Affiliation of authors: Department of Cell and Cancer Biology, Medicine Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD.
Correspondence to: Leonard Neckers, Ph.D., National Cancer Institute, 9610 Medical Center Dr., Suite 300, Rockville, MD 20850 (e-mail: len{at}helix.nih.gov).
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ABSTRACT |
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INTRODUCTION |
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Since Hsp90 is considered to be a novel molecular target for anticancer therapeutics (12), we have focused our attention on discovering less toxic agents that
are capable of
inhibiting Hsp90 function in a manner similar to that seen with the ansamycins and radicicol.
Both drugs
bind to an atypical nucleotide-binding pocket in the amino terminus of Hsp90 that shares
homology to
the adenosine triphosphate (ATP)-binding domain of the bacterial DNA gyrase B protein (16-21). Since the nucleotide-binding site of gyrase B is the target of the
coumarin
antibiotics, including novobiocin, chlorobiocin, and coumermycin A1 (Fig. 1),
we investigated whether these agents could also interact with Hsp90 and, if so, whether they
could
affect its chaperone activity. Since novobiocin, in particular, has been a clinically utilized
antibiotic with
tolerable toxicity, we were particularly interested in this compound's potential as an
anti-Hsp90
agent.
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MATERIALS AND METHODS |
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SKBR3 and MCF7 human breast carcinoma cells were purchased from the American Type Culture Collection (Manassas, VA), and v-src-transformed NIH 3T3 mouse fibroblasts were obtained from the National Cancer Institute, Bethesda, MD. The cells were maintained in Dulbecco's modified Eagle medium (DMEM) with 10% fetal bovine serum (Whittaker Bioproducts, Walkersville, MD), 1 mM glutamine, and 10 mM HEPES (pH 7.3) at 37 °C in an atmosphere of 5% carbon dioxide. When they were approximately 70% confluent, the cells were treated with various agents. Stock novobiocin, etoposide, and doxorubicin (Sigma Chemical Co., St. Louis, MO) solutions were prepared in distilled water. Chlorobiocin and geldanamycin (both from the Developmental Therapeutics Program, National Cancer Institute) and coumermycin A1 (Sigma Chemical Co.) were prepared in 100% dimethyl sulfoxide (DMSO).
Preparation of Novobiocin-Sepharose 6B and the Solid-Phase Novobiocin-Binding Assay
Novobiocin-Sepharose was prepared as follows: Three grams of epoxy-activated Sepharose 6B (Sigma Chemical Co.) was thoroughly washed and then swollen in 100 mL of distilled water for 1 hour at room temperature. The resin was washed further with coupling buffer (0.3 M sodium carbonate [pH 9.5]). The gel was mixed with 400 mg of novobiocin (sodium salt; Sigma Chemical Co.) in 10 mL of coupling buffer and incubated at 37 °C with gentle rotation for 20 hours. The excess ligand was washed away with coupling buffer, and the remaining epoxy-active groups were blocked with 1 M ethanolamine in coupling buffer for 12 hours at 30 °C with gentle shaking. The gel was thoroughly washed sequentially with coupling buffer, 0.5 M NaCl in coupling buffer, distilled water, 0.5 M NaCl in 0.1 M sodium acetate (pH 4), and again in distilled water; it was then equilibrated in 25 mM HEPES (pH 8) containing 1 mM EDTA, 10% ethylene glycol, and 200 mM KCl and kept at 4 °C protected from light (22).
Cell lysates were prepared in TNESV buffer (i.e., 50 mM Tris-HCl (pH 7.4), 1% Nonidet P-40, 2 mM EDTA, 100 mM NaCl, and 1 mM sodium orthovanadate), containing 1 mM phenylmethylsulfonyl fluoride, 20 µg of aprotinin, and 20 µg of leupeptin per milliliter. Total protein (300 µg per assay) was incubated with novobiocin-coupled resin (100 µL) in TNESV buffer, with or without previous addition of various drugs or ATP, for 1 hour at 4 °C with gentle rotation. The beads were then thoroughly washed with ice-cold TNESV buffer. Bound proteins were eluted by being boiled in reducing loading buffer and were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by either silver staining (Bio-Rad Laboratories, Hercules, CA) or western blotting with appropriate antibodies.
