previously by Andrade et al. (24). The secondary antibody was fluorescein isothiocyanate-labeled goat anti-human IgG (Tago, Burlingame, CA). Nuclei were stained with the DNA-binding dye 4',6-diamidino-2-phenylindole dihydrochloride (Molecular Probes, Inc.) according to the manufacturer's instructions.
Tubulin Assays
Assessment of the inhibition of tubulin polymerization and the evaluation of the inhibition of [3H]colchicine binding to tubulin were performed as described previously (25). In all experiments, tubulin without microtubule-associated proteins (20) was used. In brief, for inhibition of assembly, 10 µM (1.0 mg/mL) tubulin was preincubated with various concentrations of drug (4% [vol/vol] dimethyl sulfoxide as drug solvent) and 0.8 M monosodium glutamate for 15 minutes at 30 °C. The reaction mixture was placed on ice, and guanosine 5'-triphosphate (0.4 mM) was added. Reaction mixtures were transferred to cuvettes at 0 °C in Gilford 250 spectrophotometers (Beckman-Gilford, Fullerton, CA), baselines were established, and the temperature was increased to 30 °C with electronic temperature controllers (over a period of about 60 seconds). The IC50 value is the drug concentration required to inhibit 50% of the assembly, relative to an untreated control sample, after a 20-minute incubation. It should be noted that the bulk of polymer formed in the presence of glutamate consists of sheets of parallel protofilaments. The drug effects in this system are similar to those observed with a preparation containing tubulin and microtubule-associated proteins (i.e., microtubule proteins). The chief advantage of the glutamate system is that it unambiguously establishes tubulin as the drug target. For the colchicine-binding assay, reaction mixtures contained 1.0 µM (0.1 mg/mL) tubulin and 5.0 µM [3H]colchicine and were incubated for 10 minutes at 37 °C before filtration through a stack of two DEAE-cellulose filters. At this time in reaction mixtures without inhibitor, binding is 40%-50% of maximum, so that the inhibition of the rate of colchicine binding to tubulin can be measured accurately.
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RESULTS |
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In an initial screen performed by the National Cancer Institute's
Developmental Therapeutics Program, the mean 50% growth-inhibitory
concentration (GI50) of indanocine was less than or equal to
20 nM. In 29 of 49 cell lines, including a
doxorubicin-resistant breast cancer line, the GI50 for
indanocine was less than the lowest concentration tested (10
nM). Because the indanone is hydrophobic, its activity toward
the multidrug-resistant cells was surprising. To confirm this result,
we compared the effects of indanocine on the growth of the following
pairs of parent and corresponding multidrug-resistant lines (Table
1): MCF-7 and MCF-7/ADR, MES-SA and MES-SA/DX5,
MDA-MB-321 and MDA3-1/GP170+3-1, HL-60 and HL-60/ADR, CEM and
CEM/VLB100, KB-3-1 and KB-GRC-1, and MV522 and MV522/Q6 cells. The
multidrug-resistant cell lines have different multidrug resistance
mechanisms, including alterations of gp170 (mdr1 gene), gp180 (MRP
gene), and the glutathione transferase
isoform. In several of the
cell lines tested, the antiproliferative concentrations of indanocine
were equivalent or lower in the multidrug-resistant cells than in the
corresponding parent cells. Three of the cell lines tested (i.e.,
MCF-7, MES-SA, and HL-60) showed collateral sensitivity; i.e., the
multidrug-resistant cell line was substantially more sensitive to the
growth-inhibitory effects of indanocine than the parental cell line. An
example of collateral sensitivity is shown in Fig. 1,
where HL-60 and
HL-60/ADR cells were plated in a 96-well plate and then treated for 3
days with decreasing (1 : 2 dilutions) concentrations
of indanocine (from 1 µM) or paclitaxel (from 10
µM). The MTT assay was then performed at day 3. To prove
that P-glycoprotein expression did not confer resistance to the
indanone, we compared its effects on two carcinoma cell lines, KB-3-1
and MV522, and their corresponding transfectoma clones that
overexpressed P-glycoprotein (the mdr1 gene product), KB-GRC-1 and
MV522/Q6 (22). These transfectomas were resistant to paclitaxel, as
expected, but retained complete sensitivity to indanocine (Table 1
).
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The results obtained with the actively growing parent and
multidrug-resistant cell lines led us to test indanocine in
stationary-phase cell lines. As determined by flow cytometry after
propidium iodide staining, up to 81% of stationary MCF-7/ADR cells (1
week in confluent culture) were in the G1 phase of the cell
cycle (Fig. 2, B; middle panel). Remarkably,
indanocine treatment of stationary-phase multidrug-resistant cells, but
not parental cells, resulted in cell death (IC50 = 32
nM) (Fig. 2,
A). The cytotoxic effect of indanocine in
noncycling MCF-7/ADR cells was confirmed by the detection of an
apoptotic sub-G0/G1 population by flow cytometry
and by the activation of caspase-3 (Fig. 2,
B; left panel). Parental
(wild-type) MCF-7 cells were similarly growth arrested but did not show
apoptotic features (data not shown). In addition, normal peripheral
blood lymphocytes exposed to 1000-fold higher concentrations of
indanocine for 72 hours showed no loss of viability (data not shown).