Preparation of Geldanamycin-AffiGel Beads
Geldanamycin was derivatized and immobilized as previously reported (12). Briefly, 1,6-hexanediamine was added to geldanamycin (10 mM in chloroform) at a 10-fold molar excess and allowed to react for 2 hours. After aqueous extraction, 17-hexamethylenediamine-17-demethoxygeldanamycin was dried, redissolved in DMSO, and reacted with AffiGel 10 resin (Bio-Rad Laboratories). Before use, the geldanamycin-AffiGel beads were washed in TNESV buffer and blocked with 1% bovine serum albumin.
Preparation of Radicicol-Sepharose Beads
Twenty-five milligrams of the radicicol derivative KF66658 (Kyowa Hakko Kogyo Co., Ltd., Tokyo, Japan) was dissolved in tetrahydrofuran and added to 1 mL of epoxy-activated hydroxyapatite-Sepharose beads (Pharmacia LKB Biotechnology AB, Uppsala, Sweden). After 5 days of end-over-end mixing at room temperature, the beads were transferred into tetrahydrofuran/methanol (1 : 1, vol/vol) and then 20 mg of acetic anhydride was added. After end-over-end mixing at room temperature for 1 hour, two volumes of 1 M Tris-HCl (pH 7.5) were added for 15 minutes. Finally, the resin was washed three times in TNESV buffer lacking vanadate and, before use, was blocked in 1% bovine serum albumin.
Preparation of Hsp90 Constructs
Full-length chicken Hsp90 complementary DNA (cDNA) and the 380-728
carboxyl-terminal chicken Hsp90 deletion fragment, both subcloned in pGEM-7Z vector
(Promega
Corp., Madison, WI), were gifts of Dr. David Toft (Mayo Clinic, Rochester, MN), and their
method of
preparation was described previously (23). The amino-terminal
1-222
fragment of chicken Hsp90 was obtained from full-length chicken Hsp90 cDNA by polymerase
chain
reaction with the use of two primers designed to contain BamHI and EcoRI
restriction sites, and the DNA fragment was then subcloned in pBluescript II SK vector
(Stratagene
Cloning Systems, La Jolla, CA).
In Vitro Transcription and Translation
Recombinant proteins were expressed from 1 µg of plasmids by in vitro transcription and translation with the use of the TnTTM Coupled Rabbit Reticulocyte Lysate Kit (Promega Corp.) in the presence of translation-grade [35S]methionine (1458 Ci/mmol; ICN Pharmaceuticals, Inc., Costa Mesa, CA), using the appropriate DNA polymerase and following the manufacturer's instructions. The material from in vitro translation reactions (12-24 µL) was incubated with various concentrations of novobiocin or geldanamycin. After mixing at 4 °C for 30 minutes, 40 µL of resin with immobilized geldanamycin or 100 µL of novobiocin resin was added, and the mixture was incubated for 60 minutes at 4 °C while rotating. Resins were washed three times with TNESV buffer and boiled in sample buffer. After separation by SDS-PAGE, proteins were visualized by silver stain or autoradiography.
Western Blotting
Cells were lysed with TNESV buffer containing protease inhibitors. Total protein (50 µg) was separated on 10% polyacrylamide gels containing SDS, transferred to a nitrocellulose membrane by electroblotting, and blocked for 2 hours with a solution containing 5% nonfat dry milk, 10 mM Tris-HCl (pH 7.5), 2.5 mM EDTA (pH 8), 50 mM NaCl, and 0.05% Tween 20. The membranes were probed with the indicated primary antibodies and then by secondary antibodies conjugated to horseradish peroxidase, and the signal was detected with the use of chemiluminescence reagents (Pierce Chemical Co., Rockford, IL).
Mononuclear Cell Preparation
Thirty milliliters of human blood collected in a heparinized syringe was layered on a 20-mL Ficoll-Paque (Pharmacia LKB Biotechnology AB) cushion and centrifuged for 30 minutes at 1000g at 20 °C to separate the mononuclear cells. After isolation, the cells were washed twice with sterile phosphate-buffered saline (PBS), collected by centrifugation at 1000g for 10 minutes at 20 °C, and then incubated in DMEM supplemented with 10% fetal bovine serum. After 6 hours, the mononuclear cells were treated with novobiocin and further incubated at 37 °C overnight.
Treatment of Mice With Novobiocin
Mice were maintained in accordance with the guidelines of the National Institutes of Health. Healthy C57BL/6 mice weighing approximately 15 g were placed on a 5-day treatment schedule consisting of twice-daily intraperitoneal injections of novobiocin (100 mg/kg body weight) formulated as a 15-mg/mL solution in sterile water. The animals were killed 3 hours after the last injection. Their spleens were removed, minced, and briefly sonicated while suspended in cold PBS and centrifuged at 450g for 2 minutes at 4 °C. The supernatant (isolated cells) was then separated, and the cells were collected by centrifugation at 900g for 10 minutes at 4 °C. Cells were lysed with TNESV buffer containing protease inhibitors. The amount of total protein was assessed by the Bradford method (Bio-Rad Laboratories). Cell extracts were subjected to denaturing PAGE, electrotransferred, and blotted for Raf-1 protein. Films were scanned into a Power Macintosh 9500 computer, and the optical density of the Raf-1-specific bands was determined with the use of National Institutes of Health Image Software.
Statistical Methods
The means and standard deviations were determined and the statistical analysis was performed with the use of Microsoft Excel 98. P values reported are two-sided.
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RESULTS |
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We immobilized novobiocin in a manner previously shown not to
interfere with its ability to bind to topoisomerase II, the eukaryotic
homologue of bacterial DNA gyrase B (22). Immobilized
novobiocin bound in a hydrophobic manner (i.e., binding was resistant
to multiple washes in 0.6 M NaCl) to either pure Hsp90 (not
shown) or Hsp90 present in cell lysate (Fig. 2, A
and B). Preincubation of the lysate for 30 minutes with either excess
soluble novobiocin, chlorobiocin, coumermycin A1, or ATP inhibited, in
a dose-dependent manner, subsequent Hsp90 binding to
novobiocin-Sepharose (Fig. 2,
A and C). It is interesting that we
failed to observe efficient competition between either soluble
geldanamycin or radicicol and immobilized novobiocin for binding to
Hsp90 (data not shown), although soluble novobiocin was able to compete
with both geldanamycin- and radicicol-affinity beads for binding of
Hsp90 (Fig. 3)
.
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Intrigued by the fact that neither geldanamycin nor radicicol could
effectively compete Hsp90 binding to immobilized novobiocin, we
investigated the possibility that novobiocin bound to a unique location
on Hsp90. Both crystallographic and biochemical studies of the
amino-terminal region of Hsp90 (18-20) have revealed the
location of the geldanamycin-binding site to be uniquely contained
within the first 221 amino acids. Thus, an amino-terminal fragment
(designated 1), consisting of amino acids 1-221 of chicken Hsp90,
binds immobilized geldanamycin [see (18) and Fig.
4
]. To our surprise, however, this fragment did not
bind to novobiocin-Sepharose beads (Fig. 4
). In contrast, a
COOH-terminal fragment of Hsp90 (designated
2, representing amino
acids 380-728) that does not bind to immobilized geldanamycin does
bind efficiently to novobiocin-Sepharose, and the binding is
efficiently competed by excess soluble novobiocin (Fig. 4
). These data
indicate that the novobiocin-binding site on Hsp90 is different from
the previously described amino-terminal geldanamycin/nucleotide-binding site.
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A number of client protein kinases and transcription factors are recovered from cells associated with Hsp90. Both geldanamycin and radicicol dissociate these complexes and cause the destabilization and depletion of associated client proteins (5,10,11). Because novobiocin binds to a unique site on Hsp90, we wished to test its ability and the ability of structurally related coumarin antibiotics to manifest similar effects.