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The study described above demonstrated that stationary MCF-7/ADR
cells, but not wild-type MCF-7 cells, were sensitive to treatment with
indanocine. To test this observation in another cell line pair that
displayed collateral sensitivity to indanocine in the
multidrug-resistant derivative line, we selected HL-60 and HL-60/ADR
cells because of the exquisite sensitivity of HL-60/ADR cells to
indanocine. In the experiment shown in Fig. 3, we tested the ability of
indanocine to activate caspase-3 in parental and multidrug-resistant
HL-60 cells. Caspase-3, considered an "executioner" caspase, is
implicated in the last and irreversible phase of the apoptotic caspase
pathway and is activated by upstream "initiator" caspases, such as
caspase-8 and caspase-9 [reviewed in (26)]. Caspase activity
was measured by use of the fluorogenic caspase-3-specific substrate
DEVD-AMC. HL-60/ADR cells incubated with 10 nM indanocine
showed a time-dependent increase in caspase-3 activity compared with
untreated cells, reaching a maximum at 24 hours. In contrast, wild-type
HL-60 cells showed only a slight increase in caspase-3 activity, about 25% of
that obtained in the multidrug-resistant cells (Fig. 3,
A).
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Effect on Tubulin Polymerization
Indanocine did not change the flow cytometry profile of
stationary-phase cells stained for DNA, other than to cause the
appearance of hypodiploid apoptotic cells in multidrug-resistant
cultures (Fig. 2). However, concentrations of the drug that inhibited
cell proliferation caused a rapid increase in the number of cells in
G2/M phases in growing cultures (data not presented).
Antimitotic drugs usually interfere with cellular microtubules by interacting with tubulin (3). Using glutamate-induced assembly of purified tubulin (containing no
microtubule-associated proteins) as our assay, we found (Fig. 4, A) that
indanocine inhibited tubulin assembly in a manner comparable to that of nocodazole rather than
inducing
polymerization as would paclitaxel. This observation led us to perform a quantitative analysis
(Fig. 4,
B), in which we found that indanocine was nearly as potent as combretastatin A-4 (a gift of Dr.
G. R.
Pettit, Arizona State University, Tempe, AZ) as an inhibitor of tubulin assembly. We measured
the
extent of tubulin assembly after a 20-minute incubation at 30 °C and determined that the IC50 of combretastatin A-4 was 1.20 ± 0.03 µM (mean
± standard
deviation; n = 4) and that the IC50 of indanocine was 1.7 ± 0.1
µM (n = 3). Both compounds practically eliminated the binding of 5 µM
[3H]colchicine to 1 µM tubulin when present at 5 µMcombretastatin A-4 inhibited 98% ± 4% of colchicine
binding (n
= 4), and indanocine inhibited 95% ± 2% of colchicine binding (n
= 4). The effects of various concentrations of the two drugs on colchicine binding are
shown in
Fig. 4,
C. Neither agent inhibited the binding of [3H]vinblastine to tubulin
(single experiment).
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COS-1 and Hep-G2 cells were grown on glass coverslips and treated
with various concentrations of indanocine, nocodazole, or vinblastine
sulfate. The microtubule network was then visualized by indirect
immunofluorescence with an anti-ß-tubulin antibody, and the
microfilament network was stained with fluorescein
isothiocyanate-coupled phalloidin. COS-1 and Hep-G2 cells were used for
these studies because of their clearly defined microtubule and
microfilament networks, respectively. Untreated cells had extensive
microtubule systems with perinuclear organizing centers (Fig.
5, a and b) and had microfilament bundles and stress
fibers that were predominantly aligned with the major axis of the cell
(Fig. 6,
a and b).
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Indanocine-treated Hep-G2 cells had a rounded shape, and the microfilament cytoskeleton in
these
cells was disorganized, characteristic of treatment with a depolymerizing agent (Fig. 6, c). After a
1-hour incubation in 0.5 µM indanocine, some cells had a characteristic rounded
shape,
but other cells had normal microfilament bundles (Fig. 6,
d). A similar
effect
was observed after
exposure to nocodazole (Fig. 6,
e) or vinblastine sulfate (Fig. 6,
f). This is probably an indication that
the microfilament breakdown observed at 5 µM indanocine is not a direct effect
but
rather is a consequence of the rapid and potent disruption of the microtubule network.