We treated SKBR3 cells and v-src-transformed NIH 3T3 fibroblasts with increasing
concentrations of these drugs for 16 hours and then assessed the protein levels of Raf-1, p185erbB2, mutant p53, and p60v-src. The level of p185erbB2
was
reduced by 80% relative to untreated controls after overnight treatment with 800 µM novobiocin and by 40% compared with controls after overnight treatment with
300 µM drug (Fig. 5, A). Raf-1, p60v-src, and
mutant p53
levels were also markedly reduced after 16 hours of novobiocin treatment. In v-src-transformed
NIH
3T3 fibroblasts, the level of p60v-src protein was reduced by 50% after
exposure
to 600 µM novobiocin (Fig. 5
, B). Similar to geldanamycin
(5),
novobiocin lowered mutant p53 protein levels in SKBR3 cells while depleting Raf protein to an
undetectable level in the same cells (Fig. 5,
A). As controls for protein
loading
and nonspecific toxicity,
the steady-state levels of scinderin, an actin-associated protein, and glucose-regulated protein 78
(BiP
or Grp78), an Hsp70 family chaperone localized to the endoplasmic reticulum, were monitored;
they
were found not to be altered by the doses/exposure times of novobiocin used in these
experiments (Fig.
5,
B). General interference with protein synthesis is not a likely
explanation for
these results, since
overnight cycloheximide treatment of SKBR3 cells did not affect the steady-state levels of the
proteins
affected by novobiocin (data not shown). Finally, although novobiocin inhibits topoisomerase II
at the
concentrations used (100-1000 µM), the drug had no effect on the stability of this
protein
(Fig. 5,
A).
|
To determine whether the effects of novobiocin described above could
be observed with structurally related coumarin antibiotics, we tested
whether chlorobiocin and coumermycin A1 could deplete Hsp90 client
proteins. Both p185erbB2 and Raf-1 were depleted by these
drugs in a manner similar to novobiocin treatment, although at lower
drug concentrations (Fig. 6, A and B). Maximal depletion of Raf-1 and
p185erbB2 occurred with 500 µM chlorobiocin and
100 µM coumermycin A1, respectively. Scinderin, the
related actin-binding protein gelsolin, and the chaperone Hsc70 all
were unaffected by either of these drugs (Fig. 6,
A
and B).
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Effect of Novobiocin on Raf-1 in Normal Human Peripheral Blood Cells and in Mouse Splenocytes
To determine whether novobiocin could affect Raf-1 in human
peripheral blood mononuclear (PBM) cells, we cultured freshly prepared,
nonstimulated PBM cells with novobiocin for 14 hours and found Raf-1
protein to be depleted in a dose-dependent manner (Fig.
7, A). Gelsolin levels remained unaltered, even at
the highest drug concentration tested, and treated cells remained
viable as assessed by trypan blue dye exclusion.
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DISCUSSION |
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While the ansamycins and radicicol are exciting lead compounds with promising clinical utility, they may have a low therapeutic index, and these drugs display in vivo toxic effects that appear to be unrelated to their Hsp90 antagonism. Therefore, we have been searching for other compounds capable of interfering with Hsp90 function in a manner similar to that of the ansamycins. In 1997, Bergerat et al. (17) described a glycine-rich ATP-binding motif in bacterial DNA gyrase, and these investigators reported that Hsp90 contained a highly homologous region in its amino-terminal domain. This domain is fully contained within the ansamycin-binding pocket, and it has been shown to be the binding site for ATP, geldanamycin, and radicicol (16,18-21). The homology between DNA gyrase and Hsp90 nucleotide-binding domains prompted us to investigate whether topoisomerase (gyrase) ATP-binding site inhibitors might also bind to and interfere with the function of Hsp90.
We show here that novobiocin and two related coumarin antibiotics, chlorobiocin and
coumermycin A1 (see Fig. 1), bind to Hsp90. Thus,
novobiocin-Sepharose is able to affinity
precipitate Hsp90 from a cell lysate in a manner inhibited by excess ATP, and soluble
novobiocin can
compete with either immobilized geldanamycin or radicicol for binding to Hsp90. Since neither
soluble
geldanamycin nor radicicol is able to compete with novobiocin-Sepharose for Hsp90 binding, we
thought it most likely that the novobiocin-binding site must be adjacent to or overlapping with,
but not
identical to, the ansamycin/radicicol-binding pocket. Such a hypothesis is consistent with a report
(29) that the DNA gyrase B novobiocin-binding site overlaps, but is not
identical to, the gyrase ATP-binding motif. It is intriguing, however, that the binding data that we
have
obtained with Hsp90 deletion mutants suggest that novobiocin specifically interacts with a
previously
unrecognized domain in the carboxy-terminal portion of Hsp90, quite distinct from the
amino-terminal
site where geldanamycin binds. The hypothesis that the binding sites for novobiocin and
geldanamycin
are spatially separated suggests a mechanism by which novobiocin binding to Hsp90 can
interfere with
the binding of geldanamycin or radicicol, since the amino and carboxy termini of the chaperone
are
thought to interact closely with each other in solution (30). Indeed, a
molybdate-influenced domain in the extreme carboxy terminus of Hsp90 has recently been
suggested to
regulate Hsp90 conformation and to affect geldanamycin binding (31).
Although novobiocin appears to bind to a site on Hsp90 that is different from the geldanamycin/radicicol-binding site, it, like geldanamycin and radicicol, is able to interfere with the chaperone function of Hsp90 and to deplete tumor cells of a series of Hsp90-dependent signaling proteins. Thus, in SKBR3 breast cancer cells, a 16-hour exposure to novobiocin reduced p185erbB2, mutated p53, and Raf-1 protein levels in a dose-dependent fashion, with maximal activity occurring at 500-800 µM novobiocin. The p60v-src protein was also reduced in v-src-transformed NIH 3T3 cells after a 16-hour exposure to 600 µM novobiocin. Although the novobiocin analogues chlorobiocin and coumermycin A1 were similarly effective at depleting Hsp90-dependent signaling proteins, other topoisomerase inhibitors that do not bind topoisomerase at its nucleotide-binding domain, such as etoposide and doxorubicin, were inactive in this regard. The actin-associated proteins scinderin and gelsolin, the Hsp70 family member BiP (Grp78), and the constitutively expressed Hsp70 homologue Hsc70 were not affected by novobiocin or its analogues.
Novobiocin is a well-studied antibiotic, whose pharmacokinetics and toxicity profile are clearly understood. In humans, doses of 4 g/day (well below the maximum tolerated dose) yield a plasma level of 200-300 µg/mL or higher, 2 hours after oral administration, corresponding to a drug concentration of 300-500 µM (32,33). Since our results demonstrated that novobiocin can deplete Raf-1 in human PBM cells exposed to the drug at these concentrations in vitro, we examined whether in vivo activity could also be documented. None of the mice receiving intraperitoneal bolus injections of novobiocin twice daily for 5 days displayed any visible signs of toxicity, and their motor activity or body weight was not affected (data not shown). However, seven of 10 treated mice displayed significantly reduced levels of splenic Raf-1 protein (29% of control) when compared with vehicle-treated animals.
Although the plasma clearance half-life of novobiocin in mice is only 80 minutes, its plasma clearance half-life in humans has been measured to be 6 hours (32), and high doses of drug (4-6 g) can be readily administered (24). The importance of reducing the expression of particular signal transducers for improved response to chemotherapeutic agents is now appreciated. For example, recent clinical trials (34) have shown that HER-2-Neu antibody treatment nearly doubled the response rate to either paclitaxel or cyclophosphamide/doxorubicin regimens in patients with metastatic breast cancer. Our current findings suggest that novobiocin, or other coumarin antibiotics, should be further investigated for their in vivo anti-Hsp90 activity. These agents may represent a clinically better tolerated alternative to ansamycins or radicicol for depletion of Hsp90-dependent kinases and other signaling proteins, and their use might prove beneficial in combination with standard chemotherapy. Our data also suggest that pharmacologic interference with Hsp90 function at sites other than the chaperone's amino terminus is possible and should be further explored.
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Manuscript received March 13, 1999; revised November 12, 1999; accepted November 18, 1999.
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