After treatment of Hep-G2 cells with 100 nM indanocine, the subcellular
localization of
the mitotic apparatus (as shown by human autoantibodies against the type 1 nuclear mitotic
apparatus
protein) was determined by immunofluorescence. In control cells undergoing mitosis, the type 1
nuclear
mitotic apparatus protein was localized at the poles of the mitotic spindle (Fig. 7, a). In cells exposed to 100 nM indanocine and arrested in the M phase, type
1
nuclear mitotic apparatus protein was distributed in spots scattered over the nucleus (Fig. 7,
b). A
similar effect was observed with 3.3 µM nocodazole (Fig. 7
, c).
Paclitaxel treatment did
not interfere with the subcellular distribution of the type 1 nuclear mitotic apparatus protein,
although it
affected the formation of a functional mitotic spindle (Fig. 7,
d).
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DISCUSSION |
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Modification of the apoptotic machinery has been proposed as an explanation for the de novo and acquired cross-resistance to multiple antineoplastic agents. It has been shown that the Bcl-2 protein may protect cancer cells from drug-induced apoptotic cell death (18,30,31). Microtubule-disrupting drugs, such as vincristine, vinblastine, and colchicine, and microtubule-stabilizing drugs, such as paclitaxel and doxetaxel, induce growth arrest, which is followed by phosphorylation and inactivation of Bcl-2, which eventually leads to apoptotic cell death in the G2/M phase of the cell cycle (32-34). In contrast, cells in the stationary phase are generally resistant to many of these agents, and phosphorylation of Bcl-2 in the G0/G1 phase is generally not observed. This property limits the utility of tubulin-binding drugs for the treatment of malignant tumors containing only a few proliferating cells (i.e., tumors with a low S-phase fraction).
As with other microtubule-damaging drugs, indanocine arrested the growth of multidrug-sensitive cancer cells at the G2/M boundary and induced apoptotic cell death. The nanomolar concentrations of indanocine that induced apoptosis in multidrug-resistant cells did not kill wild-type G1-phase cancer cells or quiescent normal peripheral blood lymphocytes. Nanomolar concentrations of indanocine forced stationary, multidrug-resistant cells into the apoptotic program. That these cells were arrested in the G0/G1 phase of the cell cycle was confirmed by cytofluorometric analysis. Apoptosis, in these cells, was confirmed by the appearance of a subdiploid-DNA flow-cytometry peak and by caspase-3 activation. Compared with their respective parental lines, five multidrug-resistant cell lines displayed higher or indistinguishable sensitivity to indanocine toxicity.
The cell lines hypersensitive to indanocine have modified various systems for multidrug resistance. MES-SA/DX5 cells overexpress P-glycoprotein, and MCF-7/ADR cells overexpress P-glycoprotein and also have an embryonic |gp isoform of glutathione transferase (35). HL-60/ADR cells express the MRP/gp180 protein. The other two cell lines that we examined, with unaltered sensitivity to indanocine, only express P-glycoprotein.
The fact that sensitivity to indanocine was retained by all of the multidrug-resistant cells tested, including both KB-3-1 and MV522 transfectomas that overexpress the P-glycoprotein, suggests that this agent acts independently of the P-glycoprotein hydrophobic multidrug transporter and/or that the cytoskeletal disorganization induced by the indanone interfered with P-glycoprotein functions. In other experiments, indanocine did not alter the rate of rhodamine efflux from loaded cells (data not shown). Thus, it seems probable that the uptake and regulation of indanocine do not depend on or directly influence the P-glycoprotein multidrug transporter.
Several different microtubule-disrupting agents have been developed that do not depend on the 170-kd P-glycoprotein and that display antiproliferative activity against multidrug-resistant cancer cells (23,36). It should be of interest to determine whether any of these agents, like indanocine, are cytotoxic to noncycling mdr1-expressing cells. Such comparative studies could answer the question whether the mechanism of action of indanocine is related only to inhibition of microtubule function. If indanocine-induced cell death involves another intracellular target, then other microtubule-disrupting agents with antiproliferative activity toward multidrug-resistant cells should not be able to induce apoptosis in the G0/G1 phase.
The observation that indanocine kills noncycling, multidrug-resistant cells has practical implications. The low percentage of cycling cells in many human solid tumors limits the potential of antimitotic drugs. The combination of a drug that is selectively cytotoxic to nondividing, multidrug-resistant cells and an antineoplastic agent that kills tumors with abnormalities of cell cycle checkpoints could represent an exceptionally effective approach to eradicating malignant cells while sparing most normal tissues. Thus, we suggest that indanocine and related indanones be considered lead compounds for the development of chemotherapeutic strategies for drug-resistant malignancies.
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NOTES |
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Manuscript received June 29, 1999; revised November 4, 1999; accepted November 18, 1999.
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